Venkatachalam/aging_reg_dataset · Datasets at Hugging Face

MYC

4609

protein coding

TRE293

--

Aging

Prevent

SA--gal activity assay//Western blot

TRE293 cells treated with c-Myc shRNA demonstrated significant changes in senescence and induced the p53/p21CIP1 pathway.

BAG2

Downregulation

Western blot//BrdU assay//SA-¦Â-gal activity assay

In cells that demonstrated high levels of BAG2 in response to SP1 over-expression, transfection of c-Myc was shown to abrogate the BAG2 mRNA levels in a concentrationdependent manner.

p53-p21

Upregulation

Western blot

TRE293 cells treated with c-Myc shRNA demonstrated significant changes in senescence and induced the p53/p21CIP1 pathway.

Human

HL

cellular senescence

22,146,591

Gene

0

1

0

1

0

NLRX1

79671

protein coding

HCCLM3

--

Hepatocellular carcinoma

Accelerate

CCK-8 assay//SA--gal activity assay

We found that NLRX1 overexpression could hinder cell proliferation in HCCLM3 cells, while knocking down NLRX1 in Huh7 resulted in significant higher proliferation rates. NLRX1-OE significantly increased SA-¦Â-gal activity, indicating a higher proportion of aging in NLRX1-OE cells compared to cells transfected with vector.

p21

Upregulation

Western blot//RT-PCR

We found that NLRX1-OE could effectively upregulate P21 expression, and expressions of downstream targets of P21 including CDK1 and CDK2 were significantly repressed due to high level of P21 expression caused by NLRX1 overexpression, while knocking down NLRX1 in Huh7 resulted in increased expression of CDK1 and CDK2.

PI3K-Akt

Downregulation

Western blot

NLRX1-OE markedly inhibited the phosphorylation of mTOR, indicating that NLRX1 suppresses PI3K-AKT signaling.

Human

HL

cellular senescence

29,482,578

Gene

0

1

0

1

0

PRKAA1

5562

protein coding

NIH-3T3

--

Aging

Prevent

SA--gal activity assay

Next, we observed that when the H2O2-treated cells were incubated with medium containing Met and BBR, there was a dose-dependent decrease in the percentage of SA-¦Â-Gal-positive cells, which was similar to that caused by rapamycin treatment.As expected, when combined with CC, the effects of Met or BBR on senescence prevention were largely blunted when CC was coexisted, as indicated by the remarkable increase in SA-¦Â-Gal-positive cells.

LC3//p62

Downregulation//Downregulation

Western blot

With Western blot assay, we found that both Met and BBR alleviated the H2O2-induced accumulation of the LC3 and p62 proteins, and this alleviation was consistent with the effect of rapamycin.

mTOR

Downregulation

Western blot

Moreover, we found that BBR treatment significantly suppressed mTOR phosphorylation in senescent cells, similar and even stronger than that induced by rapamycin and insulin as an mTOR activation control.

Human

L

delay aging

26,890,602

Gene

0

1

0

IGFBP7

3490

protein coding

MDA-MB-231

--

Breast cancer

Accelerate

Cell morphological analysis//Flow cytometry//SA--gal activity assay

IGFBP7 treatment of MDA-MB-231 cells increased the percentage of cells in G1 (73.86%) compared to untreated cells (54.14%), with a consequent decrease in the percentage of cells in S phase (15.85% in IGFBP7-treated cells versus 33.8% in untreated controls).We found that MDA-MB-231 cells treated with IGFBP7 for 2 days underwent morphological changes characterized by cell aggregation with strong expression of senescence-associated ¦Â-galactosidase .

p21//p53

Upregulation//Upregulation

Western blot

In agreement with this, we found that IGFBP7-treated MDAMB-231 cells had higher p21 and p53 expression than untreated cells.

p38 MAPK

Upregulation

Western blot

Levels of phosphorylated p38 MAPK were dramatically increased by IGFBP7 treatment.

Human

L

apoptosis

21,997,538

Gene

0

1

0

1

0

1

0

MEF2A

4205

protein coding

HUVEC

Human umbilical vein

Cardiovascular disease

Prevent

Cell viability assay//SA--gal activity assay

The percentage of senescent phenotype cells in MEF2A overexpression group was significantly lower than that in the empty plasmid group and the MEF2A mutant group (pc-gab-MEF2A-MV30).MEF2A overexpression in HUVECs significantly increased cell viability.

p53

Downregulation

Western blot

The level of p53 was significantly decreased?in the MEF2A-overexpression HUVECs.

PI3K-p-AKT-SIRT1

Upregulation

qRT-PCR//Western blot

PIK3CA, PIK3CG, and SIRT1 mRNA levels were significantly up-regulated in the MEF2Aoverexpression HUVECs, and the protein levels of PIK3CA, PIK3CG, SIRT1, and p-Akt notably increased .

Human

L

Others

31,182,679

Gene

0

1

0

1

0

MIR584

693169

ncRNA

MGC803,SGC-7901

--

Gastric cancer

Accelerate

SA--gal activity assay//Western blot

The miR-584- 5p-mimic-transfected cells showed strong blue SA-¦Â-gal staining, while only scattered SA-¦Â-gal-positive cells were detected in miR-584-5p-inhibitor-transfected group compared with the corresponding control cells.The expression of p53, p21 and p16 was enhanced after miR584-5p-mimic-transfection. Conversely, the expression of p53, p21 and p16 was reduced after miR-584-5p-inhibitor transfection.

WWP1//p53

Downregulation//Upregulation

SA-¦Â-gal activity assay//Western blot//Flow cytometry

Intriguingly, co-transfection of MGC803 cells with miR-584-mimic and either LV-WWP1 or LV-NC showed that ectopic expression of WWP1 effectively reversed the promotion of cellular senescence resulting from miR-584-5p overexpression. Similarly, the effects of miR584-5p knockdown in SGC7901 were counteracted by WWP1 downregulation. The induction of SA-¦Â-Gal positively stained cells by miR-584-5p was also reduced by treatment of pifithrin-¦Á, indicating the involvement of p53 in miR-584-5p-induced cellular senescence.In accordance with the analysis of GC tissues, Western blot analysis showed an inverse correlation between WWP1 expression and miR-584-5p expression in GC cell lines and GES-1 cells.The expression of p53, p21 and p16 was enhanced after miR-584-5p-mimic-transfection.

TGF¦Â

Activation

Western blot//Immunofluorescence

Western blot analysis of the expression of WWP1 and key proteins involved in TGF¦Â signaling pathway showed that the protein levels of T¦ÂR1, Smad2, Smad4, and the CDK inhibitor p15 were increased after miR-584-5p-mimic transfection, while the opposite effects were observed after miR-584-5p-inhibitor transfection.However, the expression of T¦ÂR1, Smad2, Smad4, and p15 was decreased in LY364947 treated cells compared with that of the control group. Furthermore, immunofluorescence assays revealed a dramatically positive correlation between the levels of miR-584-5p and TGF¦Â.

Human

HL

cellular senescence

28,431,583

Gene

0

1

0

1

0

TP53

7157

protein coding

EJ

--

Aging

Accelerate

Knockdown//SA--gal activity assay

The adenovirus-mediated expression of p53 in EJ p53-null human bladder cancer cells promoted remarkable morphological changes and senescence-associated ¦Â-galactosidase (SA-¦Â-gal) activity, which are features of premature senescence phenotypes,within 6 days. Irreversible growth arrest, which is characteristic of cellular senescence, was also confirmed by a marked reduction in cell growth and decreased number of cells in the S-phase upon p53 expression.

p21//Akt

--//Activation

SA-¦Â-gal activity assay//Western blot

P53-mediated Akt activation and the inhibition of p53-induced SA-¦Â-gal activity by LY294002 treatment were also observed in H1299 human lung cancer cells .Akt and p21 separately regulate p53-induced ROS increases and cell cycle arrest, respectively.suppression of the induction of p21 expression successfully inhibited the p53-induced increase in SA-¦Â-gal activity and morphological changes. In addition, the p53-induced loss of proliferation and cell cycle arrest were inhibited by the suppression of p21 induction .We used a p21 shRNA retrovirus to specifically suppress the p53-mediated induction of p21 expression and cormnfied that its expression was effectively suppressed using Western blot analyses.

NF-¦ÊB-NOX4

--

SA-¦Â-gal activity assay//ChIP//RT-PCR

NOX4 knockdown resulted in the significant suppression of p53-induced SA-¦Â-gal activity but did not affect the loss of proliferation.Importantly, the chromatin immunoprecipitation (ChIP) assay results indicated that NF-kB binding to the putative binding site on the NOX4 promoter was enhanced by p53 expression, but this NF-kB binding was inhibited by BAY 11-7082 treatment. Furthermore, Akt inhibition by either LY294002 or Akt inhibitor IV treatment si gnificantly inhibited the increase in NF-kB binding in response to p53 expression, indicating that Akt activity is required for NF-kB binding to the NOX4 promoter.

Human

L

cellular senescence

28,691,365

Gene

0

1

0

1

0

NAMPT

10135

protein coding

MEF

--

Aging

Prevent

SA--gal activity assay

Over- expression of NAMPT in MEF cells slows down cellular senescence.Percentage of cells that is positive for SA- ¦Â- Gal activity in all Wt- MEF cells was dramatically increased from passage 6, consistent with the timing of CCP in Wt- MEF cells.we measured the expression level of several senescence marker genes. p16INK4a, p19ARF, and p21CIP1 are cell cycle inhibitors and their expression levels increase when cells enter senescence.

SIRT1

Upregulation

qRT-PCR

We finally examined whether SIRT1 activity is enhanced in response to NAMPT over- expression. Although SIRT1 has been shown to regulate cellular proliferation and senescence, this function is dependent on the robustness of the intrinsic NAMPT activity and NAD+ content. SIRT1 is also vital in regulating FOXO- mediated activation of antioxidant enzymes including SOD2 and Catalase. As we shown above, NAD+ content in Tg- MEF was higher than that in Wt- MEF cells. SIRT1 activity in Tg- MEF was approximately threefold higher than that in Wt- MEF cells, in line with the observed SOD2 and catalase up regulation.

--

Human

L

cellular senescence

29,178,516

Gene

0

1

0

SIRT1

23411

protein coding

WI-38

--

Aging

Prevent

BrdU assay//SA--gal activity assay

As observed for the growth curves,expression of SIRT1 alone does not significantly affect the percentage of cells in S phase or the percentage of SA-¦Â-gal-positive cells when compared with control infected cells. However, expression of PML IV results in a striking decrease of BrdU-positive cells and a huge increase in SA-¦Â-gal Activate cells, as reported previously. Double-infected PML IV¨CSIRT1 cells exhibit a significant increase in the percentage of cells in S phase as compared with PML-infected cells, in addition to a marked decrease in the percentage of SA-¦Â-gal-positive cells.

p53

Binding

Pull-down assay

We performed pull-down assays with either full-length GST¨Cp53 or a series of GST¨Cp53 fusions expressing only the N-terminal, middle or C-terminal regions of the protein, and in vitro translated SIRT1. We observed specific binding of SIRT1 to full-length p53 as well as to GST¨Cp53(290¨C393), while weaker binding was seen with GST¨Cp53(90¨C290) and no binding occurred with the N-terminal p53 fusion.

--

Human

HL

cellular senescence

12,006,491

Gene

0

1

0

1

0

CDK5

1020

protein coding

Saos-2

--

Aging

Accelerate

Knockdown//SA--gal activity assay

SiCDK5 or dnCDK5 partially inhibited this activity, and interestingly, CDK5 overexpression enhanced the number of SA-¦Â-Gal-positive cells.

Rac1

Downregulation

SA-¦Â-gal activity assay

In the presence of activated Rac1, most pRb induction of SA-¦Â-Gal was compromised, a phenotype that was exacerbated by expression of dnCDK5, roscovitine, or siCDK5 treatment . Coexpression of CDK5 with pRb and RacV12 resulted in a partial rescue of SA-¦Â-Gal expression, suggesting that CDK5 might be antagonizing some aspect of Rac1 function. However, in cells transfected with RB and RacN17, coexpression of dnCDK5 and siCDK5 or roscovitine treatment in no way inhibited pRbinduced SA-¦Â-Gal expression.

--

Human

L

cellular senescence

15,024,070

Gene

0

1

0

1

0

MORC3

23515

protein coding

WI-38

--

Aging

Accelerate

SA--gal activity assay//Western blot

The percentage of SA-¦Â-Gal¨Cpositive cells infected with the MORC3-retrovirus vector (51.0¡À5.1%) was significantly greater than that of controls infected with vector alone (2.3¡À0.8%). MORC3 expression also strongly induced p16, another senescence marker.

p53

Activation

Luciferase reporter assay

FLAG-MORC3 expression enhanced transcriptional activation by endogenous p53 of a reporter containing 12 p53-responsive elements (PG12S) in a dose-dependent manner, but not activation of a reporter containing 14 mutated responsive elements (MG14S).

--

Human

L

cellular senescence

17,332,504

Gene

0

1

0

1

0

IGFBPL1

347252

protein coding

MCF-7

--

Aging

Accelerate

Cell counting//Flow cytometry//SA--gal activity assay

Cell proliferation of MCF-7 cells was severely inhibited following IGFBP-rP1 transfection,whereas there were indistinguishable differences in cell proliferation between the control vector-transfected and the untransfected cells. On day 7, the IGFBP-rP1-transfected MCF-7 cells had an enlarged flat morphology,characteristic of senescent cells. At the same time, a significant increase in senescence-associated-¦Â-galactosidase (SA-¦Â-gal) activity was detected in IGFBP-rP1-transfected MCF-7 cells, as compared with control cells transfected with empty vectors or the cells that had not been transfected at all. Addition of CM from IGFBP-rP1-transfected cells to untransfected MCF-7 cells blocked cell proliferation and increased SA-¦Â-gal activity, whereas CM from the control vector (pEGFP-N1)-transfected or untransfected cells failed to inhibit cellular proliferation and induce senescence.A significant increase in the cell population at the G /G1phase of the cell cycle was detected in IGFBP-rP1-transfected MCF-7 cells, which is one of the typical phenotypes in cellular senescence.

p21

Upregulation

Knockdown//Western blot//Cell counting//SA-¦Â-gal activity assay

Basal and IGFBP-rP1-inducible p21 levels deceased dramatically in p21 knockdown MCF-7 cells.Accordingly, IGFBP-rP1-mediated pRb dephosphorylation was substantially reduced in these cells. We also examined cell proliferation and cellular senescence in response to IGFBP-rP1 in parental and p21 knockdown MCF-7 cells. The results showed that cell proliferation block and increased SA-¦Â-gal activity in response to IGFBP-rP1 were partially reversed by p21 knockdown. These results suggest that cellular senescence induced by IGFBP-rP1 is mediated at least in part by p21 up-regulation.

--

Human

L

cellular senescence

22,392,074

Gene

0

1

0

1

0

1

0

GADD45G

10912

protein coding

SK-Hep-1,SMMC-7721

--

Hepatocellular carcinoma

Accelerate

Cell morphological analysis//SA--gal activity assay

In agreement with previous observation, we confirmed that the induction of GADD45G expression in Sk-Hep1 cells (Tet-on-GADD45G-Sk-Hep1) and SMMC-7721 cells (Tet-on-GADD45G-SMMC-7721) resulted in characteristic morphological changes common in senescent cells and a dramatic increase in the proportion of SA-¦Â-Gal-staining positive cells.

SIP1

Upregulation

qRT-PCR//Western blot

Surprisingly, we found that the levels of SIP1 mRNA were significantly elevated in the cells upon GADD45G induction.The change in SIP1 mRNA expression was also reflected at the protein levels in Sk-Hep1 and SMMC-7721 cells upon GADD45G induction. In addition, GADD45G-induction of SIP1 expression similarly occurred in H-Ras V12-transformed mouse p53-/- liver progenitor cells (LPC-H-Ras V12 cells).

--

Human

HL

cellular senescence

26,378,039

Gene

0

1

0

1

0

HMGA2

8091

protein coding

WI-38

--

Retinoblastoma

Accelerate

SA--gal activity assay//Western blot//qPCR

We found that the ectopic overexpression of HMGA2 led to elevated SA-¦Â-galactosidase staining and acquisition of the SAHF-like chromatin foci in the nuclei. Meanwhile, the expression levels of the senescence-related proteins, such as p16, p21, p53 and phosphorylated p53, were prominently increased. The transcription level of HDM2 that reduces the stability of p53 protein was decreased together with a slightly up-regulated p14ARF, an inhibitor of HDM2, in WI38 cells expressing HMGA2 . Apparently, these results indicate that HMGA2 can induce the senescence phenotypes in WI38 cells.

E2F

Downregulation

Western blot

Surprisingly, both HMGA2 and Rb were detected in the same protein complex in our model (data not shown), together with the down-regulation of E2F target genes in WI38 cells in response to HMGA2 overexpression.

Rb

--

Western blot//qPCR//RT-PCR//Immunofluorescence

To further test whether Rb is a crucial factor in SAHF formation induced by HMGA2, E7 protein was used as the specific inhibitor of Rb , and the effect of E7 protein on the degradation of Rb was confirmed by Western blotting . Interestingly, we found that E7 protein was able to partly abolish the SAHF formation induced by HMGA2, as revealed by cellular immunofluorescence and decreased percentage of cells with SAHF .Furthermore, as another inhibitor targeted to Rb, SV40 had a similar effect to decrease the HMGA2-induced SAHF formation .

Human

L

cellular senescence

23,969,248

Gene

0

1

0

1

0

1

0

RRAD

6236

protein coding

NHF

--

Aging

Prevent

Knockdown//SA--Gal activity assay

We found that RRAD knockdown increased the ratio of senescent cells in both H2O2- and IR-induced senescence as determined by SA-¦Â-gal staining assay and BrdU incorporation assay.

p53//NF-¦ÊB

--//--

Knockdown//CHIP-Seq//qRT-PCR

We established p53 knockdown NHFs by a lentiviral based approach for the following functional assay. In OIS, p53 knockdown diminished H-Ras-induced RRAD up-regulation.p53 knockdown also abolished H 2O2-induced RRAD expression.In a published ChIP-Seq data set of IMR-90 cells, we noted that both p53 and NF-¦ÊB bind to the RRAD promoter region in adjacent sites.

--

Human

HL

cellular senescence

30,391,675

Gene

0

1

0

1

0

ING1

3621

protein coding

2BS

--

Aging

Accelerate

Immunofluorescence//SA--gal activity assay//SAHF

The results showed that 2BS cells overexpressing of p33ING1befficiently induced the expression of SA-¦Â-gal activity, flattened senescent cell morphology, and SAHF DNA staining pattern which is indicated by co-localization with H3K9Me3.

p16

Upregulation

qPCR//Western blot//Luciferase reporter assay

qPCR and Western blot results showed that introduction of p33ING1b upregulated p16INK4amRNA and protein levels.Moreover, p16INK4apromoter activity was upregulated by p33ING1b.The results showed that cells treated with p33ING1balone revealed strong SA-¦Â-gal staining and p16 increasing, whereas cells doubly treated with p33ING1b and p16-shRNA reversed the senescence phenotypes. As expected, p16-shRNA-infected cells displayed young phenotypes.

--

Human

L

cellular senescence

21,896,275

Gene

0

1

0

1

0

1

0

TWIST

100303576

protein coding

SW48

--

Aging

Prevent

BrdU assay//SA--gal activity assay

Higher percentage of SA-¦Â-gal-positive cells was found in the Sh-TWIST transfectants (filled columns) compared with the controls (open columns) in response to both agents in a dose-dependent manner. This was associated with a decreased cell proliferation rate examined by BrdUrd incorporating assay.These results suggest that suppression of TWIST expression promotes cellular senescence in PrEC.

p14//cH2AX

Downregulation//Upregulation

Western blot

We also found that the p14ARF expression levels were much lower in the TWIST over-expressing cells, either before or after exposure to H2O2 and CP (right panels), compared with the vector control, confirming the negative effect of TWIST on p14ARF. In addition, the p53 and p21 levels were lower in the TWIST over-expressing cells compared with the vector control, especially after drug treatment. Furthermore, we found that the level of cH2AX was higher in the TWIST over-expressing cells either before or after drug treatment compared with the vector control, which was associated with lower levels of phosphorylated Chk1/2 proteins, especially after drug treatment.

Mdm2-p53

--

Western blot

Western blotting analysis showed that the expression levels of p14ARF were much higher in the Sh-TWIST transfectants after exposure to H2O2 or CP in a dose-dependent manner. In contrast, the level of MDM2 was decreased, which was associated with up-regulation of p53 and p21, suggesting that inactivation of TWIST promotes p14ARFMDM2-p53-mediated DNA damage response pathway.

Human

L

cellular senescence

17,690,110

Gene

0

1

0

1

0

CAV1

857

protein coding

MEF

--

Aging

Accelerate

SA--gal activity assay//Western blot

In contrast, wild type MEFs over-expressing caveolin-1 developed a senescent phenotype 4 days after oxidative stress, as determined by senescent-associated ¦Â-galactosidase staining and p21 upregulation.

SIRT1

Binding

Pull-down assay

To determine whether Sirt1 binds directly to caveolin-1, we performed GST pulldown assays using recombinant Sirt1. We found that human recombinant Sirt1 bound to caveolin 1 (residues 82¨C101)-GST.The incubation with the purified GST-Cav-1 (82¨C101) peptide inhibited Sirt1 activity by- 45% compared to GST alone.

p53

--

Pull-down assay//IP//Western blot

We found that human recombinant Sirt1 bound to caveolin 1. However, Sirt1 strongly interacted with caveolin-1 72 hours after stimulation of WI-38 cells with sublethal doses of hydrogen peroxide that we have previously shown induce premature senescence.In contrast, free radicalinduced acetylation of p53 was significantly inhibited in cells lacking caveolin-1. Interestingly, downregulation of Sirt1 by siRNA enhanced oxidant-induced acetylation of p53 in caveolin-1 null MEFs to levels seen in wild type MEFs.

Human

L

cellular senescence

25,512,378

Gene

0

1

0

1

0

NQO1

1728

protein coding

2BS

--

Aging

Accelerate

BrdU assay//Cell proliferation assay//SA--gal activity assay//qRT-PCR

NQO1 depletion resulted in continuous cell growth compared with corresponding control lentiviral vector (Cont) infected cells . In addition, the morphology changed during the OIS, with senescent cells exhibiting the typical enlarged and flattened shape. Five days after introducing RasG12V, NQO1 depleted 2BS cells showed a different morphology compared with control 2BS cells. Accordingly, we found that 2BS cells with NQO1 depletion reduced with SA-¦Â-gal staining after expressing RasG12V. We observed that knockdown of NQO1 resulted in an increased percentage of 2BS cells with BrdU incorporation after expressing RasG12V. This result suggests that NQO1 depletion delayed the onset of RasG12V-induced cellular senescence.

p53

Binding

Western blot

To test the relevance of NQO1-p53 binding in OIS, p53 was immunoprecipitated in 2BS cells. Western blot analysis verified that NQO1 specifically interacted with p53 in premature senescence.

Nrf2-Keap1

--

Western blot//CHIP//qPCR//Knockdown//Luciferase reporter assay

MAF and KEAP1 were immunoprecipitated from 2BS cells, and western blot analysis verified that during OIS NRF2 was dislocated from KEAP1 and bound with MAF, suggesting that OIS activates NRF2 and results in NRF2-mediated up-regulation of NQO1. Next, we detected whether NRF2 bound to the endogenous NQO1 promoter by chromatin immunoprecipitation (ChIP) assay. Interaction between NRF2 and the NQO1 promoter region was detected by qPCR. As expected, interaction between NRF2 and NQO1 promoter only observed in senescent 2BS. We found that knockdown of NRF2 diminished its interaction with the NQO1 promoter. In addition, we also determined whether NRF2 affected NQO1 promoter activity with ARE by the luciferase reporter assay.

Human

HL

cellular senescence

26,078,718

Gene

0

1

0

1

0

1

0

1

0

RSL1D1

26156

protein coding

2BS

--

Aging

Prevent

SA--gal activity assay

No SA-¦Â-gal activity was observed in CSIG transfected 2BS and early-passage cells, whereas CSIG-CT transfected 2BS cells were strongly stained, as were late-passage (senescent) 2BS control cells.

PTEN

Downregulation

Western blot

As expected, CSIG overexpression diminished PTEN abundance.

--

Human

L

cellular senescence

26,686,419

Gene

0

1

0

S100A13

6284

protein coding

IMR-90

--

Aging

Accelerate

Knockdown//SA--gal activity assay

S100A13 overexpression promotes, whereas S100A13 knockdown delays replicative cellular senescence in IMR9 cells. S100A13 mutant significantly decreased the percentage of SA-¦Â-gal positive staining cells.

IL-1

Upregulation

Flow cytometry//Immunoblotting

FACS analysis of cell surface-bound IL-1¦Á showed that S100A13 overexpression significantly enhanced IL-1¦Á levels compared to empty vector control cells.

--

Human

L

cellular senescence

30,670,674

Gene

0

1

0

1

0

TRIB2

28951

protein coding

LoVo

--

Colorectal cancer

Prevent

Cell morphological analysis//Flow cytometry//Knockdown//SA--gal activity assay

TRIB2 knockdown cells showed higher rates of flat and enlarged senescence-like morphology.Cell cycle distribution of the cells transfected with TRIB2-specific siRNA were determined by flow cytometry.TRIB2 knockdown dramatically increased the G0/G1-phase ratios and reduced the S-phase ratios. The level of SA-¦Â-gal (a biomarker of senescence) was measured, and data showed that the percentage and strength of SA-¦Â-gal-positive cells were significantly increased in TRIB2 knockdown condition.

--

AP4-p21

--

Western blot//RT-PCR//Luciferase reporter assay//IP//CHIP

Silencing TRIB2 dramatically up-regulated p21 expression, inhibited cell growth, induced cell cycle arrest and enhanced cellular senescence. The luciferase assay indicated TRIB2 inhibited p21 promoter activities in an AP4-dependent manner. We then performed CCK8 and SA-¦Â-gal staining analysis to examine the effects of AP4 on TRIB2-mediated functions, and found that silencing AP4 could significantly block TRIB2 overexpression-promoted SW48 and LoVo cells growth and enhance TRIB2 overexpression-inhibited cellular senescence. We carried out co-IP assay using SW48 cells or HEK 293T cells co-transfected with Flag-AP4 and His-TRIB2. AP4 and TRIB2 were found to precipitate with each other.The results showed that overexpression of AP4 suppressed and co-transfection of TRIB2 further suppressed the activities of p21 promoter , which indicated TRIB2 enhanced the function of AP4.

Human

L

cellular senescence

30,541,550

Gene

0

1

0

1

0

1

0

1

0

LBR

3930

protein coding

TIG-7

--

Aging

Prevent

Knockdown//SA--Gal activity assay

We also found that the cells transfected with the LBR knockdown vector were stained with SA-¦Â-gal, which observation suggested that knockdown of LBR induced cellular senescence in TIG-7 cells.

--

Human

L

cellular senescence

30,615,890

Gene

0

1

0

1

0

PTEN

5728

protein coding

MCF-7

--

Aging

Prevent

Knockdown//SA--Gal activity assay

The SA-¦Â-Gal positivity of PTEN KO MEFs was dramatically attenuated in Torin1-treated cells compared to Rapa-treated cells.

--

p53-p21

--

Western blot

PTEN-depleted MCF7 cells displayed prematurely senescent phenotypes in a p53/p21-dependent manner.

Human

L

cellular senescence

30,337,688

Gene

0

1

0

1

0

MDM2

4193

protein coding

HCT116

--

Werner syndrome

Accelerate

SA--Gal activity assay

Ectopic expression of WRN attenuates the senescent phenotype induced by Etoposide,as shown by senescence-associated ¦Â-galactosidase (SA-¦Â-Gal) activity.

WRN

--

IP

WRN and MDM2 association was detected in co-transfected 293T cells by reciprocal immunoprecipitations.

--

Human

L

cellular senescence

30,532,073

Gene

0

1

0

ASPH

444

protein coding

FOCUS,HuH-7,HepG2

--

Hepatocellular carcinoma

Prevent

Knockdown//SA--Gal activity assay

Knockdown or knockout of ASPH, caused substantial increase in ¦Â-gal staining as a marker for the cells undergoing senescence.

GSK3¦Â

--

Western blot//IP//Immunoblotting

We found a significant increase of phospho-GSK3¦Â(inactivated form) with equal protein expression of GSK3¦Â when comparing the shASPH to the shLuc control.Direct protein-protein interaction between GSK3¦Âand ASPH was demonstrated by co-immunoprecipitation (IP).Immunoblotting with anti-GSK3¦Â revealed that GSK3¦Â bound to ASPH following co-transfection with WT-GSK3¦Âand ASPH.

--

Human

L

cellular senescence

26,683,595

Gene

0

1

0

1

0

RXRA

6256

protein coding

MRC-5

--

Aging

Prevent

Knockdown//SA--Gal activity assay

RXRA knockdown decreased cell proliferation as we observed a drop in the expression of the cell proliferation marker Ki©\67 and a decrease in the number of cells .Importantly, RXRA knockdown also caused an increased SA©\¦Â©\galactosidase activity.

p53

--

Knockdown//Western blot

Using the same approach,we further showed that cellular senescence induced by RXRA knockdown was also dependent on MCU and p53.

ITPR2-MCU

--

Knockdown//qRT-PCR//Western Blot//SA-¦Â-Gal activity assay

To assess whether this senescent phenotype was dependent on ITPR2, we knocked down ITPR2 together with RXRA.ITPR2 knockdown impaired the upregulation of CDKN1A and GDF15 . The proliferation arrest and the increase in SA©\¦Â©\galactosi-dase activity triggered by RXRA knockdown were alsoimpaired. Similar observations were made using either a siITPR2 pool or individual siRNAs targeting ITPR2. Using the same approach, we further showed that cellular senescence induced by RXRA knockdown was also dependent on MCU and p53.

Human

HL

cellular senescence

30,216,632

Gene

0

1

0

1

0

LRRK2

120892

protein coding

SH-SY5Y

--

Parkinson¡¯s disease

Accelerate

Western blot

Up-regulated ¦Â-galactosidase protein levels in G2019S-mediated phosphomimetic p53 and overexpression of p21 or G2019S LRRK2.

--

p53-p21

Upregulation

Western Blot//SA-¦Â-Gal activity assay

We observed a significant increase in ¦Â- gal protein level with the expression of T304/377D p53 (TD-p53) compared to that in the vector and WT-p53 in dSH-SY5Y cells . In addition, the overexpression of p21 and G2019S LRRK2 in dSH-SY5Y cells resulted in significant increases in ¦Â-gal protein expression. We found that ectopic expressions of eitherred fluorescence protein (RFP)-TD-p53 or RFP-p21 elevated GlycoGreen fluorescence by 2¨C3 folds.

Human

L

cellular senescence

30,712,480

Gene

0

1

0

TSHB

7252

protein coding

--

Adipose

Aging

Prevent

RT-PCR//Telomere length assay

In euthyroid morbidly obese subjects,both SAT and VAT TSHB gene expression was negatively associated with markers of AT senescence (TNF , TP53, BAX and DBC1). Confirming these findings, SAT TSHB mRNA was positively linked to telomere length in a subgroup of consecutive subjects , suggesting decreased senescence with less exposure to thyroid hormones. Furthermore, SAT TSHB mRNA was negatively correlated to specific senescence-associated glucosylceramides.

--

Human

L

cellular senescence

30,448,824

Gene

0

1

0

1

0

FXR1

8087

protein coding

UMSCC74A,UMSCC74B

--

Oral cancer

Prevent

Flow cytometry//Knockdown//SA--Gal activity assay

To investigate the underlying mechanism of reduction in number of viable cells following FXR1 KD, we performed cell cycle analysis of the FXR1 KD and control cells by flow cytometry. FXR1 KD induces cell cycle arrest in G /G1 providing evidence that FXR1 might regulate cell division in oral cancer.Silencing of FXR1 in multiple oral cancer cells also promotes senescence by positive SA-¦Â-gal staining.

TERC

--

Luciferase reporter assay

When full-length and truncated TERC (28 base deletion at 5¡¯-end,TERCmut£© are used for luciferase assay,TERC exhibits increased luciferase activity in control cells compared to TERCmut. However, in FXR1 KD cells, TERC exhibits a decreased luciferase activity compared to TERCmut indicating that FXR1 binds to specific G-rich sequences in these RNAs and possibly controls their turnover.Silencing of FXR1 reduces the level of TERC and subsequently interferes with the telomerase activity.

p53-p21

--

RT-PCR//Western Blot

Upregulation ofp53 and p21 protein levels are observed in the absence of FXR1 in WT p53 cell line.RT-PCR¡¢Western Blot£ºoverexpressed FXR1 in HNSCC destabilizes p21 mRNA and reduces its protein expression.concurrent overexpression of p21 and silencing of TERC significantly promoted senescence evidenced by staining of SA-¦Â-gal.

Human

HL

cellular senescence

27,606,879

Gene

0

1

0

1

0

1

0

CDKN2A

1029

protein coding

DFC

--

Aging

Accelerate

SA--Gal activity assay//Western blot

P16 gene silencing reduces the number of senescent cells.Only the protein expression of P16 in DFCs increased successively and significantly while the expression of all other examined cell cycle proteins including that of P21 was down-regulated significantly at higher cell passages.

--

Human

HL

cellular senescence

28,770,470

Gene

0

1

0

1

0

SIRT6

51548

protein coding

2BS

--

Aging

Prevent

Colony formation?assay//Flow cytometry//RT-PCR//Western blot

Western blot analysis revealed that the expression of SIRT6 was high in young cells, but decreased significantly during cellular senescence. Consistently, RT-PCR analysis revealed that mRNA levels of SIRT6 decreased in senescent cells .Another hallmarker of senescent cells is the ability of colony formation. We observed that 2BS cells overexpressing SIRT6 formed more colonies than control cells. However, SIRT6 shRNA-infected cells showed less colonies compared to control cells .It was obvious that SIRT6 shRNA-infected 2BS cells entered G1 cell cycle arrest.

p27

--

Western blot//RT-PCR//SA-¦Â-gal activity assay//Co-IP//Pull-down assay

In addition,SIRT6 silencing did not alter the protein levels of p16, p21, p53 and PTEN, but markedly increased p27 protein levels. Moreover, the real-time PCR analysis demonstrated similar p27 mRNA levels in both SIRT6-overexpressing and knockdown cells, suggesting that SIRT6 might decrease the p27 protein levels but not the mRNA levels.2BS cells co-expressing pLPC,pBabe-p27 exhibited G1 arrest and higher SA-¦Â-gal ctivity. Concurrently, the senescent markers of SIRT6-p27 appeared to be comparable with that of pLPC-pBabe control cells.Thus,the decrease of p27 mediated by SIRT6 is required for the restriction of cellular senescence. Colocalization experiments showed that both SIRT6 and p27 mainly colocalized in the nucleus. To further confirm the interaction of p27 and SIRT6, we performed a GST pull down assay using in vitro transcribed and translated p27 or SIRT6 and purified GST-SIRT6 or GST-p27.SIRT6 displayed a strong association with p27 in vitro.

--

Human

HL

cellular senescence

27,794,562

Gene

0

1

0

1

0

1

0

1

0

TIMELESS

8914

protein coding

2BS

--

Aging

Prevent

Western blot//qRT-PCR

The expression of circadian protein TIMELESS was down-regulated during the onset of OIS, while other members of circa-dian clock showed no significant changes in their expression.The downregulation of TIMELESS was also confirmed inreplicative senescence and H2O2 induced senescence,suggesting TIMELESS downregulation might be a commonevent happened during cellular senescence.

E2F1

--

Knockdown//Luciferase reporter assay//CHIP//qRT-PCR//Western blot

Chromatin immunoprecipitation (ChIP) assay was utilized to confirm the binding of E2F1 to the endogenous TIMELESS promoter.Interaction between E2F1 and TIMELESS promoter region was detected by qRT-PCR.As expected, interaction between E2F1 and TIMELESS promoter was observed in young 2BS cells.Furthermore, the mutation in E2F1 binding site of TIMELESS promoter region significantly reduced the luciferase activity in young 2BS cells.Knockdown of E2F1 by a sh-E2F1 lentiviral vector significantly down-regulated TIMELESS mRNA level in young 2BS cells.All above data support that E2F1 could bind to TIMELESS promoter and regulated the expression in young 2BS cells.

--

Human

L

cellular senescence

30,100,061

Gene

0

1

0

1

0

TSLP

85480

protein coding

BEAS-2B

--

Asthma

Accelerate

SA--gal activity assay//Western blot

Specifically,senescent cells increased >15 fold upon treatment with 1.5ng/ml TSLP.There was increased expression of SA-¦Â-gal and decreased Ki67 expression in BEAS-2B cells upon TSLP treatment.

--

Human

L

cellular senescence

24,167,583

Gene

0

1

0

1

0

UHRF1

29128

protein coding

2BS,IMR-90

--

Aging

Prevent

CCK-8 assay//EdU assay//Knockdown//SA--gal activity assay

Cell proliferation rates decreased in UHRF1 shRNA-infected cells. Growth curves of UHRF1 shRNA-infected 2BS cells or IMR9 cells were determined by CCK8 assay.The percentage of cells incorporating EdU also decreased in UHRF1 shRNA-infected 2BS cells or IMR9 cells. SA-¦Â-gal activity staining increased in UHRF1 shRNA-infected cells. Representative microscopic view of 2BS cells or IMR9 cells infected with the indicated vectors with SA-¦Â-gal activity staining.

PIM1

--

IP

In reciprocal immunoprecipitations, PIM1 was observed presentingin UHRF1 immunoprecipitates.

--

Human

HL

cellular senescence

28,394,343

Gene

0

1

0

1

0

1

0

1

0

WNT7A

7476

protein coding

A549

Lung

Lung cancer

Accelerate

CCK-8 assay//SA--gal activity assay

Histological analysis of the lung sections of the wild-type and Wnt7a-null mice showed no difference in basal staining for SA-¦Â-gal activity. However, a large number of SA-¦Â-gal-positive cells, predominantly in the tumor area, were observed in lung sections of the wild-type mice after urethane treatment, whereas Wnt7a-null mice,on the contrary, showed reduced or no SA-¦Â-gal staining.

--

SKP2-p27-pRb

--

Western Blot

Consistent with the changesin SKP2 and p27kip1 expression, the levels of phosphorylated(p-Rb-S780) retinoblastoma proteins in Wnt7a-null mice were dramatically increased as well . Cyclin D1, which is critical for Ser780 phosphorylation of Rb ,was also upregulated in Wnt7a-null mice, along with CDK2 andCDK6, components of the CDK/cyclin complex . Similarresults were obtained when using the lung lysates of wild-typeand Wnt7a-null in FVB/NJ mice.

Human

HL

cellular senescence

25,728,679

Gene

0

1

0

1

0

SPHK1

8877

protein coding

Adipose stromal cell

--

Aging

Prevent

BrdU assay//SA--gal activity assay

A SPHK1 inhibitor, significantly reduced the incorporation of BrdU into cellular DNA compared to control cells,indicative of the lowered proliferative ability.As with high-passage cells,SPHK1-depleted or SKI-treated hAD-SCs underwent accelerated cellular senescence as evidenced by higher SA-¦Â-gal activity.

S1P

--

SA-¦Â-gal activity assay//BrdU assay

To test this hypothesis, we investigated whether SPHK1 knockdown-induced cellular senescence could be attenuated by inhibition of ceramide synthesis or exogenous supple- mentation with S1P during cell culture. BrdU incorporation assays showed that the proliferation impeded by shRNA- mediated silencing of SPHK1 could not be recovered by single treatment with S1P or fumonisin B1 (FB1), a ceramide synthesis inhibitor, and even by co-treatment with both , indicating the irreversibility of cellular senescence. In contrast, the elevated activity of SA-¦Â-gal in SPHK1-silenced cells was reduced by simultaneous co-treatment with S1P and FB1, but not by single treatment with either one alone, suggesting that cellular senescence accelerated by SPHK1 depletion may result from increase in ceramide levels with concurrent decrease in S1P levels.

--

Human

L

cellular senescence

30,885,289

Gene

0

1

0

1

0

IGF1

3479

protein coding

VSMC

--

Aging

Accelerate

SA--gal activity assay

We observed a strong induction of SA-¦Â-gal activity following IGF-I stimulation in the confluent cells

--

ROS-p53

--

Immunoblotting//SA-¦Â-gal activity assay

We examined the involvement of ROS in the induction of cellular senescence by IGF-I.Treatment with an ROS scavenger, N-ace-tylcysteine (NAC), significantly suppressed the IGF-I-induced increase in SA-¦Â-gal activity. Furthermore, NAC treatment also suppressed the induction of cH2AX, p53, and p21 proteins, indicating that ROS are involved in IGF-I-induced cellular senescence. Treatment with an alternative ROS scavenger,Trion, also produced similar results (data not shown). In addition,we used p53-/- MEFs to clarify the role of p53 in IGF-I-induced cellular senescence; in the absence of p53, IGF-I-induced SA-¦Â-gal activation and p21 augmentation were inhibited.

Human

L

cellular senescence

22,877,754

Gene

0

1

0

SETD8

387893

protein coding

PC-3

--

Aging

Prevent

Flow cytometry//Knockdown//SA--gal activity assay

In PC3 cells harboring SETD8-specific siRNAs,we detected a greater extent of ¦Â-gal-positive staining,cell proliferation arrest,nuclear area enlargement,G2/M arrest .Consistent with its known pro-proliferation role,overexpression of SETD8 in the absence of dox treatment elevated the rate of cell growth.In dox-treated cultures undergoing senescence,introduction of ectopic SETD8 lessened cellular senescence induction to a large extent, as evidenced by the reversal of ¦Â-gal-positive staining,cell proliferation arrest,and upregulation of senescence markers p21,IL-6,andIL-8.

PPARc//p21

--//Downregulation

qRT-PCR//SA-¦Â-gal activity assay//CHIP//Flow cytometry

In support of its role as an upstream regulator, ectopic overexpression of PPARc led to an increase in the expression of SETD8 and H4K20me1, and conversely a decline in the p21 mRNA levels.To further demonstrate the role of PPARc in controlling senescence, we showed that overexpression of PPARc alleviated the extent of dox-induced cellular senescence, in terms of ¦Â-gal-positive staining , cell proliferation arrest, and SASP marker induction. Via ChIP-qPCR assay on the PPARc-overexpressing cells, we consequently found that the chromatin binding of PPARc increases on SETD8 promoter upon ectopic expression.Although RNAi-mediated abrogation SETD8 in cycling cell expectedly induced cellular senescence, based on the proportions of ¦Â-gal-positive cells and G2/M cells, simultaneous depletion of p21 notably suppressed these phenotypes. Further in line with this possible functional antagonism, concurrent depletion of p21 and SETD8 also lessened the progressive cell proliferation stall and nuclei enlargement, as compared with cells with down-regulated SETD8 only.

--

Human

HL

cellular senescence

28,514,051

Gene

0

1

0

1

0

1

0

YPEL3

83719

protein coding

MCF-7,U2OS

--

Aging

Accelerate

Colony formation assay//SA--gal activity assay

Using a colony formation assay,both cell lines showed considerably fewer colonies when expressing YPEL3 compared with cells not induced to express YPEL3. Growth suppression was also observed after shorter time periods of YPEL3 induction. Due to difficulties in the long-term growth of YPEL3 expressing cells, the inability to detect apoptosis, and observed morphologic changes, we examined the possibility that induction of YPEL3 would trigger cellular senescence.Two hallmarks of cellular senescence in human cells are the detection of increased acidic ¦Â-galactosidase activity and the appearance of foci within the nuclei of senescent cells . A clear increase (¡«38%) in cellular senescence was observed in U2OS/YPEL3-TetR cells exposed to tetracycline.

p53

--

Dual-Luciferase reporter assay//Immunoblotting

The level of YPEL3 mRNA induction was significantly reduced in doxorubicintreated Hct116 cells lacking p53 .Cotransfection of the YPEL3-luciferase reporter with wild-type p53 in either Hct116?/?p53 or H1299 cells led to an increase in YPEL3 promoter activity .Having established that the YPEL3 promoter could be activated by p53 when cloned into a plasmid reporter construct, we next set out to test whether p53 protein associated with the YPEL3 promoter in vivo. p53 chromatin immunoprecipitation assays were performed with Hct116 cells undamaged (non-treated) or damaged with doxorubicin. As expected, p53 was shown to bindin vivoto the YPEL3 promoter in DNA-damaged Hct116 cells.

--

Human

HL

cellular senescence

20,388,804

Gene

0

1

0

1

0

IFNG

3458

protein coding

HUVEC

--

Aging

Accelerate

Flow cytometry//MTT assay//SA--gal activity assay

Cell proliferation decreased slightly with IFN-¦Ã treatment in time- and dose-dependent manners. A decrease in cell proliferation by IFN-¦Ã treatment for 3 days was statistically significant (p < 0.05). The induction of cellular senescence by prolonged treatment with IFN-¦Ã was confirmed by an increase in SA-¦Â-gal-positive cells in the treated cells compared to the control cells . IFN-¦Ã treatment increased the G0/G1 cell population and decreased the S and G2/M cell population, suggesting that IFN-¦Ãinhibited cell proliferation through G0/G1 arrest in the cell cycle.

p53

--

Knockdown//SA-¦Â-gal activity assay//Western blot

Prolonged stimulation of IFN-¦Ã in the p16-knockdown (p16sh) cells increased SA-¦Â-gal activity staining to a level similar to that in control cells. In contrast, no increase in SA-¦Â-gal staining was observed in the p53-knockdown (p53sh) cells following prolonged stimulation with IFN-¦Ã. We reproducibly noticed an increase in the number of SA-¦Â-gal-positive cells in p16-/-MEFs, but not in IFN-¦Ã-treated p53-/-MEFs, after prolonged stimulation with IFN-¦Ã. Therefore,these results suggest that IFN-¦Ã-induced cellular senescence is mediated through a p53-dependent pathway.To confirm that p53 activation by IFN-¦Ã treatment was mediated through DNA damage signaling,the ATM level was down-regulated using siRNAs against ATM,followed by treatment with IFN-¦Ã for 6 days. Increases in p53 and phospho-p53 levels induced by IFN-¦Ã treatment were repressed by knockdown of ATM.

--

Human

L

cellular senescence

19,071,156

Gene

0

1

0

1

0

1

0

HDAC4

9759

protein coding

2BS

--

Aging

Prevent

Flow cytometry//Knockdown//MTT assay//SA--gal activity assay//Western blot//qRT-PCR

The overexpression and knockdown of HDAC4 were confirmed by western blot. Overexpression of HDAC4 resulted in only scattered senescence-associated ¦Â-galactosidase (SA-¦Â-gal) activity, which is a biomarker for senescent cells. The formation of senescence-associated heterochromatin foci (SAHF), which is another hallmark of senescent cells, was not observed when HDAC4 was overexpressed. Continuous cell growth and a decrease in the proportion of 2BS cells in the G0/G1 phases were evident in HDAC4 overexpressing cells compared with corresponding empty control vector-infected cells. In contrast, HDAC4 knockdown induced more robust senescence phenotypes, which displayed an elevated SA-¦Â-gal activity, prominent heterochromatin foci indistinguishable from those in senescent 2BS cells, pronounced cell growth arrest, and accelerated G1 cell cycle arrest compared with corresponding empty control vector-infected cells.

SIRT1

--

Knockdown//RT-PCR//Western blot//IP

We first analysed the expression patterns of SIRT1 using western blot and RT-PCR subjected to such perturbations.The protein levels of SIRT1 were markedly enhanced in HDAC4-transfected HeLa cells compared with the vector-transfected cells. In addition, we silenced HDAC4 expression in HeLa cells. Not unexpectedly, there was a marked decrease in the SIRT1 protein level in these HDAC4 silenced cells.Moreover,the RT-PCR analysis demonstrated no alteration at the SIRT1 mRNA levels in either HDAC4-overexpressing or knockdown cells, indicating that HDAC4 might normally function to increase the SIRT1 protein levels, but not at the mRNA level.To gain further insight into the interplay between HDAC4 and SIRT1, cellular interactions between HDAC4 and SIRT1 were confirmed using immunoprecipitation (IP) assays.

--

Human

L

cellular senescence

26,414,199

Gene

0

1

0

1

0

1

0

1

0

1

0

1

0

ACLY

47

protein coding

HDF

--

Aging

Prevent

Cell morphological analysis//Cell proliferation assay//Knockdown//SA--gal activity assay

ACLY knockdown cells showed apparent growth retardation and a typical senescent morphology (flat and enlarged in size). The proliferation assay and senescence-associated -¦Â-galacto-sidase staining showed that knockdown of ACLY in HDF cells significantly reduced their proliferation rate. A large proportion of ACLY knockdown cells were positive for senescence-associated-¦Â-galactosi-dase staining.

--

p53-AMPK

--

Knockdown//SA-¦Â-gal activity assay//qRT-PCR//DAPI staining//Immunofluorescence//Pull-down assay

Initially, we silenced p53 in ACLY knockdown HDF cells, and found that additional knockdown of p53 completely abrogated ACLY silencing-induced cellular senescence. we observed an increased p53 protein level in ACLY knockdown HDF cells by confocalmicroscopy and increased mRNA levels of p53 downstream target genes by real-time quantitative PCR.The direct interaction of ACLY with AMPK a2 was confirmed by an in vitro glutathione S-transferase(GST) pulldown assay . Immunofluorescent staining of over-expressed AMPK and endogeneous ACLY showed that about 26% and 37% of the signal co-localized in HDF and HCT116 cells, respectively .An in vitro kinase assay using immunoprecipitated AMPK a2 showed an apparent reduction of AMPK activity by ACLY.

Human

L

cellular senescence

25,367,309

Gene

0

1

0

1

0

1

0

PIR

8544

protein coding

WM266-4,IGR-37

--

Aging

Prevent

Cell morphological analysis//Colony formation assay//Knockdown//SA--gal activity assay//Western blot

The growth rate was significantly reduced in both mela-noma cell lines stably expressing shPIR constructs,compared with that of cells bearing the empty pSICO-R. A colony formation assay was also performed to measure the growth rate at low density. The number of colonies formed by shPIR cells was significantly reduced in both cell lines.The shPIR-transduced cell lines exhibited changes in size and morphology compatible with cellular senescence (flattened and enlarged phenotype). We therefore performed a senescence-associated ¦Â galactosidase(SA-¦Â-galactosidase) assay and found that PIR knockdown in WM266-4 cells resulted in an increase from 2.1% positivity in control cells to 41.3%, 16.6%, and 37.1% in shPIR#7, #16, and #19 transduced cells, respectively.

--

Human

L

cellular senescence

21,514,450

Gene

0

1

0

1

0

1

0

1

0

1

0

CUL4B

8450

protein coding

NHF,U2OS

--

Aging

Prevent

BrdU assay//Knockdown//SA--gal activity assay//Western blot

At day 5 post exposure to 1 Gy of ionizing radiation (IR), more than 9 % of normal human fibroblasts (NHFs) became senescent, as determined by assays of senescence-associated ¦Â¨Cgalactosidase (SA- ¦Â-gal) and BrdU incorporation. At this time point, there was a remarkable reduction in CUL4B at protein level. CUL4B was efficiently knocked down by RNA interference (RNAi). We observed that the percentage of senescent cells, as determined by SA- ¦Â-gal staining and BrdU incorporation, was significantly higher in shCUL4B cells than in shNeg cells after H2O2 treatment.

--

p53-ROS

Downregulation

Knockdown//Flow cytometry//qRT-PCR//BrdU assay//SA-¦Â-gal activity assay//Western blot

We used nutlin-3, which inhibits MDM2-mediated degradation of p53 and thus stabilizes p53, to activate p53 in NHFs and measured the level of ROS. Two days after nutlin-3 treatment the intracellular ROS level was higher in NHF-shCUL4B cells than in NHF-shNeg cells . Importantly, nutlin-3 failed to elevate the ROS level in CUL4B and p53 double knockdown cells (NHF-shCUL4B&shp53), while H2O2 treatment, as a positive control, caused an increase in the level of ROS in those cells. These results suggest that persistent activation of p53 alone may lead to an elevation in ROS level.We observed that while the percentage of senescent cells was higher in NHF-shCUL4B cells than in NHF-shNeg cells when treated with H2O2, the enhancement in senescence caused by CUL4B depletion was abolished in CUL4B and p53 double knockdown cells (NHF-shCUL4B & shp53), indicating that the enhancement in stress-induced senescence in CUL4B knockdown cells is dependent on p53. p53 activation, p53 transactivating activity and ROS production in response to H 2O2 treatment were significantly attenuated in PLOC-CUL4B cells .

Human

L

cellular senescence

25,464,270

Gene

0

1

0

1

0

1

0

1

0

IGFBP5

3488

protein coding

HUVEC

--

Aging

Accelerate

Flow cytometry//MTT assay//RT-PCR//SA--gal activity assay//Western blot

The levels of IGFBP-5 mRNA and protein in old cells were down-regulated by gene silencing using IGFBP-5 micro-RNA lentivirus. Transduction with IGFBP-5 micro-RNA lentivirus caused 65% decrease in IGFBP-5 levels. Repression of IGFBP-5 levels in old cells caused morphological changes similar to young cells and a decrease in SA-¦Â-gal activity. Treatment of young cells with rhIGFBP-5 decreased cell proliferation both time- and dose-dependently. Furthermore, prolonged treatment with rhIGFBP-5 increased SA-¦Â-gal staining and caused a characteristically enlarged and flattened morphology.To confirm that rhIGFBP-5¨Ctreated cells entered senescence-related cell cycle arrest, we analyzed DNA content by flow cytometry with PI staining. Most cells stimulated with rhIGFBP-5 for 2 d were arrested in G1 phase, which is one of the typical phenotypes in cellular senescence.

p53

--

Knockdown//Western blot//MTT assay

To further confirm that the p53 activation by IGFBP-5 might be mediated through DNA damage signaling, ATM level was down-regulated using siRNAs against ATM and the effects of IGFBP-5 overexpression were measured. Increases in p53 and phospho-p53 levels induced by IGFBP-5 up-regulation were repressed by knockdown of ATM. Furthermore, inhibition of cell proliferation induced by IGFBP-5 up-regulation was rescued by knockdown of ATM.These results suggested that IGFBP-5¨Cinduced cellular senescence is mediated by posttranslational modification of p53 such as phosphorylation and acetylation through DNA damage signaling.

--

Human

HL

cellular senescence

17,804,819

Gene

0

1

0

1

0

1

0

1

0

1

0

PLY

5601825

protein coding

BV-2,C6

--

Aging

Accelerate

Flow cytometry//SA--gal activity assay

Senescence of glial cells (BV-2 and C6) was evident by SA-¦Â-gal staining and morphological changes such as enlargement of the cell body and flat shape. Exposure to PLY for 24 h significantly increased the number of SA-¦Â-gal positive cells compared with untreated control cells. PLY induced BV-2 cell cycle arrest in the G0/G1 phase during the 24 h exposure.

--

MAPK-NF-¦ÊB

Activation

SA-¦Â-gal activity assay//Western blot//Co-IP

Phosphorylation of ERK1/2, p38 MAPK, and JNK was increased in PLY-treated BV-2 cells .NAC inhibited MAPK activities by pretreatment with for 2 h followed by incubation with PLY for 12 h . Increased expression of PAI-1 in PLY-induced BV-2 cells was significantly decreased by the MAPK inhibitors. In addition, treatment with MAPK inhibitors significantly reduced the number of SA-¦Â-gal positive cells. PLY treatment enhanced the expression of p65 subunit of NF-kB in a time-dependent manner.We also investigated whether the interaction between SIRT-1 and p65 proteins is affected by PLY treatment, given the SIRT-1 interacts with p65 in modulating cell fate in response to various signaling events. The interaction was confirmed by a co-immunoprecipitation assay. SIRT-1 and p65 physically interacted with each other at 4 h or 24 h in PLY-treate BV-2 microglial cells . Altogether, these results suggest that PLY induces cellular senescence via the SIRT-1 and NF-kB interaction in BV-2 cells.All the MAPK pathway inhibitors differentially reduced the expression of p-SIRT-1 and NF-kB enhanced by PLY.

Human

L

cellular senescence

28,232,188

Gene

0

1

0

1

0

MCAM

4162

protein coding

CD1462,CD146+

--

Aging

Prevent

Immunoblotting//Knockdown//SA--gal activity assay//TRAP assay//qRT-PCR

Specifically,the growth of CD1462cells ceased at P12, whereas CD146+ cells continued to grow until P15. In qPCR analysis, CD146+ cells showed high expression levels of stemness genes compared with those observed in CD1462 cells . Similarly, telomerase activities of CD146+ cells were significantly higher than those of CD1462 cells . Western blot analysis indicated that CD1462 cells showed enhanced expression of pho-p53 and p16 relative to that observed in CD146+ cells, as well as significantly augmented SA-¦Â-gal activity and cell areas .Cells with silenced CD146 expression exhibited a reduced duration of proliferation during the culture period and lower growth rates, compared with the respective measures made in na?ve cells or scrambled siRNA-transfected cells. SA-¦Â-gal activities and cell areas were significantly increased in CD146 siRNA transfectants.

--

Human

L

cellular senescence

26,941,359

Gene

0

1

0

1

0

1

0

1

0

1

0

TFAP4

7023

protein coding

RPE

--

Aging

Accelerate

Cell morphological analysis//SA--gal activity assay

The morphological changes in AP4-expressing cells, including enlarged nuclei and flattened cytoplasm, were characteristic of cellular senescence .When AP4 expression was induced by adding DOX to the media, the proliferation profiles of RPE-AP4 cells started to differ on Day 12 ARC from the cells without receiving DOX. AP4-expressing cells stopped proliferating, and the status of these cells remained constant for at least additional 12 days.To quantify the extent of AP4-induced senescence, the number of blue SA-¦Â-gal-positive cells was counted.The results showed that substantially more AP4-expressing cells than control cells were SA-¦Â-gal positive.

--

p14-p53

Activation

qRT-PCR//Immunofluorescence

Immunofluorescence microscopy was used to investigate the expression of p14ARF, p53,and Rb proteins in AP4-induced senescent RPE cells. p53 protein was expressed primarily in the large senescent AP4-expressing cells but not in DN-AP4-expressing cells.A similar expression pattern was observed in p14ARF, which appeared as small speckles distributed throughout the nucleoplasm but not the nucleolus of AP4-expressing cells .To determine whether AP4 can regulate p53 at the transcriptional level, the levels of p53 mRNA were quantified using qRT-PCR. The p53 gene has two promoters, P1 and P2 ,which are the transcription starting sites of the p53 gene and produce transcripts of different lengths.

Human

HL

cellular senescence

26,439,195

Gene

0

1

0

1

0

AGR2

10551

protein coding

LNCaP,DU 145,PC-3

--

Aging

Prevent

Flow cytometry//MTT assay//SA--gal activity assay//Western blot//qPCR

Forced expression of AGR2 in LNCaP or DU145 cells for 72 h resulted in a marginal increase in the cell viability as measured by MTT assays. V alidation of the AGR2 expressions at mRNA and protein levels was performed by quantitative PCR and western blot assays. AGR2 expressions were markedly increased in the transfected LNCaP or DU145 cells compared with the vector-transfected cells. Reduced endogenous AGR2 in PC-3 cells led to decreased cell viability, whereas no decreases were observed in cells transfected by control oligonucleotides.Transfection of LNCaP, PC-3 and DU145 cells with AGR2i led to significantly increase in the percentage of cells in G /G1phase compared with the control cells.

--

Human

HL

cellular senescence

22,467,239

Gene

0

1

0

1

0

1

0

1

0

1

0

PLK1

5347

protein coding

HDF,HUVEC

--

Aging

Prevent

BrdU assay//Cell counting//Cell morphological analysis//SA--gal activity assay//Western blot

Replicative senescence in cells was confirmed by increases in SA-¦Â-gal staining and the levels of p53, p21, and p16 proteins as well as enlarged, flattened cell morphology . The expression levels of PLK1 mRNA and protein decreased in old cells compared with young cells. PLK1 immunoreactivity was stronger in young cells than in old cells and PLK1 was localized in the nucleus or chromosomes of young cells under mitosis . Furthermore, the level of PLK1 protein also decreased during adriamycin-induced premature cellular senescence.Following transduction of old cells with recombinant PLK1 adenovirus, upregulation of PLK1 protein levels was confirmed by Western blot analysis. PLK1 overexpression in old cells increased cell proliferation , BrdU incorporation, and PDs . PLK1 upregulation decreased SA-¦Â-gal staining in a dose-dependent manner.

--

p53

--

Knockdown//Western blot//Cell counting//SA-¦Â-gal activity assay

Downregulation of p53, p21, and PLK1 was confirmed by Western blotting. However, we could hardly detect the p16 protein in our experimental condition because young cells were known to express very low level of p16 protein in normal condition. Instead, we confirmed the knockdown of p16 by transfecting old cells with the p16 shRNA vector since old cells show an increase in the p16 level. Although p16 shRNA¨Ctreated cells showed senescence phenotypes induced by PLK1 knockdown, such as decreased cell proliferation and increased SA-¦Â-gal staining, p53 shRNA¨Ctreated cells did not.

Human

HL

cellular senescence

23,525,475

Gene

0

1

0

1

0

1

0

1

0

1

0

HJURP

55355

protein coding

HDF,HUVEC

--

Aging

Prevent

Cell counting//DAPI staining//Flow cytometry//RT-PCR//SA--gal activity assay//Western blot

Ectopic expression of HJURP in senescent cells increased cell proliferation in a time-dependent manner and resulted in morphological changes similar to young cells and a decrease in SA-¦Â-gal staining activity.In addition, the upregulation of HJURP decreased the cell population in the G1 phase of the cell cycle, which is increased in old cells and cells with control vector and elevated the G2/M cell populations, which is declined in old cells and control vector. Because upregulation of HJURP levels partially reversed cellular senescence, we examined the effects of HJURP knockdown on senescence phenotypes in young HDFs and HUVECs by transfection of HJURP siRNAs. HJURP knockdown was confirmed by RT-PCR and Western blotting . Although the levels of pA TM, p53, and p21 were elevated, the levels of pRb and CENP-A were repressed by HJURP knockdown.

--

p53

--

Knockdown//Western blot//RT-PCR//SA-¦Â-gal activity assay

To determine the pathway involved in the regulation of cellular senescence by HJURP reduction, we knocked down p53 or p16 levels in young cells and measured the effects of HJURP downregulation on cellular senescence. The levels of HJURP, p53, and p16 were confirmed by RT-PCR and Western blotting. HJURP knockdown decreased cell proliferation in p16 siRNA cells but not in p53 siRNA cells. Similarly, depletion of HJURP increased SA-¦Â-gal staining activity in p16 siRNA cells but not in p53 siRNA cells.

Human

HL

cellular senescence

23,292,286

Gene

0

1

0

1

0

1

0

1

0

1

0

1

0

ERRFI1

54206

protein coding

2BS

--

Aging

Accelerate

BrdU assay//SA--gal activity assay//SAHF//Western blot

The Mig-6 overexpression was confirmed by Western blot analysis in the pLVX-Mig-6 cells. The results show that the pLVX-Mig-6 cells overexpressing Mig-6 showed reduced DNA synthesis.The pLVX-Mig-6 cells displayed the flat morphology, stained positively for senescence-associated-¦Â-galactosidase (SA- ¦Â-gal) and formed senescence-associated heterochromatin foci (SAHF) 12 days after lentiviral infection (8 days post-selection). Moreover, molecular markers of senescence, such as increased p53, p21, p16 and decreased p-Rb levels, were detected after Mig-6 overexpression.

FOXO3a

--

Knockdown//RT-PCR//Immunobloting//Western blot

Ly294002 treatment dramatically increased Mig-6 expression in young cells. In addition, RNA interference was used to deplete FOXO3A protein in RasV12-induced senescent cells. The amount of FOXO3A protein in the cells was reduced by at least 80% by a specific shRNA as compared with the control shRNA. FOXO3A knockdown reduced Mig-6 mRNA and protein levels in these senescent cells. Results showed that Mig-6 or FOXO3A knockdown partially restored the response of Ras-induced senescence to EGF , which was suggested by the Akt phosphorylation levels.

EGFR-ERbB

Downregulation

Western blot//SA-¦Â-gal activity assay

Western blot analysis showed that levels of the phosphorylated EGFR (p-EGFR) were evidently decreased,but its total expression levels were not obviously altered in the senescent cells compared with those in the young cells.Deletion of ErbB-2-binding domain did not affect the phosphorylation levels of p-EGFR,p-Akt (phosphorylated Akt) and p-Erk (phosphory-lated Erk), and greatly reduced the ability of Mig-6 to enhance SA-¦Â-Gal staining compared with the wild-type Mig-6. The result showed that as pcDNA-EGFR amount increased, SA-¦Â-Gal activity decreased with a dosedependence,suggesting that EGFR can an-tagonise of Mig-6 induction effect of cellular senescence.AG1478 (but not AG9)treatment resulted in the decreased EGFR phosphorylation and the increase of SA-¦Â-Gal activity, suggesting that the suppression of ErbB signalling only can elicit a cellular senescence. Subsequently, the EGFR inhibitor AG1478 treatment resulted in the increased Mig-6 expression , but not reduced Mig-6 expression.

Human

HL

cellular senescence

23,746,120

Gene

0

1

0

1

0

1

0

1

0

ING1

3621

protein coding

IMR-90

--

Aging

Accelerate

BrdU assay//Cell morphological analysis//SA--gal activity assay//Western blot

Enforced expression of ING1 caused a dramatic reduction in proliferation, as shown by a decrease in thymidine incorporation or in the number of BrdU-positive cells.The inverse correlation between ING1 expression and BrdU incorporation could also be observed in individual cells by immunofluorescence. Eighty-five percent of AU5-positive cells were BrdU-negative, while 65% of AU5-negative cells or vector-infected cells were BrdU-negative, confirming the antiproliferative effect of ectopic ING1.ING1-expressing fibroblasts also displayed distinctive markers of cellular senescence,such as SA-Beta Gal activity , and flat enlarged morphology.

--

p53

--

Western blot//SA-¦Â-gal activity assay

To investigate the functional connection between p33ING1 and p53 in the context of senescence, we inactivated the p53 pathway in these cells, using the E6 oncoprotein from human papilloma virus. Ectopic expression of p33ING1 failed to induce a significant cell-cycle arrest in fibroblasts expressing E6 .Similarly, the induction of the senescence marker SA-¦Â-Gal by p33ING1 was largely abolished in E6-expressing cells. In agreement with previous reports, inactivation of the p53 pathway was not sufficient to bypass senescence by RasV12 in these cells, which requires additional inactivation of the Rb pathway. Interestingly, ING1 increased the level of p21CIP1, a p53 target associated with senescence. The induction of p21CIP1 by ING1 was lost in E6-expressing cells .

Human

HL

cellular senescence

21,078,114

Gene

0

1

0

1

0

1

0

1

0

SIRT6

51548

protein coding

HepG2

--

Aging

Prevent

SA--gal activity assay

Inhibiting the upregulation of SIRT6 enabled TGF-¦Â1?H2O2?HOCl to efficiently induce cellular senescence.

--

ERK//Smad//p38 MAPK

--//--//--

SA-¦Â-gal activity assay

Consistent with the promoting effect of Smad and p38 MAPK pathways on the expression of SIRT6, inhibiting each of these pathways enabled TGF-¦Â1?H2O2?HOCl to induce the cellular senescence of HCC cells. Transforming growth factor-¦Â1?H2O2?HOCl could induce sustained and enhanced activation of the ERK pathway.Consistently, TGF-¦Â1?H2O2?HOCl could efficiently induce cellular senescence, if the ERK pathway was inhibited. In this situation, the effect of TGF-b1?H2O2?HOCl on cellular senescence was also attenuated by inhibiting the NF-jB,Smad, and p38 MAPK pathways, which was consistent with the effect of suppressing the upregulation of SIRT6. However, if SIRT6 expression was suppressed by shRNA, TGF-¦Â1?H2O2?HOCl could similarly induce cellularsenescence, even though the ERK pathway was activated .

Human

L

cellular senescence

25,683,165

Gene

0

1

0

MYC

4609

protein coding

RPE

--

Aging

Accelerate

Knockdown//SA--gal activity assay

The SA-¦Â-gal activity assay was performed on these two cell clones with or without 4-OHT treatment on Day 24. With the addition of 4-OHT, the average number of SA-¦Â- gal-positive cells per unit area was increased in both the cells with LL-c-Myc (clone 4) and ML-c-Myc (clone 3). In order to determine whether long-term post-confluent cells entered into senescence rather than cellular quiescence, we replated the confluent RPE LL-c-Myc cells (clone 4) treated with OHT for 24?days, and measured their SA-¦Â-gal activity at different time points. The percentage of ¦Â-gal-positive cells after 2 and 6?days was 85.6 and 87.3%, respectively, suggesting that the replated cells were not proliferating, and the confluent cells were substantially senescent rather than quiescent. Collectively, we concluded that low level of c-Myc expression (LL-c-Myc) induced cellular senescence rather than quiescence in post-confluent RPE cells during long-term culture.After infecting with lentivirus expressing shRNA, all the cells expressing high level of c-Myc (HL-c-Myc) eventually died after 8?days, while HL-c-Myc cells infected with scrambled lentiviral shRNA control could survive for 12?days. For ML-c-Myc cells,it took 24?days for the cells infected with scrambled lentiviral shRNA to die completely.

AP4//p53

--//--

Knockdown//Western blot

The expression of AP4 increased evidently in the samples that incubated with 4-OHT for the initial 14?h, while c-Myc-ER level was relatively high. Both AP4 and c-Myc-ER protein were subsequently decreased during longer incubation, which might probably due to protein degradation. After normalized to the signal of actin, the expression of AP4 increased sevenfold during the first 14?h upon the activation of c-Myc,suggesting that RPE c-Myc-ER clones were functional and could be used for further studies.Western blot analysis showed that differential c-Myc expression in the cell clones induced a corresponding level of AP4 protein.In the cells with LL-c-Myc, the expression of p53 decreased more with a larger decrease in AP4 level, indicating that p53 expression was regulated by AP4, which is consistent with our previously finding .In this study, knockdown of AP4 expression brings about a decrease in p53 expression, but the decrease in the expression of p53 was weak in RPE cells when c-Myc level is higher.

--

Human

L

cellular senescence

29,188,535

Gene

0

1

0

1

0

CDKN2A

1029

protein coding

Lymphoma cell line

--

Acute leukemia

Accelerate

Knockdown//SA--gal activity assay

Acute knockdown of p19ARF expression by shRNA resulted in decreased SA-beta-galactosidase activity.

--

Human

HL

cellular senescence

25,784,651

Gene

0

1

0

1

0

ATF7IP

55729

protein coding

IMR-90

--

Aging

Prevent

DAPI staining//EdU assay//Knockdown//SA--gal activity assay

MCAF1 knockdown severely impaired cell proliferation and reduced incorporation of a thymidine analog EdU;MCAF1 knockdown cells were positive for SA-¦Â-gal activity.DAPI staining showed that approximately 3 % of MCAF1 knockdown cells displayed nuclear foci that resemble SAHF.

--

Rb

--

Western blot

We also found that the cdk inhibitors p16 and p21 were upregulated at both mRNA and protein levels, and RB protein was dephosphorylated in MCAF1 knockdown cells.

Human

HL

cellular senescence

23,935,871

Gene

0

1

0

1

0

1

0

1

0

TNFSF13

8741

protein coding

SW480

--

Colorectal cancer

Prevent

SA--gal activity assay

These results indicated cellular ¦Â-galactosidase activity, a symbol of cell ageing, had enhanced compare with that of no pGshAPRIL-transfection.

--

HSPG

--

Western blot//SA-¦Â-gal activity assay

By contrast, high levels of SA-¦Â-gal activity were exhibited in pGshAPRIL-transfected plus TACI-Fctreated groups or those transfected with pGshAPRIL (P< 0.01). SA-¦Â-gal activity was shown to increase in groups with pGshAPRIL and heparin combined treatment and so did in pGshAPRIL, TACI-Fc add heparin treatment groups (P<0.05).

Human

L

cellular senescence

19,466,596

Gene

0

1

0

GNG11

2791

protein coding

Fibroblast

--

Aging

Accelerate

Cell morphological analysis//SA--gal activity assay

We transfected plasmid encoding GNG11 (pG11-sense),they stopped growing 1 week about after transfection, exhibited an enlarged and flat cell shape, and were clearly stained with senescence-associated ¦Â-galactosidase.

--

ERK1/2

Activation

Western blot

Both ERK1/2 were significantly activated 5 days after transfection, whereas little or no activated forms of p38 and JNK were detected.

Human

L

cellular senescence

17,092,487

Gene

0

1

0

1

0

MDH1

4190

protein coding

HDF

--

Aging

Prevent

Cell morphological analysis//Knockdown//SA--gal activity assay

We found that about 60 % of SA-¦Â-Gal-positive cells appeared among MDH1 knockdown cells, but only 10 % among control cells. shMDH1-infected young HDFs also became flattene and enlarged£»cell numbers in cultures of MDH1 knockdown HDFs were decreased by about 50 % compared to control cells plated at the same density and cultured for the same duration .

SIRT1

--

Knockdown//Western blot//RT-PCR

SIRT1 RNA levels were decreased in MDH1 knockdown cells.

--

Human

L

cellular senescence

22,971,926

Gene

0

1

0

1

0

1

0

SMARCB1

6598

protein coding

Lac-hSNF5

--

Malignant Rhabdoid Tumor

Accelerate

BrdU assay//DAPI staining//Flow cytometry//SA--gal activity assay

We found that hSNF5 induction dramatically impaired cell proliferation, even when cultured in the presence of 1 % serum . In contrast, addition of IPTG had no effect on Lac-empty control cells lacking the hSNF5 gene. Similar results were obtained with independently isolated Lac-hSNF5 cell lines (data not shown). We also assessed the effect of hSNF5 on the colony-forming capacity of Lac-hSNF5 cells, and found that hSNF5 expression dramatically reduced the number of colonies formed, after seeding these cells at a very low density.We next determined which stage of the cell cycle is affected by hSNF5.Expression of hSNF5 leads to impaired DNA synthesis, indicated by the strongly reduced incorporation of BrdUrd. FACS analysis revealed that the hSNF5-expressing cells accumulate in G1, suggesting that the cell cycle block affects entry into S-phase.

--

p16-pRb

--

Western blot

Western immunoblotting revealed a strong up-regulation of p16INK4aprotein levels following hSNF5 expression, whereas no increase or even a small reduction of p14ARFwas observe.Because p16INK4a is a specific inhibitor of the Rb-regulating cyclin D1-CDK4 complex, we determined the phosphorylation status of pRb in Lac-hSNF5 cells. In support of a role of hSNF5.

Human

L

cellular senescence

14,604,992

Gene

0

1

0

1

0

1

0

1

0

WWP1

11059

protein coding

2BS

--

Aging

Prevent

Flow cytometry//Knockdown//SA--gal activity assay

Senescence markers for WWP1-overexpressing and WWP1 shRNA-infected cells were then monitored at the same time points as their corresponding control cells. The WWP1 shRNA-transfected 2BS cells displayed enlargement, flattening, and the accumulation of vacuolated cytoplasmic inclusions. However, no significant morphological changes were observed in the WWP1-transfected cells, which were similar to young 2BS cells. Most of the WWP1 shRNA-transfected cells showed strong blue SA-¦Â-gal staining similar to senescent cells . In contrast, only scattered SA-¦Â-gal-positive cells were found in WWP1-transfected cells compared with the corresponding control cells. 2BS cells overexpressing WWP1 showed increased S and reduced G1 compartments. It was obvious that WWP1 shRNA-infected 2BS cells entered G1cell cycle arrest.

--

p27

--

Knockdown//RT-PCR//Western blot

The protein levels of p27Kip1were markedly depressed in WWP1-transfected HeLa cells as compared with the vector-transfected cells, whereas the protein levels of p16INK4a and PTEN were invariable. In addition, we silenced WWP1 in HeLa cells. Similarly, there was no detectable change in the protein levels of p16INK4a or PTEN, but a marked increase in the p27Kip1 protein level was found. Moreover, the RT-PCR analysis demonstrated no alteration in p27Kip1mRNA levels in both WWP1-overexpressing and knockdown cells, which indicated that WWP1 might normally decrease the p27Kip1 protein level but not the mRNA level. Similar results were observed in 2BS cells.

Human

HL

cellular senescence

21,795,702

Gene

0

1

0

1

0

1

0

ATP6V0A2

23545

protein coding

TIG-1

--

Aging

Prevent

EdU assay//RT-PCR//SA--gal activity assay

We evaluated the functional role of ATP6V A2 in cellular senescence of the TIG-1 cells. The mRNA expression of senescence markers p21 and p16, and protein expression of senescence markers p21, p16, phospho p38 and ¦ÃH2AX significantly increased in ATP6V A2-silenced young and old TIG-1 cells. When DNA replication was assayed by examining 5-ethynyl-2¡ä-deoxyuridine (EdU) incorporation in a pulse-label experiment, there was a steady decrease in EdU incorporation in ATP6V A2-silenced young TIG-1 cells and old TIG-1 cells. Growth was greatly reduced in ATP6V A2-silenced young TIG-1 cells and old TIG-1 cells. The activity of senescence-associated -¦Â-galactosidase (SA-¦Â-Gal) was augmented in ATP6V A2-silenced young TIG-1 cells and old TIG-1 cells.

--

Human

L

cellular senescence

26,611,489

Gene

0

1

0

1

0

1

0

KIF2C

11004

protein coding

HDF,HUVEC

--

Aging

Prevent

RT-PCR//SA--gal activity assay//Western blot

We tried to investigate the role of MCAK/Kif2C in cellular senescence.Cellular senescence of HDFs and HUVECs was confirmed by increases in SA-¦Â-gal activity and p53 levels .Decreased expression levels of MCAK/Kif2C were shown in old cells by RT-PCR, real-time PCR, and Western blotting.

--

p53

--

Knockdown//RT-PCR//Western blot

Knockdown of p53, p16, and MCAK/Kif2C were confirmed by RT-PCR and Western blotting. A decrease in cell proliferation and an increase in SA-¦Â-gal activity by MCAK/Kif2C knockdown were not observed in p53 siRNA cells, but were evident in p16 siRNA cells. Senescent-like cell morphology was also shown in p16 siRNA cells, but not p53 siRNA cells . These results support the view that premature cellular senescence induced by MCAK/Kif2C knockdown might be regulated via a p53-dependent pathway.

Human

HL

cellular senescence

23,098,759

Gene

0

1

0

1

0

1

0

CREG1

8804

protein coding

Fibroblast

--

Li-Fraumeni Syndrome

Accelerate

Cell proliferation assay//MTT assay//Western blot

The expression level of CREG1 fits the criteria of senescence-associated gene, decreased during immortalization and increased in replicative and 5-aza-dC-induced senescence justifying further investigation of the role of CREG1 in cellular senescence.We found that CREG1 overexpression decreased cell growth in immortal LFS cells when compared with pIRES empty vector transfected cells demonstrated by both MTT and cell counting proliferation assays.

p16

Upregulation

Cell cycle analysis//SA-¦Â-gal activity assay//Western blot//Cell morphological analysis

Cell cycle analysis showed an increase in the G0-G1 cell population and a decrease of S-phase population in cells cotransfected with CREG1 and p16INK4a compared to cells transfected with either CREG1 or p16INK4a alone.In p16-transfected cells and more so in cells cotransfected with CREG1 and p16INK4a, we observed cell characteristics indicative of senescence, a large and flat morphology with an increase of SA-¦Â-galactosidase staining.We found that cells coexpressing CREG1 and p16INK4a showed a marked decline in protein levels of cyclin A and B .

--

Human

HL

cellular senescence

21,263,217

Gene

0

1

0

1

0

1

0

TOPBP1

11073

protein coding

MEF,3T3

--

Aging

Prevent

DAPI staining//Flow cytometry//SA--gal activity assay

TopBP1-ablated cells underwent morphological changes including enlargement and flattening of the cells on the culture dishes.TopBP1-ablated cells were positive with respect to SA-¦Â-gal staining,which detects senescent cells.FACS analysis of TopBP1-ablated cells indicates a reduction of G1-phase cells containing 2N DNA and a reduction of S-phase cells as well as an increase of G2/M-phase cells containing 4N DNA content, relative to wild-type cells.

--

Human

L

cellular senescence

21,149,450

Gene

0

1

0

1

0

1

0

GRSF1

2926

protein coding

WI-38

--

Aging

Prevent

Immunofluorescence//SA--gal activity assay//Western blot

Immunofluorescence analysis confirmed that GRSF1 levels declined in senescent cells and Western blot analysis further established that GRSF1 levels decreased early in senescence SA-¦Â-gal analysis revealed greater numbers of positive (blue) senescent cells after GRSF1 depletion, supporting the notion that lowering GRSF1 promoted cell senescence.

IL-6

--

qRT-PCR//ELISA

The levels of IL6 mRNA and secreted IL6 were significantly higher in GRSF1-silenced WI-38 fibroblasts, as determined by RT-qPCR analysis and AlphaLISA£¬ respectively.

--

Human

L

cellular senescence

30,086,537

Gene

0

1

0

1

0

1

0

PRKAA1

5562

protein coding

BEAS-2B

Lung

Chronic obstructive pulmonary disease

Prevent

Cytokine assay//Immunocytochemistry//Western blot//qRT-PCR

Transfection of AMPK¦Á1/¦Á2 siRNA increased the mRNA of p16, p21 and p66shc, but reduced klotho gene expression in BEAS-2B cells.We also determine the expression of p16 and p21, markers of cellular senescence, in lung tissues using Western blot and immunohistochemistry.

SOD2//SIRT3

Upregulation//Upregulation

Immunocytochemistry//Western blot

In response to elastase, the abundance of SOD2 and SIRT3 proteins was significantly reduced as compared to saline control.The expression of SOD2 and SIRT3 proteins was augmented by prophylactic treatment of metformin, whereas Compound C further caused reduction of SOD2 and SIRT3 proteins in emphysematous lungs.

--

Human

L

cellular senescence

28,186,975

Gene

0

1

0

1

0

1

0

1

0

SLC25A5

292

protein coding

U2OS,MCF-7

--

Aging

Prevent

BrdU assay//Cell morphological analysis//Knockdown//SA--gal activity assay

Down-regulation of ANT2 by siRNA led to a decreased BrdU incorporation indicating inhibition of cell proliferation .Further decrease in BrdU incorporation was observed after the combined treatment. Although short term knock-down of ANT2 did not enhance senescence associated ¦Â-galactosidase staining, enhanced vacuolization, typical of senescent cells, was observed in cells with combined treatment.

NF1/Smad

--

IP//CHIP//qRT-PCR//Flow cytometry

Induction of the NF1/Smad complexes and binding of NF1 to the ANT2 NF1-dependent repressor elements followed the same time course as ANT2 repression in U2OS cells.

TGF¦Â

--

qRT-PCR//Flow cytometry

These events are also closely correlated with the expression and secretion of TGF-¦Â suggesting that formation of the NF1/Smad complexes is initiated through TGF-¦Â signaling.

Human

L

cellular senescence

25,220,407

Gene

0

1

0

1

0

1

0

1

0

TP53

7157

protein coding

MCF-7,A549,HCT116

--

Aging

Accelerate

Cell morphological analysis//Cell proliferation assay//SA--gal activity assay

Consistent with these observations, we found that overexpression of human p53¦Â increased markers of cellular senescence in a variety of human tumor cell lines (MCF-7, A549, HCT116), including enlarged cell size, decreased cell proliferation, and elevated senescence-associated ¦Â-galactosidase (SA-¦Â-gal) activity.

--

hSMG-1-RPL26-SRSF7

--

SA-¦Â-gal activity assay//Knockdown

Further, modulation of the hSMG-1-RPL26-SRSF7 pathway required for IR-induced p53¦Â alternative splicing also affected cellular senescence markers as well. Knock-down of hSMG-1, which activates this splicing pathway, increased the percentage of SA-¦Âgalactosidase positive cells and knock-down of SRSF7, a positive splicing factor for p53¦Â, suppressed IR-induced SA-¦Â-galactosidase induction.

Human

HL

cellular senescence

28,288,992

Gene

0

1

0

1

0

SRSF3

6428

protein coding

Fibroblast

--

Aging

Prevent

Knockdown//SA--gal activity assay

The cells transfected with siSRSF3 no. 1 and siSRSF3 no. 2, but not those with a control siRNA (siControl), underwent a senescent growth arrest rapidly (within 7 days) with concomitant induction of the SA-¦Â-gal activity.

tp53

Downregulation

RT-PCR//qRT-PCR//Western blot//Knockdown

Knockdown of SRSF3 also increased p53b protein level, reminiscent of its accumulation in replicative senescence. When the alternative splicing patterns of TP53 were examined by conventional RT-PCR and qRT-PCR,mRNA levels of p53b were remarkably upregulated upon SRSF3 knockdown, while the levels of fulllength p53 mRNA were moderately decreased.

--

Human

HL

cellular senescence

22,777,358

Gene

0

1

0

1

0

EZH2

2146

protein coding

SK-Mel-119,UCD-Mel-N,SK-Mel-173

--

Melanocytic nevi

Prevent

Cell morphological analysis//Flow cytometry//Immunofluorescence//Knockdown//SA--gal activity assay//SAHF//qRT-PCR

Growth of EZH2 siRNA cells was significantly decreased compared to control-transfected cells. EZH2 siRNA cells displayed a flattened, enlarged morphology and expressed SA-¦Â-Gal. In addition to SA-¦Â-Gal expression, additional senescence markers following extended EZH2 depletion (6 days) include SAHFs and the heterochromatin-associated H3K9me3 modification as well as an increase in cell size, as evaluated by tubulin immunofluorescence. Flow cytometric analysis demonstrated that the proportion of cells in the S phase decreased in EZH2 siRNA cells with a concomitant increase of cells in the G1 phase, a significant difference in all 3 cell lines tested. In SK-Mel-173 cells, we also noticed a slight increase in the proportion of cells in the G2 phase.

p21

--

Western blot

However,depletion of EZH2 elevated expression of p21 in 5/6 cell lines.

--

Human

L

cellular senescence

21,383,005

Gene

0

1

0

1

0

1

0

1

0

1

0

1

0

1

0

TAGLN

6876

protein coding

HepG2

--

Aging

Accelerate

Colony formation assay//SA--gal activity assay

SM22a overexpression significantly induced the inhibition of cell growth of HepG2 cells. Conversely, when SM22a-overexpressing cells were transfected with siRNA, which suppresses the expression of SM22a, the cell growth was partially restored to that of control cells.These results suggest that SM22a is closely involved in regulation of cell growth and senescence.

MT-1G

Upregulation

RT-PCR//Western blot

In two selected SM22a-overexpressing clones, the levels of MT-1G were also elevated.

p16-pRb

Activation

RT-PCR//Western blot

SM22a overexpression resulted in a significant elevation of p16INK4a when analyzed by RT-PCR and Western blot. In wild-type HepG2 cells, serine 780 and 807/811 residues of pRB were highly phosphorylated. However, SM22a overexpression in these cells significantly inhibited the phosphorylation of serine 780 and 807/811 residues in pRB, which may prevent the detachment of E2F from pRB and thus suppress cell proliferation. Phosphorylation of serine 608 residue in pRB was also significantly inhibited. On the other hand, when SM22a-overexpressing cells were transfected again with siRNA to suppress SM22a expression, serine 608, 780, and 807/811 residues in pRB were re-phosphorylated, thereby restoring cell growth to a certain level.

Human

L

cellular senescence

20,705,054

Gene

0

1

0

1

0

SLC13A3

64849

protein coding

WI-38

--

Aging

Accelerate

Cell morphological analysis//SA--gal activity assay

In addition, the volume of WI38/hNaDC3 cells in PD37 showed hypertrophy compared to that of control and empty vector groups.The SA-beta-gal (a biomarker of cell senescence) staining results showed that the percentage of staining positive cells in WI38/hNaDC3 group (98.5¡À 5.6%) was significantly greater than that in the WI38/con (22.3 ¡À 2.4%) and WI38/neo group (23.4 ¡À2.18%) (P< 0.05) in 37 PD.

--

SIRT1

Downregulation

Fluorescence microscopy

To determine whether NaDC3 induces aging through inhibiting SIRT1 pathway, SIRT1 deacetylase activity was detected by fluorescent assay. The results showed that the activity was down-regulated in the WI38/hNaDC3 group (9256 ¡À 675), compared with that in the control (20,569 ¡À 1035) and the WI38/neo groups (18,972 ¡À1158) (P < 0.05).

Human

L

cellular senescence

20,813,124

Gene

0

1

0

1

0

PRDX3

10935

protein coding

HTR8,Human primary trophoblast

--

Intrahepatic cholestasis of pregnancy

Prevent

Flow cytometry//ROS assay//SA--gal activity assay

As expected, knockdown of PRDX3 significantly induced mitochondrial dysfunction, as indicated by overproduction of ROS, loss of mitochondrial membrane potential and decline in ATP content .Subsequently, the cell cycle distributions of these cell strains were analysed with flow cytometer. Compared to the sh-scr cells, shPRDX3-1# and shPRDX3-2# cells exhibited prolonged G0/G1 phase, indicative of growth arrest.In our study, PRDX3-knockdown cells showed a significantly higher number of SA-¦Â-gal stained cells compared with sh-scr control cells at basal level.

p16//p21//p38

--//--//--

qRT-PCR//Western blot//HC assay

QRT-PCR assay revealed that mRNA levels of p21WAF1/CIP and p16INK4A were increased in PRDX3-knockdown cells compared with sh-scr cells.Immunoblot assay also confirmed the corresponding change of protein levels of p21WAF1/CIP and p16INK4A in PRDX3-knockdown cells,and in ICP placentas by immunoblot and IHC assays.Indeed, we observed elevated p38-MAPK phosphorylation in PRDX3-knockdown cells.

--

Human

HL

cellular senescence

27,958,341

Gene

0

1

0

1

0

1

0

UBE2E3

10477

protein coding

RPE-1

--

Aging

Prevent

ELISA//EdU assay//Flow cytometry//SA--gal activity assay//Western blot

Depletion of UBE2E3 led to a loss of proliferation, as indicated by a failure of siUBE2E3 cells to incorporate 5- ethynyl-2 -deoxyuridine (Edu), a marker of newly-synthesized DNA ,an increase in cell size,a robust expression of senescence-associated ¦Â-galactosidase , a decrease in intracellular HMGB1 content,redistribution of lamin B from its primary localization at the nuclear envelope to a punctate, nucleoplasmic distribution and a concomitant decrease in lamin B1 mRNA expression,increased transcription of the tumor suppressor p16INK4a, and increased secretion of the pro-inflammatory cytokine IL-6 .

--

p53-p21

--

SA-¦Â-gal activity assay//EdU assay

Using a combination of co-knockdowns and measuring both SA-¦Â-Gal expression as well as Edu incorporation in the same cells, 4 days after siRNA administration, we found that silencing the expression of either p53 or p21CIP1/WAF1 mitigated the siUBE2E3- induced SA-¦Â-gal expression whereas silencing either p21CIP1/WAF1 or p16INK4a mitigated the cell cycle exit.

Human

L

cellular senescence

29,879,550

Gene

0

1

0

1

0

1

0

1

0

1

0

FAHD1

81889

protein coding

HUVEC

--

Aging

Prevent

Knockdown//SA--gal activity assay//Western blot

Furthermore,depletion of FAHD1 induced premature senescence in HUVEC, as shown by an increased percentage of cells staining positive for senescence-associated ¦Â galactosidase (SA-¦Â-gal) .We found that the levels of both senescence marker proteins were significantly down-regulated in FAHD1-depleted cells.These findings suggest that FAHD1 is required for cell proliferation in human endothelial cells and its inactivation induces premature senescence.

p53//p21/WAF1

--//--

Western blot

Of note, we observed slightly increased p53 protein levels in FAHD1-depleted cells, which were accompanied by a significant increase in the level of the cdk inhibitor p21Cip1/Waf-1, which is known to contribute to the senescence-associated proliferation arrest.

--

Human

L

cellular senescence

28,286,170

Gene

0

1

0

1

0

1

0

KCNH7

90134

protein coding

A375

--

Melanoma

Accelerate

Flow cytometry//Western blot

We found that, in agreement with our previous investigation, stimulation of Kv11.3 strongly upregulated both senescent markers in melanoma cells. However, in contrast with the effect of NS1643 in breast cancer cells in which we detected an arrest of the cell cycle in G0/G1 phase, analyses of the cycle progression in A375 melanoma shows that stimulation of Kv11.3 channel significantly increased the population of cells in the G2/M phase compared to untreated cells suggesting that NS1643 determined an arrest of the cell cycle in the G2/M phase of the cell cycle.

--

Raf-MEK-ERK

--

Western blot

Interestingly, we found that concomitant to the effect of NS1643 on cell cycle proteins, NS1643 strongly inhibited both MEK and ERK activities as indicated by the decreased phosphorylation of these proteins.

Human

L

cellular senescence

26,942,884

Gene

0

1

0

1

0

CIP2A

57650

protein coding

HCC

--

Hepatocellular carcinoma

Prevent

Flow cytometry//Knockdown//SA--gal activity assay//Western blot

The percentage of G0/G1 phase was significantly increased in CIP2A shRNA transfected cells, compared with empty vector group.Our data revealed that the SA-¦Â-gal positive cells were significantly increased in CIP2A shRNA transfected HCC cells.

--

Human

HL

cellular senescence

29,175,329

Gene

0

1

0

1

0

1

0

1

0

SFRP1

6422

protein coding

IMR-90

--

Aging

Accelerate

BrdU assay//Cell morphological analysis//Immunostaining//RT-PCR//SA--gal activity assay//SAHF

We then tested whether SFRP1 upregulation induces senescence. Lentiviral SFRP1 expression in IMR-9 cells caused striking proliferation arrest as determined by the lack of BrdU incorporation and Ki-67 staining , which was accompanied by senescence phenotypes including flat and enlarged morphology , senescence-associated ¦Â gal activity , senescence-associated heterochromatic foci, and induction of genes representing senescence-associated secretory phenotype.

--

Wnt-¦Â-catenin//Rb//p53

Downregulation//Activation//--

Western blot//Luciferase reporter assay//Knockdown//SA-¦Â-gal activity assay

Lentiviral SFRP1 expression in IMR-90 cells resulted in reduced soluble ¦Âcatenin. Interestingly, etoposide treatment of IMR-90 cells, which stimulates SFRP1 secretion, also reduced soluble ¦Â-catenin levels.abolished etoposide-induced reduction of soluble ¦Â-catenin levels, suggesting that SFRP1 mediates the downregulation of Wnt signaling upon etoposide treatment. Suppression of Wnt signaling by SFRP1 and etoposide treatment was also verified by Wnt-responsive reporter assays: Wnt3 efficiently activated Super TOPFLASH reporter in IMR-90 cells, which was almost completely repressed by co-expression of SFRP1.Lentiviral SFRP1 expression in IMR-90 cells resulted in dephosphorylation of Rb, but the expression of p53 and a p53 transcriptional target, p21, remained unchanged, suggesting that SFRP1 activates the Rb pathway, but not the p53 pathway. Rb and p53 were efficiently silenced by corresponding shRNAs and upon SFRP1 expression, we observed significantly attenuated senescence induction in cells with Rb or p53 knock-down. This suggests that in addition to Rb, p53 is required for SFRP1-induced senescence even though SFRP1 does not activate the p53 pathway.

Human

L

cellular senescence

22,927,647

Gene

0

1

0

1

0

1

0

1

0

1

0

1

0

EZH2

2146

protein coding

HCT116

--

Colorectal cancer

Prevent

SA--gal activity assay

Consistently, LDMcaused senescence was reduced by overexpressed EZH2 level.

p21

--

Western blot//SA-¦Â-gal activity assay

We then checked whether EZH2 knockdown may affect p21 expression, as well as senescent phenotype. Three siRNAs caused apparent reduction of EZH2 expression in both cells, and all the siRNAs significantly increased p21 expression in SW620 cells, whereas two of them (2# and 3#) induced marked p21 expression in HCT116 cells. Although si-EZH2 (1#) failed to increase p21 expression in HCT116 cells for unknown reason, this result confirms that EZH2 negatively regulated p21 expression. Furthermore, EZH2 knockdown with siRNA (3#) caused senescent phenotype, indicating that EZH2 may mediate LDM-induced senescence as well.

--

Human

HL

cellular senescence

27,882,937

Gene

0

1

0

RRM2

6241

protein coding

SKOV3 EOC

--

Epithelial ovarian cancer

Prevent

SA--gal activity assay

Indeed,knockdown of RRM2 in SKOV3 EOC cells significantly increased SA-¦Âgal activity compared with control cells.

--

Human

HL

cellular senescence

24,200,970

Gene

0

1

0

SUV39H1

6839

protein coding

WI-38

--

Aging

Prevent

Cell viability assay//SA--gal activity assay//Western blot

In order to test this, we treated cells at different PD levels with chaetocin, a specific inhibitor of SUV39H1 .First, we tested the cytotoxicity of increasing concentrations of chaetocin in the cells for all three PD levels, and found that cell cultures of all senescence states maintained a high level of viability when treated with 5 or 1 nM chaetocin and decreased rapidly at higher concentrations . Interestingly, the PD 54 cells, which express lower levels of SUV39H1, were more tolerant to higher concentrations of chaetocin, as seen by their viability at 48 h and 96 h following chaetocin treatment. In addition to a decrease in viability, increasing concentrations of chaetocin induced greater senescence in PD 47 cells.

--

Human

HL

cellular senescence

25,063,769

Gene

0

1

0

1

0

1

0

CCN1

3491

protein coding

MyoFibroblast

--

Liver disease

Accelerate

SA--gal activity assay

Numerous senescent cells were found surrounding the central veins in CCl4-treated Ccn1flox/flox mice as judged by the expression of senescence-associated ¦Â-galactosidase (SA-¦Â-Gal) as a marker, but their numbers were reduced by £¾6 % in Ccn1¡÷Hep mice ,indicating that CCN1 is critical for senescence.

¦Á6¦Â1

Binding

SA-¦Â-gal activity assay//Cell counting//qRT-PCR

First, the CCN1-DM mutant protein, which is disrupted in its ¦Á6¦Â1 binding sites, was defective for the senescence-inducing activity since it did not suppress cell proliferation or induce SA-¦Â- Gal expression. Second, coincubation of CCN1 with a function-blocking monoclonal antibody against integrin ¦Á6 (GoH3), but not control IgG, inhibited CCN1-induced senescence.Finally, an ¦Á6¦Â1-binding peptide that competes with ligand binding to ¦Á6¦Â1 abrogated CCN1-induced senescence, whereas a mutated T1 peptide that does not bind ¦Á6¦Â1 had no effect. Moreover, CCN1 also induced cellular senescence in an ¦Á6¦Â1-dependent manner in activated human HSCs. We also isolated PFs, which expressed elastin but not desmin(data not shown), and found that CCN1 triggered cellular senescence in activated PFs as it suppressed cell proliferation and induced the expression of SA-¦Â-Gal and SASP.

RAC1-NOX1-ROS

--

RT-PCR//qRT-PCR//SA-¦Â-gal activity assay//DHC//Knockdown

CCN1 treatment specifically elevated Nox1 and Rac1 expression, but not expression of other Nox or Rac isoforms or of the Nox2 cofactor p47phox. Knockdown of either Nox1 or Rac1 by specific siRNAs inhibited CCN1-induced ROS and senescence as judged by cell proliferation and SA-¦Â-Gal staining. Knockdown of Nox4, which is also expressed in fibroblasts, had no effect. Likewise, in activated PFs CCN1 induced ROS accumulation and senescence, which also required NOX-depen- dent ROS generation. These results show that CCN1 binds to integrin ¦Á6¦Â1and induces cellular senescence through the RAC1-NOX1-ROS axis, leading to the expression of antifibrosis genes associated with the SASP to inhibit fibrosis.

Human

L

cellular senescence

23,508,104

Gene

0

1

0

MAGEA2

4101

protein coding

WI-38

--

Tumor

Prevent

SA--gal activity assay

Analysis of senescenceassociated ¦Â-galactosidase activity (SA-¦Â-Gal), a well-known marker of senescence,demonstrated that MageA2 expression significantly reduced SA-¦Â-Gal levels in PMLIV-expressing cells.

PMLIV

--

Western blot

Expression of MageA2 together with PMLIV/p300 strongly decreased PMLIV acetylation. Importantly, siRNA-mediated downregulation of MageA2 in U2OS cells stably expressing PMLIV resulted in increased levels of sumoylated PMLIV, demonstrating the involvement of endogenous MageA2 in the regulation of PMLIV sumoylation.

PML-p53

Upregulation

Cell morphological analysis//qRT-PCR//SA-¦Â-gal activity assay//BrdU assay//Western blot

To this end, normal human fibroblasts were co-transduced using retroviral vectors expressing PMLIV with an empty vector or in combination with MageA2 or alternatively MageA4. After 10 days under selective culture conditions, PMLIV transduced cells showed all the features of the senescence process, because they ceased to proliferate at sub-confluent densities and became flat and enlarged. Importantly, cells co-expressing PMLIV and MageA2 did not show major morphological changes and behaved similarly to control cells. Colocalization between MageA2 and PMLIV was observed in these cells (data not shown). Analysis of senescenceassociated ¦Â-galactosidase activity (SA-¦Â-Gal), a well-known marker of senescence, demonstrated that MageA2 expression significantly reduced SA-¦Â-Gal levels in PMLIV-expressing cells. Moreover, cells expressing MageA2 and PMLIV showed higher levels of BrdU incorporation with respect to those expressing only PMLIV. Cells overexpressing MageA2 alone behaved indistinguishably from control or MageA4 transduced cells. Importantly, MageA2 expression correlated with reduced p21 protein levels, probably due to an effect of MageA2 on efficient p53 activation.Moreover, MageA2 expression affected p53 activity in this cellular system, as assessed by quantification of p53 target genes, thus confirming that MageA2 downregulates the p53-dependent response.

Human

L

cellular senescence

22,117,195

Gene

0

1

0

FOXO3

2309

protein coding

HDF

--

Aging

Prevent

Cell morphological analysis//Knockdown//SA--gal activity assay//Western blot

Senescence Phenotypes in FOXO3a-siRNA Cells Senescent cells are resistant to mitogen-induced proliferation , express senescence-associated ¦Â-gal, and have a characteri stically enlarged, flattened morphology. We measured cell growth and senescence-associated ¦Â-gal activity in FOXO3a-siRNA cells. FOXO3a-siRNA cells had an enlarged, flattened cell morphology, like old cells. The percentage of blue cells (indicating senescence-associated ¦Â-gal activity) was higher in FOXO3a-siRNA cells than in vectortransfected cells.As expected, the levels of p53 and p21 proteins were up-regulated in FOXO3a-siRNA cells as well as in old cells .

--

Human

L

cellular senescence

15,741,276

Gene

0

1

0

1

0

1

0

1

0

AGT

183

protein coding

HUVEC

--

Atherosclerosis

Accelerate

Cell morphological analysis//Flow cytometry//SA--gal activity assay//Transmission electron microscopy

SA-¦Â-gal staining was significantly increased in Ang II-stimulated cells in contrast to the control cells which indicates that Ang II promoted cellular senescence in HUVEC.We found that the cell cycle was at G0-G1, the S phase and G2/M phase tended to disappear in Ang II-induced cells compared with the control cells. Transmission electron microscopy was used to evaluate the ultra-microstructure of HUVEC. Senescent cells appeared flattened and enlarged. Chromatin was condensed at the nuclear margin, invagination of the nuclear membrane and vacuolization of the cytoplasm identified aging cells .But the control cell appeared round and smooth in shape and possessed even chromatin.

Bcl-2//ERK

Downregulation//Activation

Immunocytochemical staining//RT-PCR//Western blot

The decrease in Bcl-2 content during aging.Exposure of cells to 10-6mol L 1 Ang II resulted in activation of ERK as manifested by an increase in ERK phosphorylation. The increased phosphorylation of ERK peaked at 12 h after exposure to Ang II and lasted for 36 h. However, the activity of p-ERK declined to the basal level at 48 h after Ang II treatment.

--

Human

L

cellular senescence

18,383,564

Gene

0

1

0

1

0

1

0

1

0

TGFB1

7040

protein coding

HCEC

--

Aging

Accelerate

Flow cytometry//SA--gal activity assay//Western blot//qPCR

Cell cycle analysis by flow cytometer showed that the HCECs accumulated at G1 phase with a concomitant depletion of S phase cells after TGF-¦Â1 exposure, suggesting that cell cycle arrest during HCECs senescence induced by TGF-¦Â1 occurred at G1 phase, while H2O2 induced an obvious G2/M phase arrest. In association with the G1 arrest, we also found TGF-¦Â1 increased the percentage of SA-¦Â-gal staining cells and concomitantly increased the expression of p16 and p21, as analyzed by real-time PCR or western blot. In order to further confirm the effect of TGF-¦Â on cellular senescence, we interrupted the TGF-¦Â signaling pathway using a specific inhibitor, LY364947. When treated with LY364947, the percentage of SA-¦Â-gal staining cells was significantly decreased, and the levels of p21 and p16 were also downregulated.

--

NF-¦ÊB

Activation

Western blot

Compared with pre-senescent cells, TGF-¦Â1 treatment led to significant reduction in I¦ÊB¦Á protein levels. I¦ÊB¦Á is a canonical and specific inhibitor of NF-¦ÊB, and its reduction suggested elevated NF-¦ÊB activity in TGF¦Â-induced senescent cells; our subsequent results confirmed this. I¦ÊB¦Á loss is the key step that immediately releases cytoplasmic NF-¦ÊB for nuclear translocation and the subsequent phosphorylation of p65, a subunit of NF-¦ÊB. Therefore, we measured the phosphorylation of p65 in HCECs treated with or without TGF-¦Â. Much more phosphorylation of p65was found in TGF-¦Â-treated HCECs than in untreated cells.

Human

L

cellular senescence

27,713,146

Gene

0

1

0

1

0

1

0

1

0

CSNK2A1

1457

protein coding

HCT116

--

Aging

Prevent

SA--gal activity assay

To detect senescence, p53-/- and wild-type HCT116 cells were treated with CKII inhibitors, apigenin and DRB, and then stained for SA-¦Â-gal activity. In the p53 positive HCT115 cells, there was a significant dosedependent increase in SA-¦Â-gal activity in response to apigenin and DRB.

p52//Rb

--//--

SA-¦Â-gal activity assay//Western blot

However, p53 negative HCT116 cells showed only slight signs of senescence demonstrating that p53 is required for the induction of senescence through CKII inhibition.The level of hyperphosphorylated RB protein decreased while hypophosphorylated form increased in wildtype HCT115 cells treated with CKII inhibitor.

p53-p21

Activation

SA-¦Â-gal activity assay//Western blot//RT-PCR

When these transfectants were stained for SA-¦Â-gal activity, p53 negative HCT116 cells exhibited an apparently lower rate of SA-¦Â-gal activity compared to p53 positive HCT116 cells. In p21Cip1/WAF1 positive HCT116 cells, there was a significant increase in SA-¦Â-gal activity in response to apigenin and DRB. However, p21Cip1/WAF1 negative HCT116 cells showed only slight signs of senescence demonstrating that p21Cip1/WAF1 is required for the induction of senescence via CKII inhibition. Cells treated with apigenin showed an increase both in the p21Cip1/WAF1 protein and mRNA levels, suggesting that CKII inhibition up-regulates p21Cip1/WAF1 expression at the transcriptional level .

Human

L

cellular senescence

19,855,935

Gene

0

1

0

BAG2

836330

protein coding

TRE293

--

Aging

Accelerate

BrdU assay//SA--gal activity assay//SAHF//Western blot

To assess the functional role of BAG2 in senescence, we generated a TRE293 cell line with inducible BAG2 (TRE293-BAG2) in which the BAG2 expression is triggered by the tetracycline regulatory (Tet-on) system. Like other senescent cells, the TRE293-BAG2 cells exhibited cell cycle arrest and slow proliferation and a significant increase in SA-¦Â-Gal staining. In a senescence cell, it is basically accepted that the abundant enhancement of senescence-associated heterochromatic foci (SAHF) with heterochromatin proteins such as HP1 represses expression of proliferation-promoting genes. The senescence induced by BAG2 was further confirmed by examining changes in the chromatin structure.

--

p21

Activation

Western blot//Knockdown//SA-¦Â-gal activity assay

We monitored the levels of typical biomarkers of senescence in TRE293-BAG2 cells, (p21CIP1, p16INK4a, or p27KIP1). Western blotting revealed no significant change in the levels of p16INK4a or p27KIP1. However, there was a strong accumulation of p21CIP1, suggesting that the senescence induced by BAG2 was likely via the p21CIP1 pathway, which was also observed in H520-BAG2 cells. This interpretation was further supported by BAG2 knock-down experiments.The level of p21CIP1 protein was reduced in the TRE293 cells treated with BAG2 small interfering RNA (siRNA). To determine if the senescence induced by BAG2 was mediated only through the p21CIP1 pathway, we transfected p21CIP1 shRNA into TRE293- BAG2 cells. We found that senescence was almost fully restored when p21CIP1 was down-regulated.

Human

HL

cellular senescence

22,146,591

Gene

0

1

0

1

0

1

0

1

0

GRK4

2868

protein coding

HEK293

--

Aging

Accelerate

Immunostaining//SA--gal activity assay

The Ki-67 protein were clearly detected within the cell nuclei of GRK4(-) cells and CNE2 cells, whereas it was barely found in GRK4(+) cells. We next assessed the SA-¦Â-gal activity in the GRK4(+) and GRK4(-) cells by a SA-¦Â-gal staining. The stained cells and total cells were counted under an inverted phase microscope and the percentage of cells staining positive was calculated.High stain positive of 99¡À0.20% in GRK4(+) cells for SA-¦Â-gal was detected , while this was markedly low to only1¡À0.15% in GRK4(-) cells.

--

Human

L

cellular senescence

28,912,086

Gene

0

1

0

1

0

TAK1

39659

protein coding

IMR-90

--

Aging

Prevent

SA--gal activity assay

The number of cells expressing SA-¦Â-gal was dramatically increased after treatment with the TAK1 inhibitor relative to the number in the control cells.

p53

--

Western blot

p53 expression in RPE cells under oxidative stress was strongly affected by pretreatment of the cells with the TAK1 inhibitor, seen by the inhibition of p38 phosphorylation . In control cells (without such pretreatment) the expression of p53 gradually increased, reaching a peak after 60 minutes, whereas in the pretreated cells, p53 expression peaked after 10 minutes and then declined.Over a longer period, TAK1 inhibition reduced p53 expression to a level slightly higher than in control cells.

--

Human

L

cellular senescence

25,118,260

Gene

0

1

0

BTG3

492479

protein coding

HCT116

--

Aging

Prevent

BrdU assay//Cell morphological analysis//Flow cytometry//Knockdown//SA--gal activity assay//Western blot

In contrast to what was observed previously in HCT116 cells,loss of BTG3 expression in IMR90 cells resulted in blunted cell proliferation, as demonstrated by a significant decrease of cells in the S phase, a moderate increase in the G1 population, a marked reduction in BrdU incorporation and RB phosphorylation. The slowed proliferation remained for at least for 2 weeks , indicating that this is not a transient growth arrest. Additionally, BTG3-knockdown cells displayed the enlarged, flattened morphology that is typical of senescent cells . The phenotype was confirmed by staining the cells for senescenceassociated ¦Â-galactosidase (SA-¦Â-Gal) activity: we observed an increase in SA-¦Â-Gal staining of at least sixfold 48 h after transfection with the BTG3-2 siRNA , which was further enhanced 1 week after transfection.

JMJD3

--

RT-PCR//Knockdown

Using RT PCR, we found that the expression of JMJD3 was increased in BTG3-knockdown cells.

ERK-AP1

--

RT-PCR//SA-¦Â-gal activity assay//Knockdown

To confirm the involvement of the ERK signaling pathway, we treated BTG3-knockdown IMR90 cells with the ERK-specific inhibitor PD98059. As a result, a significant reduction in senescence was observed when ERK was inhibited. In contrast, two other unrelated drugs, SB202190 (p38 inhibitor) and SB202474 (negative control), had no apparent effect, indicating the specificity of the treatment.

Human

HL

cellular senescence

22,020,331

Gene

0

1

0

1

0

1

0

1

0

1

0

1

0

RRN3

54700

protein coding

MCF-7,MEF

--

Aging

Accelerate

Immunostaining//SA--gal activity assay//Western blot//qRT-PCR

We found that RPL11 depletion suppressed the accumulation of p53, p21, and p16, and the number of SA-¦Â-gal-positive cells induced by TIF-IA overexpression . We depleted RPL11 in the presence of small interfering RNAs (siRNAs) specific for DKC1, RRP5, and RRS1, any of which induced p53 accumulation, p21 induction, p16 induction, and cellular senescence.We found that RPL11 depletion abrogated these events.

--

p53-p21

Activation

SA-¦Â-gal activity assay

As with the case of TIF-IA overexpression, the increased number of SA-¦Â-positive cells by the depletion of the rRNA-processing factors was counteracted by p53 or p21 depletion .

Human

L

cellular senescence

25,732,822

Gene

0

1

0

1

0

1

0

1

0

PON1

5444

protein coding

HCT116

--

Aging

Prevent

Knockdown//SA--gal activity assay

To evaluate the influence of PON1 knockdown on ageing of HDMECs, we measured cellular senescence by SA-¦Â-gal staining. Cell senescent morphology, such as an enlarged and flattened shape with an increased diameter, was observed in aged HDMECs at passage 2 and in PON1 siRNA #2-treated HDMECs at 48 h post-transfection. The number of cells positive for SA-¦Â-gal staining increased significantly compared to early passage HDMECs at passage 8 and in scrambled siRNA-treated HDMECs.

p16

--

Western blot

In Western blot, the expression of p16 in PON1 knockdown HDMECs was observed. At 48 h post-transfection, the protein expression of p16 in PON1 siRNA-treated HDMECs was significantly higher than the expression level in scrambled siRNA-treated HDMECs.

--

Human

L

cellular senescence

22,897,574

Gene

0

1

0

1

0

SMARCA4

6597

protein coding

SW48

--

Colorectal cancer

Prevent

CCK-8 assay//DAPI staining//Flow cytometry//SA--gal activity assay//Western blot//qRT-PCR

Data showed that the percentages of SA-¦Â-galpositive cells in SW48 sh-BRG1 cells significantly increased compared with sh-Con cells in both DOX treatment and nontreatment conditions .The results showed that the growth rates significantly decreased in BRG1 KD groups, suggesting that BRG1 KD inhibited cell proliferation. Cell cycle analysis by flow cytometry detection was performed, and the results revealed that BRG1 KD led to cell cycle arrest at G2-M phase. Furthermore, we examined SAHF by staining 4,6-diamidino-2-phenylindole (DAPI), which showed distinct SAHF formation in BRG1 KD cells as observed by laser confocal fluorescence microscopy scanning.

--

SIRT1-p53-p21

--

Western blot//qRT-PCR//IHC//SA-¦Â-gal activity assay//Co-IP//Immunofluorescence

Our data showed that the increase of p21 induced by BRG1 KD was abrogated by the treatment of p53 siRNA. Moreover, SA-¦Â-gal-positive cells stopped increasing after the KD of BRG1 with p53 siRNA in both DOX treatment and non-treatment conditions.The regulation of BRG1 in the p53/p21 pathway was further confirmed by using another shRNA against BRG1. These results suggested that KD of BRG1 promotes senescence by activating the p53 pathway in CRC cells.

Human

L

cellular senescence

28,182,012

Gene

0

1

0

1

0

1

0

1

0

1

0

1

0

PPARD

5467

protein coding

Primary keratinocyte

--

Skin cancer

Accelerate

BrdU assay//SA--gal activity assay

A higher percentage of BrdU labeling and lower percentage of ¦Â-gal-positive cells was noted in HRAS-expressing PPAR¦Â/¦Ä-null compared with wild-type cells. Surprisingly, ligand activation of PPAR¦Â/¦Ä decreased both the percentage of ¦Â-gal- and BrdU-positive cells in HRAS-expressing wild-type but not in PPAR¦Â/¦Ä-null cells.

RASGRP1//ILK

--//Downregulation

Western blot//qPCR//CHIP

Expression of the negative RAS regulator RASGAP120 was increased and the positive RAS regulator RASGRP1 was decreased in response to HRAS activation, respectively .Interestingly, expression of RASGAP120 was significantly higher, whereas expression of RASGRP1 was lower in HRAS expressing PPAR¦Â/¦Ä-null cells compared with wild-type cells. In response to HRAS activation, expression of mRNA encoding the negative RAS regulator Rasa4 was increased in cells from both genotypes, but relatively higher expression of Rasa4 mRNA was also observed in HRAS-expressing PPAR¦Â/¦Ä-null cells compared with wild-type cells (data not shown).Consistent with past studies,32 34 expression of ILK and PDPK1 was higher in control and HRAS-expressing PPAR¦Â/¦Ä-null cells. In silico examination of the Ilk gene revealed two potential PPREs in the second intron .Interestingly, mutating the PPAR¦Â/¦Ä binding half-site in a luciferase reporter construct containing the upstream PPRE abolished the repression of ILK by PPAR¦Â/¦Ä.

PI3K-Akt//MEK-ERK

Downregulation//Upregulation

Western blot//SA-¦Â-gal activity assay//Immunofluorescence//Histochemical staining//Cumulative frequency of mean intensity of cells

The higher levels of p-MEK and p-ERK and lower level of p-AKT in wild-type cells correlated with higher markers of senescence and this was not found in HRAS-expressing PPAR¦Â/¦Ä-null cells . Inhibition of MEK1 with PD98059 delayed HRAS-induced senescence in both wild-type and PPAR¦Â/¦Ä-null cells .Higher expression of ILK, p-AKT (S473, T308) was also found in skin tumors from PPAR¦Â/¦Ä-null mice compared with wild-type mice.

Human

HL

cellular senescence

24,213,576

Gene

0

1

0

1

0

HDAC1

3065

protein coding

UCB-MSC

--

Aging

Prevent

SA--gal activity assay

Consistently with our previous report, the inhibition of HMGA2 as well as HDAC1 and HDAC2 led to the senescence of hUCB-MSCs as shown by SA ¦Â-gal staining, in addition to significantly increased expression of p21CIP1/WAF1 and p16INK4A.

--

RT-PCR//qRT-PCR//SA-¦Â-gal activity assay

Consistently with our previous report, the inhibition of HMGA2 as well as HDAC1 and HDAC2 led to the senescence of hUCB-MSCs as shown by SA ¦Â-gal staining, in addition to significantly increased expression of p21CIP1/WAF1 and p16INK4A.

--

Human

HL

cellular senescence

20,652,617

Gene

0

1

0

HDAC2

3066

protein coding

UCB-MSC

--

Aging

Prevent

SA--gal activity assay

Consistently with our previous report , the inhibition of HMGA2 as well as HDAC1 and HDAC2 led to the senescence of hUCB-MSCs as shown by SA ¦Â-gal staining, in addition to significantly increased expression of p21CIP1/WAF1 and p16INK4A.

--

RT-PCR//qRT-PCR//SA-¦Â-gal activity assay

Consistently with our previous report [21], the inhibition of HMGA2 as well as HDAC1 and HDAC2 led to the senescence of hUCB-MSCs as shown by SA ¦Â-gal staining, in addition to significantly increased expression of p21CIP1/WAF1 and p16INK4A.

--

Human

HL

cellular senescence

20,652,617

Gene

0

1

0