Venkatachalam/aging_reg_dataset · Datasets at Hugging Face

BHLHE40

8553

protein coding

MCF-7

--

Aging

Accelerate

SA--gal activity assay//Western blot

Microscopic analysis showed that the number of SA-¦Â-galactosidase-positive colonies was increased in DEC1-expressing cells compared with that in control and mutant DEC1-expressing cells.

p53

--

Northern blot

To confirm this, Northern blot analysis was performed. We showed that DEC1 was induced by p53 but not mutant p53(R249S) in H1299 cells.

--

Human

HL

cellular senescence

18,025,081

Gene

0

1

0

1

0

SLC13A3

64849

protein coding

MRC-5,WI-38

--

Aging

Accelerate

Immunostaining//SA--gal activity assay

In the present study, the positive rate of SA-¦Â-gal staining has reached 95% in the NaDC3-infected MRC-5 cells at PD 37, which was significantly increased than those in the uninfected and control vector infected cells. When examining cellular morphology of the infected cells, we observed significant senescent morphological changes in the NaDC3-infected cells. The results by DAPI staining showed that NaDC3 could also induce similar premature cellular senescence in the WI-38 cells.

NADH

Upregulation

NAD+/NADH Quantitation kit//Confocal microscope

NaDC3 overexpression markedly increased intracellular NADH levels compared with the two controls.

--

Human

L

cellular senescence

25,384,549

Gene

0

1

0

1

0

DIRAS3

9077

protein coding

ASC

--

Aging

Prevent

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

DIRAS3 KD cells displayed a cytoplasm with thin and long processes and a significant number of these cells became flat, resembling the morphological features of the premature senescence phenotype induced by oncogenic assault in primary human fibroblasts . As demonstrated by cytochemical staining and FACS analyses using a fluorogenic substrate C12FDG,DIRAS3 KD led to a significant increase in the number of senescenceassociated ¦Â-galactosidase (SA-¦Â-GAL) positive ASCs and to a significant increase in cell size .

--

Akt-mTOR

Downregulation

Western blot//qRT-PCR//Knockdown

To investigate the effect of DIRAS3 knock-down (KD) on Akt-mTOR signaling in ASCs we employed lentivirus mediated DIRAS3 specific shRNA . DIRAS3 KD leads to increased activity of Akt-mTOR signaling in ASCs.

Human

L

cellular senescence

28,316,325

Gene

0

1

0

1

0

1

0

EZH2

2146

protein coding

SKOV3/DDP

--

Ovaria cancer

Prevent

SA--gal activity assay//Western blot

The inhibition of EZH2 expression significantly downregulated H3K27me3 expression and upregulated p14, p16, p53, and pR protein expression. This suggested that EZH2 inhibition led to the activation of the INK/ARF/Rb pathway and, thereby, induced cellular senescence. Furthermore, microscopic analyses revealed the following morphological changes in the SKOV3/DDP cells in the pSUPER-EZH2 group: increased cell volume, flat granules, and ¦Â-galactosidase staining (blue), which are characteristics of cellular senescence. These changes were not observed in the blank control group.

--

INK-ARF-Rb

--

Western blot

The inhibition of EZH2 expression significantly downregulated H3K27me3 expression and upregulated p14, p16, p53, and pR protein expression. This suggested that EZH2 inhibition led to the activation of the INK/ARF/Rb pathway and, thereby, induced cellular senescence.

Human

HL

cellular senescence

27,610,467

Gene

0

1

0

1

0

UBE3A

7337

protein coding

Human Burkitt Lymphoma cell line derived cell line

--

B-cell lymphoma

Prevent

Flow cytometry//SA--gal activity assay

Down-regulation of E6AP increased the proportion of cells in G1 with a corresponding decrease of cycling cells , resulting in a reduction in cell proliferation with no impact on cell viability. This effect coincided with a significant number of cells showing hallmarks of senescence .

--

PML

Upregulation

Western blot//Immunofluorescence

Strikingly, E6AP knockdown resulted in restoration of PML expression and formation of PML-NBs in these cells.

Human

L

cellular senescence

22,689,861

Gene

0

1

0

1

0

DNMT1

1786

protein coding

UCB-MSC

--

Aging

Prevent

MTT assay//SA--gal activity assay

DNMT inhibition by 5-AzaC treatment induced cellular senescence, as shown by SA ¦Â-gal staining , and decreased the cellular proliferation rate in a dose-dependent manner, as shown by an MTT assay.

p16//p21/WAF1//EZH2//BMI1//miR200c//miR-214

--//--//--//--//--//--

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

SA ¦Â-galactosidase activity and expression levels of p16INK4A and p21CIP1/WAF1 were increased at day 3 of 5-AzaC treatment, and prominent changes were observed after 5 days of treatment with 5- AzaC.After inhibition of DNMT by 5-AzaC treatment, we observed decreased EZH2 and BMI1 expression levels. Because the significant decrease of EZH2 and BMI1 occurs after 3 days of treatment with 5-AzaC, we investigated miRNA expression levels at 1, 3 and 7 days after treatment with 5-AzaC and found that both mature and precursor miRNAs were increased at the time points indicated. After overexpression of miR-200c and miR-214, MSCs underwent cellular senescence, as shown by SA ¦Â-gal staining, and BMI1 and EZH2, the respective targets of miR200c and miR-214 were decreased, as shown by real-time qPCR. In addition, inhibition of miR-214 using antisense oligonucleotide transfection increased EZH2 expression. Although inhibition of miR-200c did not yield consistent results, overexpression of both miRNAs decreased their respective target (BMI1 and EZH2) at the mRNA level, suggesting that overexpressed miRNA during cellular senescence regulates the expression levels of BMI1 and EZH2.

--

Human

HL

cellular senescence

21,572,997

Gene

0

1

0

1

0

SIRT1

23411

protein coding

2BS

--

Aging

Prevent

Immunostaining//SA--gal activity assay

2BS with enforced expression of wild type SIRT1 displayed lower frequency of SA-¦Â-gal staining than cells transfected with mock vector and H363YSIRT1 constructs. We observed that 2BS cells expressing SIRT1 did not develop pronounced nucleoli or DNA foci.

--

p16-Rb//ERK-S6K1

--//Activation

Western blot

Compared with mock vector and H363YSIRT1 transfected 2BS cells, p16INK4A displayed a two-fold decrease in SIRT1-transfected cells, accordingly phospho-Rb (Ser795) was increased for more than two times whereas the levels of b-actin and Rb remained unchanged in different batches of transfected cells.The levels of SIRT1 protein were declined in late passage of 2BS (63PDs), and the phosphorylations of ERK and S6K1, but not that of AKT, were also decreased in senescent cells.

Human

L

cellular senescence

18,320,031

Gene

0

1

0

1

0

TBX2

6909

protein coding

WI-38

--

Aging

Prevent

Immunostaining//SA--gal activity assay

Additionally, TBX2/PML-IV-expressing cells stained positive for Ki67 and negative for SA-¦Â-Gal when compared with DON/PML-IV cells.

PML

Binding

qRT-PCR//Western blot

We detected a robust increase both in TBX2 transcript (~4-fold) and in protein levels (B3-fold) in PML-/- MEFs when compared with PML+/+ MEFs as determined by qRT-PCR (left panel) and western blot analysis (right panel).

--

Human

HL

cellular senescence

22,002,537

Gene

0

1

0

1

0

NUAK1

9891

protein coding

WI-38

--

Aging

Accelerate

Crystal violet assay//SA--gal activity assay

NUAK1 expression was found to block cell growth and induce a characteristic senescent morphology. This growth arrest was due to premature senescence induction, as evidenced by the increased proportion of SA-¦Â-Gal-positive and SAHF-positive cells among the NUAK1-overexpressing cells, as compared with control cells .

LKB1//LATS1

Activation//Downregulation

Crystal violet staining//IP

The growth-inhibitory effect of NUAK1 was largely reverted when LKB1 activity was inhibited by either expression of a dominantnegative form or by its knockdown .NUAK1 and LA TS1by performing co-immunoprecipitation assays on extracts from cells co-expressing NUAK1 and LATS1. Interestingly, NUAK1 was detected in the LATS1 immunoprecipitate. We next repeated the immunoprecipitation experiment with endogenous proteins. Once again, NUAK1 protein was found in the LATS1 immunoprecipitate.

--

Human

HL

cellular senescence

19,927,127

Gene

0

1

0

1

0

LATS1

9113

protein coding

WI-38

--

Aging

Prevent

Crystal violet assay//Hoechst staining//SA--gal activity assay

A growth arrest was observed when compared with control infected cells, as judged using colony formation assays.Similar to constitutive NUAK1 expression, the LATS1 DN expression induced gross aneuploidies and the appearance of SA-¦Â-Gal activity.

--

Human

HL

cellular senescence

19,927,127

Gene

0

1

0

1

0

1

0

GDF15

9518

protein coding

TBE

--

Aging

Accelerate

SA--gal activity assay

Disruption of GDF15 markedly reduced CSEinduced cellular senescence, represented by less induced SA ¦Â-gal activity, less inhibition of cell proliferation, and less HMGB1 release.

--

ALK1-Smad1

Activation

Western blot

GDF15 protein time-dependently induced phosphorylation of Smad1/5/8 in primary human airway epithelial cells. Moreover, a direct binding assay of GDF15 and ALK1 protein was performed in vitro to confirm the direct interaction between GDF15 and ALK1. We found that GDF15 bound to the ALK1 receptor in a dose-dependent manner.In our pilot study, CSE significantly increased GDF15, HMGB1, p21 and Smad1/5/8 phosphorylation at day 1 in hTBE cells.

Human

HL

cellular senescence

27,093,475

Gene

0

1

0

AKTIP

64400

protein coding

Fibroblast

--

Aging

Prevent

Knockdown//SA--gal activity assay

Cell labelling with the empirical senescence marker SA-¦Âgal revealed that early passage HPFs at 11 and 13 days after shAKTIP infection exhibit a significant increase in senescent cells compared with controls.

--

Human

L

cellular senescence

27,512,140

Gene

0

1

0

1

0

PPM1B

5495

protein coding

IMR-90

--

Aging

Prevent

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

In contrast to the basal level of staining observed in the control cells, the PPM1B depleted cell lines developed a characteristic senescence-associated ¦Â-GAL staining pattern with a 10-fold increase in the percentage of stained cells. Moreover PPM1B depleted cells displayed a flattened and irregular cell shape with a bigger cell volume which is typical of senescent cell morphology . In addition, the gene expression levels of four senescence markers were examined by RT-qPCR in these cells . The transcript abundance of p21, plasminogen activator inhibitor 1 (PAI), SA-¦Âgalactosidase (¦Â-GAL) and p16 all showed a two to threefold increase in gene expression upon PPM1B depletion compared to those infected with control virus. The increase in transcript abundance is consistent with the immunoblot analysis against of PAI-1, p21 and p16, suggesting that the upregulation of senescence markers occurs at both the transcript and protein levels.

--

Human

HL

cellular senescence

24,674,756

Gene

0

1

0

1

0

1

0

RPS14

6208

protein coding

IMR-90

--

Aging

Accelerate

SA--gal activity assay//Western blot

Next, we characterized the effects of directly decreasing the ribosome biogenesis factors RSL1D1, NS, DDX21 or EBP2 using shRNAs. Depletion of these factors induced a proliferation arrest, a decrease in the proliferation markers KI67 and CENPA, and an increase in senescence-associated ¦Â-galactosidase staining (SA-¦Â-gal).

CDK4//Cyclin D1

--//--

Pull-down assay

GST pull down assays with purified proteins revealed that RPS14 binds to either CDK4 or cyclinD1 alone or to the complex of CDK4 with cyclinD1.

Rb

--

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

The E7 mutant ¦¤79 83, which binds and inhibits Rb, rescued cells from senescence after knockdown of RSL1D1, while mutants E7¦¤6 10 and E7¦¤21 24 that do not bind Rb were not efficient at inhibiting senescence after knockdown of RSL1D1. Furthermore, the kinases CDK4 and CDK6, which are able to phosphorylate and inactivate Rb29, were also capable of preventing senescence after knockdown of RSL1D1. Expression of intact CDK4, but not of a catalytically inActivate mutant (CDK4(K35M)), efficiently bypassed senescence, preventing the downregulation of Ki67 expression, as well as rescuing Rb phosphorylation and the expression of E2F targets such as CENPA and MCM6 after knockdown of RSL1D1 .

Human

HL

cellular senescence

29,941,930

Gene

0

1

0

1

0

ERBB2

2064

protein coding

--

Prostate

Prostate cancer

Prevent

BrdU assay//Ki67 staining//SA--gal activity assay

The PB-Cre4: Ptenfl/fl Her2KI tumors had significantly higher levels of proliferation than the PBCre4: Ptenfl/fl tumors, as assessed by Ki67 and BrdU . Furthermore, there was a marked down-regulation of the growth arrest and senescence markers p16 and p21 in the PB-Cre4: Ptenfl/fl Her2KI compared with the PB-Cre4: Ptenfl/fl mice. Moreover, there was a robust down-regulation of senescence-associated ¦Â-galactosidase signals in the PBCre4: Ptenfl/fl Her2KI prostates compared with PB-Cre4: Ptenfl/fl mice .

--

MEK-ERK//PI3K-AKT

Activation//--

Western blot

Tumors from PB-Cre4: Ptenfl/fl Her2KI mice showed elevated pERK1/2 activation (with total ERK1/2 levels unchanged), whereas PB-Cre4: Ptenfl/fl tumors had very low levels of pERK1/2. Heregulin treatment also increased PTEN phosphorylation, which can in turn decrease the ability of PTEN to inhibit PI3K-mediated AKT activation .

Human

HL

cellular senescence

21,930,937

Gene

0

1

0

1

0

1

0

SIRT3

23410

protein coding

WI-38

--

Aging

Prevent

DAPI staining//SA--gal activity assay//Western blot

Our results indicated that the overexpression of SIRT3 significantly reduced the number of cells positive for SA-¦Â-gal and inhibited SAHF formation compared with empty vector .Meanwhile, the accumulation of p16INK4A was decreased .

--

FOXO1

Binding

Confocal microscopy//Western blot

We found that the endogenous SIRT3 was highly expressed in the mitochondria and cytoplasm, FOXO1 was expressed in the cytoplasm and nuclei, and the SIRT3 staining strongly overlapped with that of FOXO1 in the cytoplasm. We could confirm that SIRT3 was binding with FOXO1. SIRT3 overexpression keeps FOXO1 acetylation to a very low level.To verify the direct effect of SIRT3 on FOXO1-targeted gene expression including catalase and MnSOD, we examined the expression of target genes. The results indicated that expression levels were increased in the pSIRT3 group compared with control and vector groups.

Human

L

cellular senescence

23,494,737

Gene

0

1

0

1

0

1

0

AKT1

207

protein coding

Primary leiomyoma cell

--

Uterine leiomyomas

Prevent

SA--gal activity assay//qPCR

The percentage of ¦Â-galactosidase stained DDHLM and primary leiomyoma cells with MK-2206 treatment were significantly higher than untreated cells.Approximately 32% of cells exhibited SAHF when treated with MK-2206 compared to 8% in control cells. When leiomyoma cells were treated with MK-2206, the senescence associated genes,P16,P21 and P53 were significantly upregulated .

miR-182//miR-200//HMGA2

--//--//--

Immunofluorescence//qPCR

In response to MK-2206 as well as the dual PI3K/mTOR inhibitor, BEZ235, ROS levels increased in primary leiomyoma cells. Similarly,miR-182 and miR-200a/c expression increased in leiomyoma cells treated with MK2206 in a dose-dependent manner.HMGA2 expression was significantly increased in a dose-dependent manner when AKT was inactivated by MK-2206.

--

Human

L

cellular senescence

24,476,133

Gene

0

1

0

1

0

CPT1C

126129

protein coding

PANC-1

--

Pancreatic cancer

Prevent

BrdU assay//DAPI staining//SA--gal activity assay//qPCR

Upon CPT1C loss, the number of PANC-1 cells Activately replicating DNA decreased,consistent with effective inhibition of colony formation in a cell concentrationdependent manner , indicating that PANC-1 cells lacking expression of CPT1C have reduced proliferation. In addition, strong SA-¦Â-gal activity indicated that CPT1C depletion led to the severe Y. Wang et al. senescence state of PANC-1 cells.As expected, loss of CPT1C caused PANC-1 cells to change shape and increase cytoplasmic granularities. DNA SAHF was specifically enriched in CPT1C depleted PANC-1 cells, while controls were excluded from SAHF. CPT1C-depleted PANC-1 cells also had abnormal nuclear size, when compared to control cells.Furthermore, a significant increase in IL8 mRNA levels confirmed a sharp induction of the SA phenotypic changes observed by the SA-¦Â-gal assay. Finally, the TRF length assay revealed that CPT1C depletion inhibited the elongation of telomeres by 0.8 kb in PANC-1 cells.

--

Human

L

cellular senescence

29,317,762

Gene

0

1

0

1

0

1

0

1

0

MIF

4282

protein coding

MSC

--

Aging

Prevent

Cell morphological analysis//qPCR

Moreover, when comparing cell morphology between MIF-overexpressing and the control cells, the control hMSCs displayed enlarged and flattened morphology, whereas the MIF-overexpressing hMSCs maintained spindle morphology.In addition, our real-time PCR results showed that the expression levels of p16 and p21 mRNA transcripts were significantly downregulated (P < 0.05) in MIF-overexpressing hMSCs compared to those in the control hMSCs.

--

Akt

Activation

Western blot

In addition to increased expression of MIF, our western blot results also showed that the levels of total and phosphorylated AKT were increased in MIF-overexpressing hMSCs than those in the control cells.

Human

HL

cellular senescence

24,274,936

Gene

0

1

0

1

0

IGFBP3

3486

protein coding

MCF-7,IMR-90

--

Aging

Accelerate

BrdU assay//Ki67 staining//SA--gal activity assay

Lentiviral IGFBP3 expression in MCF7 cells caused striking proliferation arrest as determined by the lack of BrdU incorporation and Ki-67 staining, which was accompanied by the induction of SA ¦Â-gal activity. Further, addition of purified recombinant IGFBP3 protein to the culture medium of MCF-7 cells resulted in dosedependent induction of SA ¦Â-gal activity, increased G1 and decreased S and G2/M fractions, reduced BrdU incorporation , and reduced mitotic phospho-histone H3 staining . Lentiviral IGFBP3 expression in IMR-90 human primary fibroblasts also resulted in abrogation of Ki-67 staining , induction of SA ¦Â-gal activity,and formation of senescence-associated heterochromatic foci.

Akt

--

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

In accordance with this finding, IGFBP3 suppressed the baseline and IGF-induced levels of phosphorylated AKT, which is a downstream target of IGF signaling. We then assessed the role of AKT in IGFBP3-induced senescence by using constitutively Activate AKT (i.e., myr-AKT). myr-AKT expression in MCF-7 cells resulted in elevation of phosphorylated AKT levels and abrogation of IGFBP3-induced senescence, suggesting that IGFBP3 induces senescence through suppression of AKT activity.

p53//Rb

--//--

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

Senescence induced by IGFBP3 was abolished by knockdown of p53 or Rb, which suggests the requirement of p53 and Rb tumor suppressor pathways for IGFBP3-induced senescence.

Human

L

cellular senescence

22,778,398

Gene

0

1

0

1

0

1

0

SERPINE1

5054

protein coding

MCF-7

--

Aging

Accelerate

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

PAI-1 knockdown resulted in concomitant decrease in extracellular IGFBP3 levels and suppression of senescence induction. These results suggest that doxorubicin-induced DNA damage enhances the secretion of PAI-1, which in turn results in elevated extracellular IGFBP3 and induction of senescence in MCF-7 cells.

t-PA//IGFBP3

--//--

Knockdown//Western blot

The reduction of IGFBP3 levels upon PAI-1 knockdown was alleviated by the addition of anti t-PA antibody to the culture medium, supporting the role for t-PA in mediating IGFBP3 reduction upon PAI-1 knockdown.Recombinant PAI-1 increased extracellular IGFBP3 levels and induced senescence in MCF-7 cells, which was abolished by knockdown of IGFBP3.

--

Human

L

cellular senescence

22,778,398

Gene

0

1

0

1

0

1

0

1

0

AGO2

27161

protein coding

WI-38

--

Aging

Accelerate

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

In AGO2-deficient cells replicative lifespan increased by ¡«3¨C4population doublings whereas E7-expressing cells showed a lifespan extension of¡«5¨C6 population doublings. In line with this finding, the fraction of AGO2- deficient cells incorporating EdU increased and senescence-associated beta-galactosidase (SA-¦Â-Gal) activity decreased transiently when compared with shC control cells.Strikingly, AGO2-overexpressing cells induced an abrupt proliferative arrest with features of senescence as shown by a decrease in the number of cells incorporating EdU and an increase in the percentage of SAHF-positive cells.

E2F//Rb1//let-7f

Binding//--//--

CHIP//Western blot//RIP

This analysis revealed, that of the top 577 E2F-responsive promoters known at present ,320 (that is 55.5%) were occupied by AGO proteins in senescent cells, as opposed to only 77 (that is 13.3%) in control cells.To test this possibility, we first carried out co-immunoprecipitation experiments between endogenous AGO2, RB1 and HDAC1 in cellular lysates prepared from RASV12-induced senescent fibroblasts.HDAC1 and AGO2 co-immunoprecipitated with each other. Moreover, both HDAC1 and AGO2 efficiently co-immunoprecipitated RB1. Similar results were obtained in Saos-2 cells undergoing RB1- induced senescence. Consistent with the physical interaction between AGO2 and RB1,AGO2 showed partial colocalization with SAHFs at their periphery, a staining pattern that was similar for the heterochromatin marker H3K27me3 and RB1 (ref. 2) and is thus congruent with the peripheral SAHF localization of E2F-target genes.The interaction between either H3K9me2 or AGO2 and let-7f was confirmed by RNA immunoprecipitation combined with small RNA complementary DNA (srcDNA) cloning and sequencing in senescent cells.

--

Human

HL

cellular senescence

22,366,686

Gene

0

1

0

1

0

1

0

YAP1

10413

protein coding

MSC

--

Osteoarthritis

Prevent

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

Phenotypic characterizations revealed that the downregulation of YAP in hMSCs also resulted in a similar premature aging phenotype. By contrast, ectopic expression of YAP rescued the premature senescence observed in YAP?/?hMSCs, as evidenced by the reduced number of SA-¦Â-gal¨Cpositive cells ,enhanced growth rate and clonal expansion ability , decreased expression of P16 and P21, lower levels of ROS , and slower in vivo decay after engraftment.

--

YAP-TEAD//YAP-FOXD1

--//--

SA-¦Â-gal activity assay//Dual-Luciferase reporter assay//Western blot//Clonal expansion assay

Similar to YAP-deficient hMSCs, TEADs KD/KO hMSCs also showed major phenotypes of premature senescence, such as an increased number of SA-¦Â-gal¨Cpositive cells, compromised clonale. Moreover, the activity of 8 ¡Á GTIIC-Luc, a YAP/TAZ-responsive reporter, decreased in both RS hMSCs and WS hMSCs . Lentiviral overexpression of YAP or FOXD1 effectively attenuated the senescent features of RS hMSCs and WS hMSCs.

Human

HL

cellular senescence

30,933,975

Gene

0

1

0

1

0

1

0

ING1

3621

protein coding

PCa

--

Prostate cancer

Accelerate

SA--gal activity assay

By conducting SA-¦Â-Gal staining in the stably transduced cells, ING1b was shown to induce cellular senescence in PCa cells . Further SA-¦Â-Gal assays were performed with different ligand treatments and indicate that ING1b enhances the level of senescent cells without synergizing with AR .

p16//p27//AR

Upregulation//--//--

Co-IP//Pull-down assay//Fluorescence microscopy

The results indicated that ING1b upregulates mRNA and protein expression levels of p16 in LNCaP-ING1b cells.Moreover, ING1b has an additive effect along with the AR antagonist Casodex on p16 mRNA induction. ING1b also stabilized p27 protein level in LNCaP-ING1b cells while not altering p27mRNA level (data not shown).In line with the previous results, AR protein was indeed detected in the immunoprecipitates with anti-ING1 antibody whereas no AR was detected in the negative control using a nonspecific antibody.AR protein could be detected with GST-ING1b eluates whereas the negative GST control did not show AR signal. Quantitative colocalization analyses of the images from cotransfected cells suggest that ING1b colocalizes with AR in the nucleus excluding the nucleolus with a high statistical significance.

--

Human

HL

cellular senescence

26,993,046

Gene

0

1

0

TGFB1

7040

protein coding

HBEC

--

Idiopathic pulmonary fibrosis

Accelerate

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

TGF-¦Â1 increased the percentage of SA-¦Â-gal staining cells and concomitantly induced the expression of p21/waf-1, as assessed by RT-PCR and Western blotting . Cell cycle analysis also showed an increase of senescent cells, as reflected by an accumulation of cells in the G0/G1phase . Furthermore, the accumulation of cells in the G0/G1phase was slightly increased after an additional 48-h incubation without TGF-¦Â, suggesting irreversible cell cycle arrest after TGF-¦Â treatment.

p21

Upregulation

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

TGF-¦Â1 increased the percentage of SA-¦Â-gal staining cells and concomitantly induced the expression of p21/waf-1, as assessed by RT-PCR and Western blotting.

--

Human

L

cellular senescence

21,224,216

Gene

0

1

0

1

0

1

0

1

0

SIRT6

51548

protein coding

HBEC

--

Idiopathic pulmonary fibrosis

Prevent

SA--gal activity assay

Overexpression of SIRT6 significantly suppressed the percentage of SA-¦Â-gal staining cells after TGF-¦Â1 treatment. In contrast, SIRT6 siRNA dramatically increased the percentage of SA-¦Â-gal staining cells, indicating that TGF-¦Â-induced intrinsic SIRT6 upregulation alone is not sufficient to completely inhibit senescence, but SIRT6 has the ability to antagonize TGF-¦Â-induced cellular senescence in HBEC.

TGF-¦Â

Downregulation

SA-¦Â-gal activity assay

Overexpression of SIRT6 significantly suppressed the percentage of SA-¦Â-gal staining cells after TGF-¦Â1 treatment. In contrast, SIRT6 siRNA dramatically increased the percentage of SA-¦Â-gal staining cells, indicating that TGF-¦Â-induced intrinsic SIRT6 upregulation alone is not sufficient to completely inhibit senescence, but SIRT6 has the ability to antagonize TGF-¦Â-induced cellular senescence in HBEC.

--

Human

L

cellular senescence

21,224,216

Gene

0

1

0

SIRT1

23411

protein coding

HDF

--

Colorectal cancer

Prevent

SA--gal activity assay

Only in c-MYC immortalized but not in primary HDFs down-regulation of SIRT1 by siRNAs induced a pronounced increase in cell size and senescence-associated ¦Â-galactosidase activity at pH 6, two markers of cellular senescence.

c-Myc

--

Western blot

HDFs immortalized by retroviral introduction of constitutive expression of c-MYC (6) showed increased expression of SIRT1 protein.

--

Human

HL

cellular senescence

22,190,494

Gene

0

1

0

DUSP3

1845

protein coding

HeLa,MeWo

--

Cervical cancer

Prevent

SA--gal activity assay

The results indicated that decreasing endogenous DUSP3 activity per se caused increased cellular senescence of both cell lines after 72 h .

--

Human

L

cellular senescence

28,389,334

Gene

0

1

0

SIRT1

23411

protein coding

Human Nucleus pulposus cell

--

Disc degenerative disease

Prevent

RT-PCR//Western blot

The expression of p16 and p21 mRNA were higher in the resveratrol group than the control; while their expression increased in the Sirt siRNA plasmid group .Western blotting results were consistent with that of the results of reverse transcription-qPCR.

--

Human

L

cellular senescence

27,792,110

Gene

0

1

0

1

0

SRSF2

6427

protein coding

HAoEC

--

Aging

Prevent

Knockdown//SA--gal activity assay

Knockdown of either SRSF2 or HNRNPD expression was sufficient to cause an increase in the numbers of senescent cells from 4 % to 6 % and 63% for HNRNPD and SRSF2 respectively .

--

Human

HL

cellular senescence

30,026,406

Gene

0

1

0

1

0

HNRNPD

3184

protein coding

HAoEC

--

Aging

Prevent

Knockdown//SA--gal activity assay

Knockdown of either SRSF2 or HNRNPD expression was sufficient to cause an increase in the numbers of senescent cells from 4 % to 6 % and 63% for HNRNPD and SRSF2 respectively .

--

Human

HL

cellular senescence

30,026,406

Gene

0

1

0

1

0

CSNK1A1

1452

protein coding

IMR-90

--

Colorectal cancer

Prevent

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

Likewise, ablation of Csnk1a1 in MEFs and mRNA depletion of the corresponding gene, CSNK1A1, in human primary fibroblasts (IMR-90 cells) resulted in a persistent DDR and a senescence phenotype.

--

p53

--

Western blot//Immunohistochemistry

By contrast, Csnk1a1Dgut mice showed marked p53 expression. Transcriptome analysis revealed that, not only the Wnt cascade, but also the p53 pathway is strongly induced byCsnk1a1 ablation . This was confirmed by monitoring the induction of several specific p53 target genes, including the anti-proliferative gene p21.

Human

HL

cellular senescence

21,331,045

Gene

0

1

0

1

0

1

0

CSF2

1437

protein coding

SH-SY5Y

--

Aging

Accelerate

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

Beta-Galactosidase Assay The average number of SA-¦Â-gal-stained SH-SY5Y cells in the GM-CSF group was 4.75¡À 0.96, which was significantly higher than that in the control group.

--

Human

L

cellular senescence

30,807,472

Gene

0

1

0

1

0

1

0

FOXM1

2305

protein coding

MEF

--

Aging

Prevent

SA--gal activity assay

Moreover, significantly higher numbers of Foxm1-/- MEFs displayed SA-¦Â-gal and senescent morphology compared with WT MEFs following exposure to 5 Gy of g-irradiation.

NBS1//P-ATM//FHRE

--// --// --

Western blot//Luciferase reporter assay

The result showed that overexpression of FOXM1 caused an induction of NBS1 levels and ATM phosphorylation in MCF-7 cells ,further confirming that FOXM1 regulates NBS1 expression and thus MRN complex formation to promote ATM activation and phosphorylation.We observed the (WT) NBS1 promoter activity was augmented by FOXM1 in a dose-dependent manner, whereas the mut NBS1 promoter had lower basal promoter activity and was not inducible by FOXM1.

--

Human

HL

cellular senescence

24,141,789

Gene

0

1

0

NBS1

821254

protein coding

MCF-7

--

Aging

Prevent

Crystal violet assay//SA--gal activity assay

Like FOXM1, NBS1-knockdown sensitized MCF-7 and MCF-7EpiRcells to long-term proliferative arrest following treatment with epirubicin. Consistently, NBS1 knockdown in MCF-7 and MCF-7EpiRcells also enhanced the number of cells exhibiting SA-¦Â-gal activity and morphology at 0 and 10nM epirubicin.

--

Human

HL

cellular senescence

24,141,789

Gene

0

1

0

1

0

SIRT1

23411

protein coding

HUVEC

--

Aging

Prevent

SA--gal activity assay

Overexpression of Sirt1 and resveratrol decreased SA-¦Âgal¨Cpositive cells and specific morphological senescent changes induced by sirolimus and everolimus.

--

Human

L

cellular senescence

19,520,256

Gene

0

1

0

SUMO2

6613

protein coding

NPC

--

Disc degenerative disease

Accelerate

SA--gal activity assay

The positive rate of cell senescence in the model and NC groups were significantly higher than that in the sham group (p < 0.05). The positive rate of cell senescence in the shRNA group was significantly lower than that in the IDD group (p < 0.05).

--

p53

--

qRT-PCR

The mRNA and protein levels of SUMO2, p53, p21, MDM2, GADD45a, MMP-2, HIF-1¦Á, and the p53 phosphorylation level of NPCs in the shRNA group were significantly lower than those in the IDD group (p < 0.05), but the mRNA and protein levels of CDK2/4 and CyclinB1 were significantly higher than that in the IDD group (p < 0.05). There was no significant difference observed between the model and NC group .

Human

L

cellular senescence

29,700,214

Gene

0

1

0

YAP1

10413

protein coding

h-PDLSC

--

Aging

Prevent

SA--gal activity assay

Staining results showed that the OE YAP group had a lower senescence rate than the OE NC group (P<0.01), which indicates that activated YAP postponed the senescence of h-PDLSCs.

CDK6//Cyclin B1//P18//p27

Upregulation//Upregulation//Downregulation//Downregulation

Western blot

In Western blotting, cyclin-dependent kinase 6 (CDK6) and cyclin B1 were upregulated, while CDK inhibitors P18 and P27 were downregulated when YAP was overexpressed.

ERK

Upregulation

Western blot

The expression of P-Msk1, which can phosphorylate ERK, increased when YAP was overexpressed. At the same time, the protein levels of P-ERK1/2 and its target proteins P-P90RSK and P-Msk1 increased in the OE YAP group.

Human

L

cellular senescence

30,123,063

Gene

0

1

0

CDKN1A

1026

protein coding

MDA-MB-231

--

Cancer

Prevent

Knockdown//SA--gal activity assay

When senescence-associated ¦Â-galactosidase staining was performed, where only cells in senescence developed blue staining, the results showed that ¦Â-galactosidase activity was greatly increased by p21 knockdown.

--

Human

L

cellular senescence

26,187,313

Gene

0

1

0

1

0

CAV1

857

protein coding

HCT116,NIH-3T3

--

Aging

Accelerate

SA--gal activity assay//Western blot

We found that only the expression of the mutant form of Nrf2 that cannot bind to caveolin-1 and is constitutively Activate (¦µ A-Nrf2) protected cells from oxidative stress induced premature senescence, as shown by reduced staining for senescence-associated ¦Â-galactosidase activity (SA-¦Â-gal) and p21Waf1/Cip1 protein expression. The stable overexpression of caveolin-1 in HCT116 cells potentiated the oxidative stress¨Cinduced up-regulation of p21Waf1/Cip1 and p16 expression and promoted the development of SA-¦Â-gal¨Cpositive cells.

--

Nrf2

Binding

SA-¦Â-gal activity assay//Pull-down assay

We found that only the expression of the mutant form of Nrf2 that cannot bind to caveolin-1 and is constitutively Activate protected cells from oxidative stress induced premature senescence, as shown by reduced staining for senescence-associated ¦Â-galactosidase activity (SA-¦Â-gal) and p21Waf1/Cip1 protein expression. We found that WT -Nrf2-His bound to caveolin 1 (residues 82¨C101)¨CGST.

Human

L

cellular senescence

23,637,463

Gene

0

1

0

1

0

GPX3

2878

protein coding

LO2,MIHA

--

Aging

Prevent

SA--gal activity assay

The cellular senescence of liver cells was induced using 500 M H2O2 treatment for 1 hour and then the liver cells were cultured with normal medium. Forty eight hours later, we found that recombinant GPx3 protein (rGPx3) significantly suppressed hepatic senescence in LO2 and MIHA cells in a dose dependent manner.

CD44//NOX4//SERPINB2

Downregulation//Downregulation//Downregulation

PCR array

48h after rGPx3 treatment, 4 down-regulated gene candidates (CD44, Nox4, IFNG, SERPINB2) were identified . Three of them (CD44, Nox4, SERPINB2) were significantly up-regulated when cellular senescence was established (after H2O2 treatment), and sharply decreased upon rGPx3 treatment.

--

Human

HL

cellular senescence

29,290,803

Gene

0

1

0

FOXM1

2305

protein coding

H929

--

Multiple myeloma

Prevent

Knockdown//SA--gal activity assay

Left shows that FOXM1 knockdown sufficed to induce ¦Â-gal activity in myeloma.

Rb

Binding

Western blot

Densitometric analysis of Western blots showed that total Rb and phosphorylated Rb (pRb) were increased in FOXM1Hi cells by 20-40%, with somewhat higher levels seen in XG1 than CAG cells. Conversely, Rb and pRb were decreased in FOXM1Lo cells (~ 30 to 50%), with the loss of the latter somewhat exceeding that of the former.

--

Human

HL

cellular senescence

30,463,534

Gene

0

1

0

1

0

IL1B

3553

protein coding

Osteoarthritic Chondrocyte

--

Osteoarthritis

Accelerate

Flow cytometry//SA--gal activity assay

In preliminary experiments (data not shown), we also confirmed the IL-1¦Â¨Cinduced premature senescence at several concentrations of IL-1¦Â(0.1, 1.0, 5.0, and 10.0 ng/ml) in vitro. The results indicated that premature senescence of chondrocytes was induced by IL-1¦Â at lower concentrations as well.

Caveolin-1//p38MAPK//CII

Upregulation//Upregulation//Downregulation

qPCR//Western blot//qRT-PCR//ELISA

Quantitative real-time RT-PCR analysis showed that both IL-1¦Â and H2O2 enhanced caveolin 1 mRNA expression in a time-dependent manner, with the peak level occurring at 3 hours after stimulation(n £½ 3). By Western blot analysis, we confirmed that both IL-1¦Â and H2O2 also induced prolonged up-regulation of caveolin 1 protein expression (n = 3).Expression levels of phospho¨Cp38 MAPK were markedly increased after IL-1¦Â stimulation, with a peak level reached at 12 hours and sustained for more than 24 hours, whereas levels of phospho¨CERK-1/2 were transiently downregulated from 6 to 12 hours.Furthermore, pretreatment with the specific p38 MAPK inhibitor SB202190 blocked the enhancement of SA¨C¦Â-gal activity induced by IL-1¦Â and H2O2.Quantitative real-time RT-PCR revealed a marked reduction in levels of expression of mRNA for CII and aggrecan in chondrocytes 3 days after stress with IL-1¦Â or H2O2. Decreased production of CII from senescent chondrocytes after administration of either stress factor was also confirmed by ELISA (n = 4).

--

Human

L

cellular senescence

16,508,959

Gene

0

1

0

1

0

CAV1

857

protein coding

Osteoarthritic Chondrocyte

--

Osteoarthritis

Accelerate

SA--gal activity assay

Pretreatment with caveolin 1 antisense ODN significantly inhibited the up-regulation of caveolin 1 expression induced by IL-1¦Â and H2O2 (n = 4) and also significantly blocked the increase in SA¨C¦Â-gal activity induced by IL-1¦Â and H2O2 (n = 4).

p38MAPK//CII

Upregulation//Downregulation

Western blot//qRT-PCR//ELISA

Compared with chondrocytes transfected with empty vectors, the chondrocytes transduced with caveolin 1 cDNA showed higher levels of phospho¨Cp38 MAPK and lower levels of phospho¨CERK-1/2. In addition, caveolin 1 overexpression impaired the ability of chondrocytes to express CII (n = 4) and aggrecan (n = 4).

--

Human

L

cellular senescence

16,508,959

Gene

0

1

0

FN1

2335

protein coding

U251,T98G

--

Aging

Prevent

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

Compared to that in the blank and NC groups, the cell senescence rate was notably increased in the siRNA-FN1 group, and the expression levels of p16 and p21 were significantly elevated (all P < 0.05).

--

PI3K-Akt

Activation

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

Compared to the pGL3- basic group, the luciferase activity of the pGL3-FN1 group was increased in both the U251 and T98G cells. Compared to blank and NC groups, the expression levels of FN1, PI3K, and AKT were all decreased in U251 and T98G cells in the siRNA-FN1 group, but the expression level of PTEN increased.

Human

HL

cellular senescence

30,048,971

Gene

0

1

0

1

0

1

0

PTTG1

9232

protein coding

MCF-7,MCF 10A

--

Breast cancer

Accelerate

SA--gal activity assay

By measuring the activity of senescence-associated b-galactosidase (SA-¦Â-Gal), we found that hPTTG1 overexpression significantly reinforced senescence in both cell lines.

IL-8//GROa

Upregulation//Upregulation

Cytokine array//ELISA

With antibody arrays, we determined that hPTTG1 overexpression led to a considerable induction in IL-8 and GROa. Because the antibody arrays were semiquantitative, we performed ELISA (enzyme-linked immunosorbent assay) analyses to confirm this result.

CXCR2-p21//NF-¦ÊB

Upregulation//Activation

Dual-Luciferase reporter assay//CHIP array

Indeed, in both cells, hPTTG1 overexpression increased the expression of the p53 and p21 . Furthermore, in hPTTG1-overexpressing MCF-7 cells, deletions of the NF-kB binding site eliminated the luciferase activity of the IL-8 and GROa promoters. These results were further confirmed by ChIP (chromatin immunoprecipitation) assay, which revealed that the binding capacities of p65 were enhanced by hPTTG1 overexpression .

Human

HL

cellular senescence

22,789,011

Gene

0

1

0

SALL1

6299

protein coding

MCF-7,MDA,E0771

--

Breast cancer

Accelerate

SA--gal activity assay

We observed that transfection of SALL1 in MCF-7, MDA, and E0771 tumor cells significantly increased the number of SA-¦ÂGal+ cells, indicating the induction of tumor cell senescence.

ATM//H2AX//53BP1//Chk2

--//--//--//--

Flow cytometry

Overexpression of SALL1 significantly induced Activate, phosphorylated ATM in MCF-7, MDA and E0771 cancer cells. We observed that transfection of SALL1, but not SALL4 or control vector also significantly induced phosphorylation of H2AX, 53BP1 and CHK2 in MCF-7, MDA and E0771 cells (Data not shown).

p38 MAPK//ERK1/2//mTOR

--//--//Activation

Western blot

We first determined the activation and phosphorylation of MAPKs, including ERK1/2, p38 and JNK in breast cancer cells transfected with SALL1 using western blot analyses. We found that transfection of SALL1 but not mutated SALL1 selectively activated ERK1/2 and p38, but not JNK, resulting in significantly enhanced phosphorylation of ERK1/2 and p38 in both MCF-7 and E0771 breast cancer cells. Transfection of SALL1 but not mutated SALL1 in both MCF-7 and E0771 breast tumor cells significantly induced the phosphorylation of mTOR, p70S6K, and 4E-BP1, further confirming the activation of mTOR signaling in tumor cells after SALL1 expression.

Human

HL

cellular senescence

29,625,565

Gene

0

1

0

DUSP1

1843

protein coding

SO-Rb5

--

Aging

Accelerate

Knockdown//SA--gal activity assay

DUSP1 plasmid and siRNA transfection enhanced and attenuated SO-Rb5 cell senescence induced by AGII, respectively, revealing that DUSP1 participated in SO-Rb5 cell senescence induced by AGII.

--

Akt

Downregulation

Western blot

DUSP1 plasmid and siRNA transfection inhibited and strengthened Akt signaling in SO-Rb5 cell senescence induced by AGII, respectively, revealing that DUSP1 participated in SO-Rb5 cell senescence induced by AGII.

Human

L

cellular senescence

30,536,303

Gene

0

1

0

1

0

CIT

11113

protein coding

Daoy ,ONS-76

--

Medulloblastoma

Prevent

Knockdown//SA--gal activity assay

In addition, CITK knocked-down cells had a senescent morphology. Accordingly, CITK shRNA induction led to strong increase of cells positive for ¦Â-galactosidase cytochemical reaction, which is typical of senescent cells.

--

Human

L

cellular senescence

29,921,697

Gene

0

1

0

1

0

KLF4

9314

protein coding

T-REx-293 KLF4,T-REx-HeLa KLF4

--

Aging

Accelerate

SA--gal activity assay//Western blot

Then we characterized senescence in KLF4 overexpressed cells. Senescenceassociated ¦Â-galactosidase activity (SA-¦Â-gal) markedly increased in cells with KLF4 overexpression . We also detected the senescence related proteins by Western Blot, and found that p21W AF1/CIP1 protein was strongly accumulated after KLF4 overexpression, accompanied by reduced expression of Cyclin E1 and increased expression of Suv39H1, but no significant variation of p53 and p16Ink4a.

p21

Upregulation

CHIP//qPCR//Western blot

We found that p21 mRNA level was induced by KLF4 overexpression, and KLF4 could bind to the promoter region of p21 gene, confirmed by ChIP assay. When p21 protein was knocked down, KLF4 induction could induce only about 8 percent of senescent cells, comparing with more than 70% senescent cells in control cells .

miR-203-Survivin-p12

--

Western blot//qPCR//Luciferase reporter assay

Protein level of survivin and mRNA expression were both inhibited by KLF4 overexpression. Additionally, p21 upregulation induced by KLF4 was significantly inhibited.In our study, survivin protein could directly bind to the distal and proximal p53 binding sites of p21 promoter in T-REx-293 KLF4 cells, as confirmed by ChIP assay. T-REx-293 cells were co-transfected with survivin and reporter plasmids, and reporter assay showed that the transcription activities of both pGL3 p21 5¡ä, pGL3 p21 3¡ä were significantly inhibited by survivin. ChIP assay showed that KLF4 could directly bind to -189bp to +11bp fragment of miR-203 gene. To test whether miR-203 can target survivin or not, pre-miR-203 precursor and miR-203 inhibitor were transfected into T-REx-293 cells, respectively.mRNA expression of survivin was inhibited by pre-miR-203, while inhibition of miR-203 could increase survivin expression, the similar result was shown in protein level.

Human

HL

cellular senescence

27,531,889

Gene

0

1

0

1

0

PIK3R4

30849

protein coding

HUVEC

--

Aging

Prevent

SA--gal activity assay

The ¦Â-galactosidase positive rates in cells of control, scrambled and Vps15-shRNA transfection groups were22.4¡À1.9%, 24.3¡À2.1% and 42.5¡À2.5%, respectively.Moreover, both the ¦Â-galactosidase positive- (23.1¡À2.7%) and apoptotic rates (10.4¡À1.1%) in cells with Vps15 reexpression were lower than in cells of Vps15 knockdown group.

--

Human

L

cellular senescence

31,356,904

Gene

0

1

0

NFKB2

4791

protein coding

Primary Fibroblast

--

Aging

Prevent

SA--gal activity assay

When analyzing siRNA depletion of NF-kB2 and RelB in NHD fibroblasts, we also observed that cells ceased to proliferate, changed morphology and after 7 days in culture, using the acidic ¦Â galactosidase assay, were found to enter a senescent state.

p53//EZH2

--//Binding

SA-¦Â-gal activity assay//Knockdown//CHIP-Seq

Importantly induction of senescence and ROS upon depletion of NF-kB2, RelB and Bcl-3 was p53 dependent.This result was confirmed by mining of ChIP-Seq data from GM12878 B-cells [40], where binding of all NF-kB subunits to the EZH2 promoter was seen.

--

Human

HL

cellular senescence

25,255,445

Gene

0

1

0

ATM

472

protein coding

HCT116

--

Colorectal cancer

Accelerate

Flow cytometry//SA--gal activity assay

We used a widely known inhibitor of the PIKKs¡ªcaffeine20, and analyzed different markers of cell senescence. Higher SA-¦Â-Gal activity and cell granularity, measured by flow cytometry, were observed in dox-treated cells than in control (untreated) ones, while pretreatment with caffeine substantially decreased the activity of SA-¦Â-Gal and cell granularity and preserved the cell proliferation capabilities.

--

Human

L

cellular senescence

29,352,261

Gene

0

1

0

1

0

ATR

545

protein coding

HCT116

--

Colorectal cancer

Accelerate

Flow cytometry//SA--gal activity assay

We used a widely known inhibitor of the PIKKs¡ªcaffeine20, and analyzed different markers of cell senescence. Higher SA-¦Â-Gal activity and cell granularity, measured by flow cytometry, were observed in dox-treated cells than in control (untreated) ones, while pretreatment with caffeine substantially decreased the activity of SA-¦Â-Gal and cell granularity and preserved the cell proliferation capabilities.

--

Human

L

cellular senescence

29,352,261

Gene

0

1

0

1

0

PRKDC

5591

protein coding

HCT116

--

Colorectal cancer

Accelerate

Flow cytometry//SA--gal activity assay

We used a widely known inhibitor of the PIKKs¡ªcaffeine20, and analyzed different markers of cell senescence. Higher SA-¦Â-Gal activity and cell granularity, measured by flow cytometry, were observed in dox-treated cells than in control (untreated) ones, while pretreatment with caffeine substantially decreased the activity of SA-¦Â-Gal and cell granularity and preserved the cell proliferation capabilities.

--

Human

L

cellular senescence

29,352,261

Gene

0

1

0

1

0

CHUK

1147

protein coding

--

Tumor tissue

Lung cancer

Accelerate

Ki67 staining//SA--gal activity assay

KrasG12D;Ikk¦Á¦¤Lu lung ADCs displayed substantially less SA-¦Â-gal staining and more Ki67 than KrasG12D ADCs.

--

Nrf2-NQO1

Downregulation

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

Indeed, KrasIKK¦ÁL cells expressed reduced IKK¦Á, NRF2, NQO1, p53, and p21 and showed attenuated cell senescence compared with Kras-CL cells.

Human

HL

cellular senescence

29,311,298

Gene

0

1

0

1

0

PTEN

5728

protein coding

MCF-7

--

Aging

Prevent

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

PTEN depleted MCF7 cells displayed prematurely senescent phenotypes in a p53/p21-dependent manner.The SA-¦Â-Gal positivity of PTEN KO MEFs was dramatically attenuated in Torin1-treated cells compared to Rapa-treated cells.

mTORC1//mTORC2//p53

--//--//Activation

Western blot//IP

The activities of both mTORC1 and mTORC2 were increased along with that of AKT during PICS.Indeed, p53 phosphorylation at S15 was abolished in PTEN-depleted cells expressing mTOR KD. We further conducted an immunoprecipitation (IP) assay with anti-p53, and observed endogenous association of mTOR with p53 during PICS . Raptor and Rictor were also found in the p53 immunoprecipitates, indicating that both mTORC1 and mTORC2 physically associated with p53.

--

Human

L

cellular senescence

30,337,688

Gene

0

1

0

1

0

1

0

TP53

7157

protein coding

U87,U2OS

--

Aging

Accelerate

EdU assay//Immunofluorescence//SA--gal activity assay

Nutlin-3a treatment strikingly induced cellular senescence in U87 and U2OS cells, as shown by positive senescence-associated ¦Â-galactosidase (SA-¦Â-gal) staining, reduction of lamin B1, and reduced 5-ethynyl-2 -deoxyuridine (EdU) incorporation, p16.

EGFR//DYRK1A

Downregulation//Downregulation

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

Indeed, ectopic expression of EGFR in U87 or U2OS cells significantly attenuated senescence induced by p53 activation.Activation of p53 by Nutlin-3a led to a significant reduction in DYRK1A.

EGFR-MEK-ERK

Downregulation

Western blot

Indeed, the level of phosphorylated (Activate) form of ERK was decreased in Nutlin-3a treated U87 and U2OS cells.

Human

L

cellular senescence

30,910,997

Gene

0

1

0

1

0

1

0

POLE2

5427

protein coding

Fibroblast

--

Aging

Prevent

Cell counting//EdU assay//Knockdown//RT-qPCR//SA--gal activity assay

To this end, we tested several DNA repair genes by knocking-down their expression in human fibroblasts. Strikingly, knock-down of POLE2, BARD1, FEN1, RAD51, EXO1, BRCA1, or BLM alone was able to induce hallmarks of premature senescence.

--

p53-p21-Rb

--

qRT-PCR

Furthermore, Nutlin-3 or AT7519 notably repressed DNA repair gene expression through a P53/ P21/RB pathway.

Human

HL

cellular senescence

29,449,545

Gene

0

1

0

1

0

1

0

1

0

1

0

BARD1

580

protein coding

Fibroblast

--

Aging

Prevent

Cell counting//EdU assay//Knockdown//RT-qPCR//SA--gal activity assay

To this end, we tested several DNA repair genes by knocking-down their expression in human fibroblasts.Strikingly, knock-down of POLE2, BARD1, FEN1, RAD51, EXO1, BRCA1, or BLM alone was able to induce hallmarks of premature senescence.

--

p53-p21-Rb

--

qRT-PCR

Furthermore, Nutlin-3 or AT7519 notably repressed DNA repair gene expression through a P53/ P21/RB pathway.

Human

HL

cellular senescence

29,449,545

Gene

0

1

0

1

0

1

0

1

0

1

0

FEN1

2237

protein coding

Fibroblast

--

Aging

Prevent

Cell counting//EdU assay//Knockdown//RT-qPCR//SA--gal activity assay

To this end, we tested several DNA repair genes by knocking-down their expression in human fibroblasts. Strikingly, knock-down of POLE2, BARD1, FEN1, RAD51, EXO1, BRCA1, or BLM alone was able to induce hallmarks of premature senescence.

--

p53-p21-Rb

--

qRT-PCR

Furthermore, Nutlin-3 or AT7519 notably repressed DNA repair gene expression through a P53/ P21/RB pathway.

Human

HL

cellular senescence

29,449,545

Gene

0

1

0

1

0

1

0

1

0

1

0

RAD51

5888

protein coding

Fibroblast

--

Aging

Prevent

Cell counting//EdU assay//Knockdown//RT-qPCR//SA--gal activity assay

To this end, we tested several DNA repair genes by knocking-down their expression in human fibroblasts . Strikingly, knock-down of POLE2, BARD1, FEN1, RAD51, EXO1, BRCA1, or BLM alone was able to induce hallmarks of premature senescence.

--

p53-p21-Rb

--

qRT-PCR

Furthermore, Nutlin-3 or AT7519 notably repressed DNA repair gene expression through a P53/ P21/RB pathway.

Human

HL

cellular senescence

29,449,545

Gene

0

1

0

1

0

1

0

1

0

1

0

EXO1

9156

protein coding

Fibroblast

--

Aging

Prevent

Cell counting//EdU assay//Knockdown//RT-qPCR//SA--gal activity assay

To this end, we tested several DNA repair genes by knocking-down their expression in human fibroblasts. Strikingly, knock-down of POLE2, BARD1, FEN1, RAD51, EXO1, BRCA1, or BLM alone was able to induce hallmarks of premature senescence.

--

p53-p21-Rb

--

qRT-PCR

Furthermore, Nutlin-3 or AT7519 notably repressed DNA repair gene expression through a P53/ P21/RB pathway.

Human

HL

cellular senescence

29,449,545

Gene

0

1

0

1

0

1

0

1

0

1

0

BRCA1

672

protein coding

Fibroblast

--

Aging

Prevent

Cell counting//EdU assay//RT-qPCR//SA--gal activity assay

To this end, we tested several DNA repair genes by knocking-down their expression in human fibroblasts. Strikingly, knock-down of POLE2, BARD1, FEN1, RAD51, EXO1, BRCA1, or BLM alone was able to induce hallmarks of premature senescence.

--

p53-p21-Rb

--

qRT-PCR

Furthermore, Nutlin-3 or AT7519 notably repressed DNA repair gene expression through a P53/ P21/RB pathway.

Human

HL

cellular senescence

29,449,545

Gene

0

1

0

1

0

1

0

1

0

BLM

641

protein coding

Fibroblast

--

Aging

Prevent

Cell counting//EdU assay//Knockdown//RT-qPCR//SA--gal activity assay

To this end, we tested several DNA repair genes by knocking-down their expression in human fibroblasts. Strikingly, knock-down of POLE2, BARD1, FEN1, RAD51, EXO1, BRCA1, or BLM alone was able to induce hallmarks of premature senescence.

--

p53-p21-Rb

--

qRT-PCR

Furthermore, Nutlin-3 or AT7519 notably repressed DNA repair gene expression through a P53/ P21/RB pathway.

Human

HL

cellular senescence

29,449,545

Gene

0

1

0

1

0

1

0

1

0

1

0

SIX1

6495

protein coding

IMR-90

--

Aging

Prevent

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

Stable silencing of SIX1 in early passage IMR90 cells markedly reduced proliferation, as assessed by bromodeoxyuridine incorporation.Most importantly, IMR90 shSIX1 cells displayed features of cellular senescence, including senescent morphology with extended vacuolized cytoplasms and prominent nucleoli, and increased number of senescence-associated ¦Â-galactosidase-positive cells.

p16//Polycomb

--//--

Western blot//Immunofluorescence//qPCR//CHIP

In agreement with the inverse correlation between SIX1 and p16 found in ER:Ras induced senescence, we observed that p16 protein and RNA levels were significantly increased in shSIX1-IMR90 cells, as did the number of p16-positive cells in immunofluorescence assays.To test if SIX1 could cooperate with Polycomb complexes in senescence, we analyzed changes in the Polycomb-associated mark H3K27Me3. ChIP showed a clear reduction of H3K27Me3 in the INK4A promoter (~5-fold) in shSIX1-senescent cells , in the absence of gross changes in the H3K27Me3 nuclear level or distribution, similar to other senescence stimuli.

--

Human

HL

cellular senescence

26,500,063

Gene

0

1

0

1

0

1

0

1

0

PIM1

5292

protein coding

2BS

--

Aging

Accelerate

Knockdown//SA--gal activity assay//SAHF

Furthermore, PIM-1 knockdown resulted in a reduced percentage of cells with senescence-associated b-galactosidase (SA-¦Â-gal) staining and SAHF formation, two well-known markers of cellular senescence.

HP1¦Ã

--

IP//Western blot//qPCR

IP with anti-PIM-1 antibody successfully pulled down HP1¦Ã, while reciprocal experiment using anti-HP1¦Ã antibody validated the interaction between PIM-1 and HP1¦Ã in senescent cells.

IL-6-STAT3

--

Luciferase reporter assay//Knockdown

Reporter analysis showed that RasV12 overexpression increased the wild-type PIM-1 promoter activity, and mutation of the STAT3 binding site dramatically attenuated the RasV12- induced promoter activity .In addition, expressing IL-6 shRNA suppresses the PIM-1 promoter activity.

Human

L

cellular senescence

25,040,935

Gene

0

1

0

1

0

1

0

GSK3A

2931

protein coding

Chang

--

Aging

Prevent

Cell morphological analysis//SA--gal activity assay

As expected, at the subcytotoxic doses of SB415286, cells acquired senescence phenotypes in dose- and time-dependent manners, as shown by the gain of SA-¦Â-gal activity and flattened, enlarged cellular morphology .

GS

--

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

GS phosphorylation was decreased when SB415286, a small molecule inhibitor of GSK3 (Coghlan et al., 2000), was applied to Chang cells at concentrations < 15 g mL 1, but rather increased at higher concentrations of SB415286 (20 and 25 g mL 1).Clearly, GS was activated by dephosphorylation in these conditions, resulting in the progressive accumulation of glycogen along with the senescence phenotypes, such as SA-¦Â gal and induction of p16 and p21.

--

Human

L

cellular senescence

18,782,348

Gene

0

1

0

1

0

GSK3B

2932

protein coding

Chang

--

Aging

Prevent

Cell morphological analysis//SA--gal activity assay

As expected, at the subcytotoxic doses of SB415286, cells acquired senescence phenotypes in dose- and time-dependent manners, as shown by the gain of SA-¦Â-gal activity and flattened, enlarged cellular morphology.

GS

--

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

GS phosphorylation was decreased when SB415286, a small molecule inhibitor of GSK3 (Coghlan et al., 2000), was applied to Chang cells at concentrations < 15 g mL 1, but rather increased at higher concentrations of SB415286 (20 and 25 g mL 1).Clearly, GS was activated by dephosphorylation in these conditions, resulting in the progressive accumulation of glycogen along with the senescence phenotypes, such as SA-¦Â gal and induction of p16 and p21.

--

Human

L

cellular senescence

18,782,348

Gene

0

1

0

1

0

EZH2

2146

protein coding

MKN28,SGC-7901

--

Aging

Prevent

Cell morphological analysis//SA--gal activity assay

In MKN28 and SGC7901 cells, a series of phenotypic changes which are the hallmarks of cellular senescence were observed after EZH2 shRNA treated, including flat and enlarged morphology and an increase in SA-¦Â- gal activity but not in AGS cells.

H3K27me3//INK4/ARF//p14//p15INK4b//p16

--//--//--//--//--

Western blot//qRT-PCR

Moreover, a significant decrease of H3K27me3 was also observed compared with negative control.Western blot analysis confirmed the activation of INK4/ARF locus leading by EZH2 depletion at protein level.For cells with EZH2 down-regulated, p14ARF, p15INK4b and p16INK4a transcript levels were increased dramatically compared with control cells .

p15-Rb

--

Western blot

Meanwhile, depletion of EZH2 increased p15INK4b expression dramatically, demonstrating that EZH2 blocks p15INK4b activation and this regulation maybe make great contribution to cellular senescence induced by EZH2 silencing in MNK28 cells. Moreover, the decreased p15INK4b protein expression was associated with increased hyperphosphorylation of its downstream, Rb.

Human

L

cellular senescence

26,004,298

Gene

0

1

0

1

0

PEBP1

5037

protein coding

A549

--

Aging

Accelerate

SA--gal activity assay//Western blot

Elimination of RKIP could increase ERK activation and suppress the cellular senescence.

p53

--

CHIP

Moreover, RKIP was a direct target of p53.

Raf-MAPK

Downregulation

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

Elimination of RKIP could increase ERK activation and suppress the cellular senescence. In contrast, suppression of Ras-Raf-ERK pathway using Raf kinase inhibitor could induce the cellular senescence .

Human

L

cellular senescence

23,814,485

Gene

0

1

0

1

0

EGF

1950

protein coding

HME

--

Aging

Prevent

SA--gal activity assay

Further analysis of HME cells cultured in the absence of EGF revealed that these cells displayed high SA-¦Â-gal activity in addition to severely impaired proliferation .

--

Human

L

cellular senescence

25,367,123

Gene

0

1

0

IFNA1

3439

protein coding

HDF

--

Aging

Accelerate

SA--gal activity assay

Consistent with these observations, the knockdown of IFN¦Á/¦ÂR¦Á subunit expression decreased the number of SA-¦Â-gal positive cells, which is indicative of the onset of cellular senescence in HDFs.

IFI16//IL-1¦Â//AIM2

--//Upregulation//--

Western blot

Similarly, the knockdown reduced basal and IFN-¦Â induced levels of IFI16 protein.Interestingly, the treatment also increased levels of the cleaved IL-1¦Â, a proinflammatory cytokine, which is indicative of the activation of an inflammasome.The knockdown of AIM2 expression in BPH-1 cells increased steady-state levels of STAT1 and potentiated IFN-g induced activation of STAT1. Importantly, the knockdown increased basal and the IFNg induced levels of the IFI16 protein.

--

Human

L

cellular senescence

21,471,287

Gene

0

1

0

IFNB1

3456

protein coding

HDF

--

Aging

Accelerate

Knockdown//SA--gal activity assay

Consistent with these observations, the knockdown of IFN¦Á/¦ÂR¦Á subunit expression decreased the number of SA-¦Â-gal positive cells, which is indicative of the onset of cellular senescence in HDFs.

IFI16//IL-1¦Â//AIM2

--//Upregulation//--

Western blot

Similarly, the knockdown reduced basal and IFN-¦Â induced levels of IFI16 protein. Interestingly, the treatment also increased levels of the cleaved IL-1¦Â, a proinflammatory cytokine, which is indicative of the activation of an inflammasomeThe knockdown of AIM2 expression in BPH-1 cells increased steady-state levels of STAT1 and potentiated IFN-g induced activation of STAT1. Importantly, the knockdown increased basal and the IFNg induced levels of the IFI16 protein.

--

Human

L

cellular senescence

21,471,287

Gene

0

1

0

1

0

CSN2

1447

protein coding

MEF

--

Aging

Accelerate

Cell proliferation assay

Expression of these antisense constructs partially bypasses growth arrest in senescence and is able to produce a moderate increase in the lifespan of MEFs.

--

Human

HL

cellular senescence

17,968,325

Gene

0

1

0

BRF1

2972

protein coding

MEF

--

Aging

Accelerate

Cell proliferation assay

Expression of these antisense constructs partially bypasses growth arrest in senescence and is able to produce a moderate increase in the lifespan of MEFs.

--

Human

HL

cellular senescence

17,968,325

Gene

0

1

0

CDK4

1019

protein coding

MCF-7

--

Aging

Accelerate

SA--gal activity assay

MCF7 cells arrested for 6 days were stained for ¦Â-galactosidase and an increase in ¦Â-gal+ cells was detected in the doxycycline + PD¨C treated cells, suggesting that these nonproliferative cells were actually senescent and could not reenter cell cycle.

--

Human

L

cellular senescence

29,330,290

Gene

0

1

0

EGFR

1956

protein coding

A549

--

Aging

Accelerate

SA--gal activity assay//Western blot

Examination of p53 wild-type cells revealed several features of senescence, including morphologic characteristics indicating premature differentiation and expression of senescence-associated ¦Â-galactosidase as well as increased levels of trimethylated histone H3K9 .

p53

--

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

Examination of p53 wild-type cells revealed several features of senescence, including morphologic characteristics indicating premature differentiation and expression of senescence-associated ¦Â-galactosidase as well as increased levels of trimethylated histone H3K9.

MEK-ERK

--

Cell viability assay//Cell Proliferation assay//SA-¦Â-gal activity assay

Using A549, ABC1, and HCC44 cells as representative examples, we observed that treatment with a MEK inhibitor indeed increased the fraction of cells with residual g-H2AX foci by 8.2% to 18.1%, which was comparable with the effects of EGFR inhibition in A549 cells. MEK inhibition also caused p21 induction, radiosensitization, cellular senescence, and did not further enhance the radiosensitizing effects of erlotinib,implicating the MEK¨CERK pathway as one common effector pathway of radioresistance downstream of EGFR.

Human

L

cellular senescence

21,852,385

Gene

0

1

0

1

0

HRAS

3265

protein coding

MEF

--

Aging

Accelerate

SA--gal activity assay//Western blot

As expected, early-passage primary MEFs expressing H-RasV12 and WT KSR1 exhibited characteristics of cellular senescence, such as reduced proliferation in culture and SA ¦Â-galactosidase activity, concomitant with the induction of p16INK4a, p15INK4b, and p53 and sustained ERK activity.

KSR1

--

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

Early-passage primary MEFs expressing H-RasV12 and WT KSR1 exhibited characteristics of cellular senescence, such as reduced proliferation in culture and SA ¦Â-galactosidase activity, concomitant with the induction of p16INK4a, p15INK4b, and p53 and sustained ERK activity.However, MEFs expressing H-RasV12 and KSR1.CBM did not undergo proliferation arrest or demonstrate SA ¦Â-galactosidase activity, similar to primary MEFs expressing only H-RasV12.

--

Human

L

cellular senescence

25,002,533

Gene

0

1

0

1

0

KIR2DL4

3805

protein coding

NK

--

Aging

Accelerate

qRT-PCR

To test if TAK1 was required for the 2DL4-mediated SASP of resting NK cells, we stimulated resting NK cells with agonist Ab to 2DL4 for 16 h in the presence or absence of 1 mM TAK1 inhibitor. Quantitative RT-PCR was used to test the relative expression of a set of 10 genes that contribute to the senescent signature of primary NK cells. Upregulation of each of these 10 genes upon stimulation of NK cells with agonist Ab to 2DL4 was blocked in the presence of 1 mM TAK1 inhibitor . These results suggest that TAK1 is required for the SASP that results from the activation of primary NK cells by 2DL4.

TAK1

--

qRT-PCR

To test if TAK1 was required for the 2DL4-mediated SASP of resting NK cells, we stimulated resting NK cells with agonist Ab to 2DL4 for 16 h in the presence or absence of 1 mM TAK1 inhibitor. Quantitative RT-PCR was used to test the relative expression of a set of 10 genes that contribute to the senescent signature of primary NK cells. Upregulation of each of these 10 genes upon stimulation of NK cells with agonist Ab to 2DL4 was blocked in the presence of 1 mM TAK1 inhibitor. These results suggest that TAK1 is required for the SASP that results from the activation of primary NK cells by 2DL4.

--

Human

L

cellular senescence

24,337,384

Gene

0

1

0

IGFBPL1

347252

protein coding

HEC-1A

--

Endometrial cancer

Accelerate

SA--gal activity assay//Western blot

SA-¦Â-galactosidase staining, a golden standard for cellular senescence, indicated that HEC-1A-IGFBP-rP1 cells had a higher proportion of SA-¦Â-galactosidase positive cells than the control cells. On the contrary, SA-¦Â-galactosidase activity significantly decreased in Ishikawa-siRNA (HEC-1A-IGFBP-rP1) cells compared with negative controls . Moreover, p-RB, a key regulator in cellular senescence, was significantly reduced in HEC-1A-IGFBP-rP1 cells than control cells while other senescence-related proteins p21. p53 and p16, increased ~four-fold. 55-fold and 24-fol in HEC-1A-IGFBP-rP1, respectively. In contrast, Ishikawa-siRNA (IGFBP-rP1) cells presented a 2.8-fold increased expression of p-RB (P<0.01) and a decrease level of p16, p21 and p53 protein than control cells.

--

ERK

Downregulation

SA-¦Â-gal activity assay

HEC-1A-IGFBP-rP1 cells with PD98059 presented more SA-¦Â-galactosidase positive cells than HEC-1A with PD98059 treatment.

Human

L

cellular senescence

29,067,463

Gene

0

1

0

1

0

PATZ1

23598

protein coding

HUVEC

--

Aging

Prevent

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

Knockdown of PATZ1 in young cells increased SA-¦Â-gal staining activity.Although cell populations in S and G2/M phases were reduced, cells in G0/G1 increased in PATZ1 siRNA cells, suggesting that PATZ1 knockdown induces G0/G1 cell cycle arrest.Upregulation of PATZ1 in old cells increased cell proliferation and repressed SA-¦Â-gal activity.PATZ1 overexpression also released old cells from G2/M cell cycle arrest, confirmed by increases in G1/G0 and S-cell populations as visualized by flow cytometry.

--

p53

--

SA-¦Â-gal activity assay//Knockdown

Downregulation of PATZ1 inhibited cell proliferation in p16INK4a-knockdown cells, but not in p53-knockdown cells.Transfection with PATZ1 siRNA didn't increase SA-¦Â-gal activity in p53-knockdown cells .

Human

HL

cellular senescence

22,052,190

Gene

0

1

0

1

0

1

0

1

0

SIRT1

23411

protein coding

SK-Hep-1,HepG2

--

Hepatocellular carcinoma

Prevent

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

Knockdown of SIRT1 reduced the number and size of SK-Hep-1 cell colonies .Cell-cycle analysis showed that significant G1arrest was observed in SK-Hep-1 and HepG2 cells. We also observed that gene silencing of SIRT1 in SK-Hep1 and HepG2 (p53 wild-type) resulted in cells that were enlarged in size, flattened in shape, and highly positive for SA-¦Â-gal staining.

--

Human

HL

cellular senescence

21,527,554

Gene

0

1

0

1

0

1

0

1

0

AKR1B1

231

protein coding

Keratinocyte

--

Aging

Prevent

Knockdown//SA--gal activity assay

In cells transfected with siRNA against AR or treated with EBPC, this increase in SA ¦Â-Gal-positive cells was further enhanced.

p53

--

Knockdown//Western blot

SiRNA-mediated knockdown of AR further increased the elevated expression of p53 induced by UVB radiation, indicating that AR prevents cellular senescence through the regulation of p53 protein expression.

--

Human

L

cellular senescence

21,182,935

Gene

0

1

0

1

0

SP1

6667

protein coding

2BS

--

Aging

Accelerate

Knockdown//SA--gal activity assay

The results showed that Sp1-overexpressed cells were strongly stained blue versus the control. However, there were only a few dispersed cells that were SA-¦Â-Gal-stained in the Sp1 knocked-down cells.

p16

--

Luciferase reporter assay//Western blot

As determined by luciferase activity, Sp1 activated the p16INK4a promoter in a dose-dependent manner in both young and senescent 2BS cells,the p16INK4a promoter activity was inhibited by MTR in a dose-dependent manner in both young and senescent 2BS cells. Further, the p16INK4a expression was also reduced at mRNA and protein levels in 2BS cells, by 66% (mRNA level) and 48% (protein level) respectively, in the senescence group with 24 hours of MTR treatment.Western Blot showed that si-Sp1 remarkably reduced the expression of Sp1, which in turn lead to a reduction of p16INK4a expression.

--

Human

L

cellular senescence

17,225,865

Gene

0

1

0

1

0

ID1

3397

protein coding

NPTX

--

Aging

Prevent

Flow cytometry//SA--gal activity assay

The AS Id-1 clones displayed several morphological changes compared with the parental NPTX cells, characterized by enlarged, vacuolized and flattened cell bodies and loss of light refractability.The SA-¦Â-gal staining further confirmed that AS Id-1 clones contained significantly more senescent cells .In addition, inactivation of Id-1 resulted in a decrease in S-phase cells , and an increase in G2/M phase cells.

p21/WAF1//p27

--//--

Western blot//RT-PCR

Compared with the pBabe control, AS Id©\1 clones had much lower Id©\1 expression, along with increased p21WAF1/Cip1 and p27KIP1 protein levels.In agreement with the Western©\blot results, the addition of TGF©\¦Â1 had a positive effect on p21WAF1/Cip1 and p27KIP1 mRNA expression and a negative effect on Id©\1 mRNA expression in AS Id©\1 clones.

--

Human

L

cellular senescence

16,686,600

Gene

0

1

0

1

0

TBX2

6909

protein coding

B16

--

Melanoma

Prevent

Flow cytometry//SA--gal activity assay

Activation of ER-dnTbx2 by 4-OHT induces a marked accumulation of cells staining positive for SA¦Â-gal activity, ~25% of clone 1 B16 HA-ER-dnTbx2-expressing cells and ~80% of the clone 2 cells.

p21

Upregulation

Western blot

Western blot analysis using anti-p21 antibodies revealed that in cells expressing HA-ER, p21 expression was barely detectable either in the presence or absence of 4-OHT.The addition of 4-OHT to the HA-ER-dnTbx2-expressing cells led to a dramatic increase in p21 expression consistent with it acting to displace the endogenous Tbx2 from the p21 promoter.with both siRNAs efficiently down-regulating Tbx2 and consequently up-regulating p21 expression comparedwith the control siRNA .

--

Human

L

cellular senescence

15,781,639

Gene

0

1

0

1

0

TNF

7124

protein coding

HUVEC

--

Aging

Accelerate

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

Cell cycle analysis revealed prolonged exposure to TNF¦Á significantly impaired proliferation of cells, as evidenced by progressive decline in abundance of BrdU-incorporating cells accompanied by a decreased percentage of Sphase and increased G1/G2-phase cells compared to control, indicating proliferative arrest.Cells exposed to TNF¦Á also developed classic senescent phenotypes, including a flattened, multi-nucleated, giant cell morphology, and concomitantly increased expression of senescence markers, including p16, p21, and cellular SA-¦Â-gal activity.

--

JAK-STAT

Activation

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

The abundance of serine-phosphorylated STAT1 in cells undergoing senescence due to TNF¦Á was sustained at day 3 and remained elevated at day 6, concomitant with the establishment of senescence .The phosphorylated forms of STAT1 and STAT3 peaked early and then slowly decayed in cells treated with conditioned medium compared to control.

Human

HL

cellular senescence

29,176,033

Gene

0

1

0

1

0

1

0

1

0

NOX4

50507

protein coding

VSMC

--

Aging

Prevent

BrdU assay//SA--gal activity assay

The number of SA-¦Â-gal-positive cells correlated inversely with the level of NOX4 expression obtained after transfection with different siRNA sequences.Moreover, cells with diminished NOX4 expression cease to proliferate since a significant decrease in the number of cells able to replicate DNA was observed already 3 days after transfection. These cells did not restart DNA replication during the next 4 days of the culture.

--

Human

L

cellular senescence

27,655,718

Gene

0

1

0

1

0

BMI1

648

protein coding

UCB-MSC

--

Aging

Prevent

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

The overexpression of BMI1 and the decreased expression of p16INK4a was confirmed via western blotting. BMI1-overexpressing hUCB-MSCs showed a reduced SA-¦Â-gal activity compared to that of the control GFP-transduced cells.The depletion of BMI1 increased SA-¦Â-gal staining and p16INK4a expression, showing the accelerated aging of these hUCB-MSCs. Cellular proliferation was also decreased in shBMI1-MSCs.

--

Human

L

cellular senescence

27,454,161

Gene

0

1

0

1

0

1

0

BAP1

8314

protein coding

HeLa-S3

--

Cervical cancer

Accelerate

SA--gal activity assay

Strikingly, retroviral overexpression of BAP1 reduced cell proliferation and induced senescence-associated ¦Â-galactosidase (SA-¦Â-gal) activity.

ASXL1/2

--

Western blot

We found that BAP1 protein levels increased with ASXL1/2 expression in a dose-dependent manner. Conversely, ASXL1/2 protein levels were also increased following overexpression of BAP1.

p53-p21

--

Western blot

We found that although the effect of BAP1 was less pronounced than the BAP1C91S?form, overexpression of this DUB induced the p53/p21 tumor suppressor pathway.Overexpression of BAP1¦¤CTD, BAP1R666-H669, BAP1C91S-¦¤CTD, or BAP1C91S-R666-H669?did not up-regulate p53/p21 indicating the requirement for ASXL1/2 in BAP1-mediated senescence .

Human

L

cellular senescence

26,416,890

Gene

0

1

0

SOX1

6656

protein coding

HONE1

--

Nasopharyngeal carcinoma

Accelerate

Flow cytometry//SA--gal activity assay

We also found that overexpression of SOX1 in HONE1 cells enhanced SA-¦Â-gal staining.Ectopic expression of SOX1 increased the number of cells in the G1/G0 phase (from 67.77?¡À?0.9% to 71.82?¡À?1.05%) and decreased the number of cells in the G2/M phase (from 12.7?¡À?0.10% to 9.3?¡À?0.10%) in HONE1 cells.

c-Myc//Cyclin D1//p21//p27//Cyclin E

Downregulation//Downregulation//Upregulation//Upregulation//Downregulation

Western blot

A decrease in both c-Myc and Cyclin D1 protein was detected in SOX1-overexpressing HONE1 cells.In HONE1 cells, SOX1 overexpression significantly enhanced the expression of p21 and p27 but suppressed the expression of Cyclin E.

Wnt-¦Â-catenin

Downregulation

Western blot//Knockdown

Furthermore, the down-regulation of ¦Â-catenin induced by SOX1-overexpression was reversed by knockdown of SOX1 using siRNA. Consistently, ¦Â-catenin was down-regulated in a dose-dependent manner with increasing amounts of SOX1.

Human

L

cellular senescence

25,427,424

Gene

0

1

0

1

0

CDCA2

157313

protein coding

IMR-90

--

Aging

Accelerate

SA--gal activity assay

CDCA2 over-expressing cells finally arrested their growth and showed SA-¦Â-gal accumulation, just like control cells.

SAmiR-494

Downregulation

Luciferase reporter assay//Western blot

In the case of CDCA2 and ID4 the reporter gene expression was significantly reduced by SAmiR-494 or SAmiR-486-5p pre-miR transfection, respectively, with a variation of relative luciferase expression similar to the positive control OLFM4 (N).We also investigated the expression profiles of the two targets by Western blot analysis, upon SAmiRs over-expression or down-regulation in PDL 33 IMR90 cells.The decrease in CDCA2 and ID4 endogenous expression levels was well detectable at protein level.

--

Human

HL

cellular senescence

24,905,922

Gene

0

1

0

RBP2

5948

protein coding

SMMC-7721,HepG2

--

Hepatocellular carcinoma

Prevent

Knockdown//SA--gal activity assay

Cell senescence was induced with RBP2 siRNA knockdown for 72 hr in cancer cells.

CDKIsp16//p21CIP2//p27//hsa-miR-212

--//--//--//--

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

The expression of known targets of RBP2, namely, the CDKIs p16ink4a, p21CIP2 and p27kip1, was markedly increased with RBP2 siRNA knockdown.siRNA knockdown of RBP2 expression greatly increased luciferase activity of p21CIP2 and slightly but significantly increased activity of p27kip1 in HepG-2 cells; ChIP assay confirmed the results.with RBP2 overexpression, the expression of p21CIP2 and p27kip1 was relatively low .RBP2 expression was markedly repressed with hsa-miR-212 overexpression. To further confirm that RBP2 was negatively regulated by hsa-miR-212, transfection of hsa-miR-212 inhibitor plasmid in the 2 cell lines decreased the expression of hsa-miR-212 and increased that of RBP2.

--

Human

HL

cellular senescence

23,922,798

Gene

0

1

0

1

0

NOX1

27035

protein coding

REF52,TIG-3

--

Aging

Accelerate

SA--gal activity assay

Nox1- and Nox4- overexpressing cells ceased to proliferate and accumulated SA-¦Â-gal during 6¨C7?days postinfection, whereas vector control displayed little or no senescence biomarker and growth inhibition.

--

p19//p38 MAPK

Upregulation//Upregulation

Western blot

Nox1 up©\regulated the expression of p19Arf?in REF52 cells.Moreover, a marked increase in the level of phosphorylated p38MAPK was detected in Nox1©\ or Nox4©\expressing cells .

Human

L

cellular senescence

23,216,904

Gene

0

1

0

NOX4

50507

protein coding

REF52,TIG-3

--

Aging

Accelerate

SA--gal activity assay

Nox1- and Nox4-overexpressing cells ceased to proliferate and accumulated SA©\¦Â©\gal during 6¨C7?days postinfection, whereas vector control displayed little or no senescence biomarker and growth inhibition.

--

p16//p38 MAPK

Upregulation//Upregulation

Western blot

Nox4 caused accumulation of p16Ink4a?in TIG©\3 cells.Moreover, a marked increase in the level of phosphorylated p38MAPK was detected in Nox1©\ or Nox4©\expressing cells.

Human

L

cellular senescence

23,216,904

Gene

0

1

0

HRAS

3265

protein coding

TIG-3,REF52

--

Aging

Accelerate

SA--gal activity assay//Western blot

H©\RasV12©\infected cells displayed a flat, enlarged morphology and had arrested growth by 7?days after infection, which was accompanied by an increase in the activity of a senescence-associated ¦Â-galactosidase.?Furthermore, oncogenic Ras elevated the expression of senescence©\related cell cycle regulatory proteins: p53, p21 and p19Arf in REF52 cells and p53, p21 and p16Ink4a in TIG-3 cells.

NOX1//NOX4

--//--

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

Nox1 siRNA and Nox4 siRNA consistently abrogated both the accumulation of SA©\¦Â©\gal and up©\regulation of p53, p21 or p16Ink4a in H©\RasV12©\transduced cells.

p53-p21//p16

Upregulation//Upregulation

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

Nox1 siRNA and Nox4 siRNA consistently abrogated both the accumulation of SA©\¦Â©\gal and up©\regulation of p53, p21 or p16Ink4a in H©\RasV12©\transduced cells.

Human

L

cellular senescence

23,216,904

Gene

0

1

0

1

0

AKT1

207

protein coding

U87

--

Glioma

Prevent

SA--gal activity assay

The cytoprotective effects of Akt overexpression were associated with a reduction in the percentage of cells expressing senescence-associated ¦Â-galactosidase activity at 10 days following temozolomide exposure.

Chk2

--

Western blot

U87MG cells expressing the AktERM+ construct, however, exhibited a statistically significant reduction in temozolomide-induced Chk2 phosphorylation (but not in total Chk2 levels) relative to that noted in cells not overexpressing Akt.

--

Human

L

cellular senescence

15,930,307

Gene

0

1

0

RB1

5925

protein coding

H1299,H460

--

Lung cancer

Accelerate

SA--gal activity assay

RB-proficient and RB-deficient H1299 and H460 cells were exposed to PD 0332991 for two weeks and analyzed for the expression of the senescence marker ¦Â-galactosidase activity. Interestingly, elevated levels of ¦Â-galactosidase activity were observed in RB-proficient cells in response to PD 0332991, while RB-deficient cells failed to induce ¦Â-galactosidase, suggesting an RB-dependent senescence program.

--

Human

HL

cellular senescence

29,311,118

Gene

0

1

0

MAP2K4

6416

protein coding

MEF

--

Aging

Prevent

Knockdown//SA--gal activity assay

As expected, MKK4-KO MEFs, in which the non-canonical NF¦ÊB pathway is inactivated, showed more prominent ¦Â-galactosidase activity than MKK4-WT when tested in a senescence-associated ¦Â-gal activity assay, indicating that MKK4 depletion increased cellular senescence.

p16//p21//p27

--//--//--

Western blot

MKK4 depletion enhanced the protein levels of cyclin-dependent kinase (CDK) inhibitors, p16, p21, and p27, which in turn induced cell growth retardation.

NF-¦ÊB

Activation

Western blot

To investigate the direct roles of the non-canonical NF¦ÊB pathway, we knocked down NF¦ÊB2 using shRNAs in MKK4-WT MEFs.As with MKK4 depletion, NF¦ÊB2 knock-down also suppressed cell growth.

Human

L

cellular senescence

28,733,031

Gene

0

1

0

1

0