Venkatachalam/aging_reg_dataset · Datasets at Hugging Face

HIF1A

3091

protein coding

SH-SY5Y,SKNBE2c,SKNAS

--

Neuroblastoma

Prevent

Knockdown//SA--gal activity assay

We observed that the vehicle-treated NBL shHIF1A and shEPAS1 cells showed greater senescence compared to the vehicle-treated NBL shCTR cells. This suggested that the decreased HIF1A and EPAS1 expression in these NBL cells might make them more prone to go into senescence, independent of the ATRA treatment.

--

Human

HL

cellular senescence

26,057,707

Gene

0

1

0

1

0

EPAS1

2034

protein coding

SH-SY5Y,SKNBE2c,SKNAS

--

Neuroblastoma

Prevent

Knockdown//SA--gal activity assay

We observed that the vehicle-treated NBL shHIF1A and shEPAS1 cells showed greater senescence compared to the vehicle-treated NBL shCTR cells. This suggested that the decreased HIF1A and EPAS1 expression in these NBL cells might make them more prone to go into senescence, independent of the ATRA treatment.

--

Human

HL

cellular senescence

26,057,707

Gene

0

1

0

1

0

NOTCH1

4851

protein coding

HUVEC

--

Aging

Prevent

SA--gal activity assay

We examined the replicative lifespan of infected cells and found that up-regulation of Notch1 prolonged the lifespan of endothelial cells along with a decrease of senescence-associated bgalactosidase (SA-¦Â-gal) activity and decreased expression of senescence-associated molecules such as p53, p21, and p16.

--

Id-1-p16

--

Knockdown//SA-¦Â-gal activity assay//Proliferation assay

To investigate whether Notch1 deletion induced endothelial cell senescence via a p16-dependent pathway, we co-infected human endothelial cells with the p16 shRNA and Notch1 shRNA vectors. Notch1 deletion led to premature senescence of mock-infected cells but not p16 shRNA-infected cells, suggesting that disruption of Notch1 promotes endothelial cell senescence via an Id1/p16-dependent pathway.

Human

L

cellular senescence

24,950,189

Gene

0

1

0

ROC1

830029

protein coding

HuH-7,HepG2,LM 6

--

Liver cancer

Prevent

Flow cytometry//SA--gal activity assay

In contrast, senescence started to occur at 72 h with continuous increase and reaching the peak at 120 h .SiROC1 induced the G2¨CM arrest, which occurred at 48?h post ROC1 silencing, and reached a peak at 96?h in HepG2 and Huh7 as well as LM6 cells (data not shown). Morphologically, when compared with control cells, ROC1-silenced cells were much larger in size with flattened shape, a feature of cell senescence.

--

Human

L

cellular senescence

22,935,614

Gene

0

1

0

1

0

PRKD2

25865

protein coding

U87,GM133

--

Glioblastoma

Prevent

Knockdown//SA--gal activity assay

Silencing of PRKD2 induced acidic senescence-associated ¦Â-galactosidase activity (SA-¦Â-gal) in U87MG (64+10.4% positive) and GM133 (41+ 5.4% positive) cells. SA-¦Â-gal activity was also observed in both primary glioma cultures (GBM2, Gli25) in response to PRKD2 silencing.

--

p53

--

qPCR//Immunoblotting

Moreover, PRKD2-deficient cells showed a distinct reduction in the expression of c-Myc on mRNA and protein level. None of these alterations were observed in p53mut GM133 cells . mRNA expression of transcription factor E2F1 was significantly attenuated in p53wt U87MG and p53mut GM133 cells.

Human

L

cellular senescence

24,463,355

Gene

0

1

0

1

0

XRCC3

7517

protein coding

NIH-3T3

--

Hypertension-induced left ventricular hypertrophy

Prevent

SA--gal activity assay

Consistent with the result of spontaneous DSBs, cellular senescence was induced to a greater degree in 3T3-241Met than in 3T3-241Thr.

--

Human

L

cellular senescence

29,626,209

Gene

0

1

0

SYT7

9066

protein coding

H23

--

Lung cancer

Prevent

SA--gal activity assay

Moreover, more beta-gal positively stained cells were found in control cells compared with SYT7 overexpressing cells, suggesting that SYT7 inhibited cellular senescence.

p16//p21//p53

Downregulation//Downregulation//Downregulation

Western blot//Knockdown//qPCR

Knocking down the expression of SYT7 increased the mRNA levels of P16, P21 and P53, and vice versa. Consistently, Overexpression of SYT7 inhibited the protein level of P16, P21 and P53, and knocking down SYT7 increased the protein level of P16, P21 and P53.

--

Human

L

cellular senescence

30,647,108

Gene

0

1

0

PAK4

10298

protein coding

U87 MG,LN-18

--

Glioblastoma

Prevent

Knockdown//SA--gal activity assay

¦Â3 knockdown also led to enhanced methylation of histone H3 in senescence-associated heterochromatin foci,increased ¦Ã-H2AX positive nuclei indicative of DNA double strand breaks,and accumulation of senescence-associted acidic ¦Â-galactosidase activity.

p21

Downregulation

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

Silencing of ¦Â3 induced a marked increased in p21 protein expression for multiple glioblastoma cells,whereas p21 levels were unchanged or even decreased following ¦Â3 knockdown in various epithelial cancer cells.

--

Human

L

cellular senescence

26,297,735

Gene

0

1

0

1

0

DPP4

1803

protein coding

WI-38

--

Aging

Accelerate

SA--gal activity assay//Western blot

As shown, markers p16 and p21 were elevated while SIRT1 levels declined in cells overexpressing DPP4, supporting the notion that DPP4 promoted cell senescence. In addition, overexpression of DPP4 in WI-38 cells elevated SA-¦Â-gal activity and decreased 3 H-thymidine incorporation.

--

Human

L

cellular senescence

28,877,934

Gene

0

1

0

1

0

LRP8

7804

protein coding

Smooth muscle cell

--

Cardiovascular disease

Prevent

Knockdown//SA--gal activity assay

In contrast, smooth muscle cells isolated from the aorta or the carotid arteries of Lrp8?/? mice displayed very slow growth rate under the same culturing conditions with significant reduction in population doubling after the first passage. The number of senescence-associated ¦Â-galactosidase positive Lrp8?/? cells after 8 days in culture with 10% FBS (¡Ö25%¨C30%) was higher than the ¡Ö5% to 8% of senescence observed in Lrp8+/+ cells.

CDC20-PP2A

--

CHIP//Western blot

We used coimmunoprecipitation to analyze the CDC20-PP2A complex formation in Lrp8+/+ and Lrp8?/? smooth muscle cells and found that binding of the regulatory PP2A-B56¦Á subunit to CDC20 does not require apoER2, while binding of the PP2A-C (catalytic subunit of PP2A) to CDC20 requires apoER2 expression . More importantly, while no direct interaction of CDC20 with apoER2 was detected, we observed binding of apoER2 to PP2AC in coimmunoprecipitation experiments with apoER2 antibodies. Since CDC20 interaction with PP2A and aurora kinase dephosphorylation are necessary steps for cell cycle exit,35,36 these data, together with the observation of a trend toward increased CDC20 level in Lrp8?/? smooth muscle cells, is consistent with the interpretation that apoER2 deficiency suppresses PP2A-C interaction with CDC20, thereby preventing its activation to cause cytokinesis impairment and cell cycle arrest.

--

Human

L

cellular senescence

31,412,739

Gene

0

1

0

1

0

CCNB1

891

protein coding

Capan©\2 PC,PC primary cell

--

Pancreatic cancer

Prevent

Knockdown//MTT assay//SA--gal activity assay

The staining results showed that cells in the blank, NC, and PFT©\¦Á + shCCNB1 groups grew at a high density and were in a diamond shape with observable nuclear division. Cells in the normal and shCCNB1 groups grew at a low density, and part of the cells was in a triangle and enlarged shape with observable vacuolar degeneration, indicating cell senescence. Cells in the PFT©\¦Á group grew at a high density with observable nuclear division. The result of ¦Â©\galactosidase staining showed that compared to that in the normal group, SI was significantly lower in other groups.. In comparison with the blank group, the shCCNB1 group had significantly decreased cell proliferation, whereas the PFT©\¦Á group had significantly increased cell proliferation (p < 0.05) and the NC and PFT©\¦Á + shCCNB1 groups exhibited no significant difference in cell proliferation (p > 0.05).

Bax//caspase©\9//Caspase©\3//p21

Downregulation//Downregulation//Downregulation//Downregulation

Western blot//qRT-PCR

The mRNA and protein expressions of Bax, caspase©\9, caspase©\3, p21, and p53 were significantly lower, and the protein expression of p©\p53 was significantly lower in other five groups (p < 0.05).

p53

Downregulation

Western blot//qRT-PCR

Compared with those of the normal group, the mRNA and protein expressions of CCNB1 and MDM2 were significantly higher, the mRNA and protein expressions of Bax, caspase©\9, caspase©\3, p21, and p53 were significantly lower, and the protein expression of p©\p53 was significantly lower in other five groups (p < 0.05).

Human

L

cellular senescence

30,069,972

Gene

0

1

0

1

0

1

0

DEK

7913

protein coding

CaSki

--

Cervical cancer

Prevent

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

Most of the CaSki¨CDEK and CaSki¨CI¦ÊB cells displayed an intense blue staining, indicative of senescence, whereas only a few positively stained cells were detected in untransfected cells. As measured by microscopic image analysis, the percentage of positive staining was 83.25 +? 9.46% in CaSki¨CDEK cells and 76.47 +? 6.31% in CaSki¨CI¦ÊB cells,whereas the positive staining in CaSki¨Cneo and untransfected cells was 23.13 +? 7.28 and 18.92 +? 6.46% respectively. The cell growth curve showed that, compared with untransfected and CaSki¨Cneo cells,the proliferation activity of CaSki¨CDEK cells was inhibited in a time-dependent manner.In CaSki¨CDEK and CaSki¨CI¦ÊB cells, more cells were blocked in the G0/G1-phase, with a corresponding decrease in the G2/M-phase. The percentage of cells in the G0/G1-phase was significantly increased in CaSki¨CDEK and CaSki¨CI¦ÊB cells compared with control cells (P < 0.05).

NF-¦ÊBp65

Upregulation

ELISA//Immunocytochemistry

The NF-¦ÊB activity determined by ELISA supported the findings observed by immunoblotting. CaSki¨CDEK and CaSki¨CI¦ÊB cells showed a significant increase in the activity of the NF-¦ÊB p65 subunit compared with control cells (P < 0.05). In accordance with this, in CaSki¨Cneo and untransfected cells, NF-¦ÊB p65 was retained uniformly in the cytoplasm and only a mild-to moderate expression was observed by immunocytochemical staining.

--

Human

L

cellular senescence

22,390,170

Gene

0

1

0

1

0

1

0

1

0

HES1

3280

protein coding

Caco-2,SW48

--

Colorectal cancer

Prevent

Knockdown//SA--gal activity assay

We knocked down Hes1 in SW48 and Caco2 cells and observed significantly more beta-galactosidase expression in Hes1 knocked down cells, suggesting more senescence in the Hes1depleted cells.

--

STAT3-MMP14

--

Western blot//qRT-PCR//Knockdown

We observed that only MMP14 expression level decreased upon Hes1 knockdown at the mRNA level in Caco2 cells. We then found knock-down Hes1 lead to MMP14 protein level decrease in SW48 cells.Hes1 depletion resulted in a decrease of STAT3 phosphorylation and no change of total STAT3 in the Caco2 and SW48 cells.

Human

L

cellular senescence

26,650,241

Gene

0

1

0

1

0

STAT5A

100135887

protein coding

MEF

--

Aging

Accelerate

Cell proliferation assay//SA--gal activity assay

We show here that primary MEFs also undergo cell cycle arrest, with markers of cellular senescence when forced to express a constitutively Activate allele of STAT5A (caS5A) or RasV12 by retroviral gene transfer.

--

JAK-STAT5//p53

Activation//Activation

SA-¦Â-gal activity assay//PML immunofluorescence//Proliferation assay

To investigate whether we could also trigger senescence by activating JAK/STAT5 signaling upstream of STAT5, we used a Tel/Jak2 fusion protein.54 Similar to caS5A,Tel/Jak2 was also sufficient to trigger senescence cell cycle arrest in MEFs .The process required p53 or the p53 activator p19ARF, because it was totally prevented in MEFs from p53 or p19ARF null mice.

Human

HL

cellular senescence

20,536,843

Gene

0

1

0

1

0

HGF

3082

protein coding

EPC

--

Aging

Prevent

SA--gal activity assay//Western blot

HGF and SOD, but not VEGF, significantly attenuated Ang II¨Cinduced senescence of EPCs as determined by senescence associated -galactosidase staining, although HGF and VEGF alone promoted EPC senescence. Moreover, HGF and SOD significantly inhibited the induction of p53, p21Cip1, and p16INK4a by Ang II.

Rac1//PIP3//Akt

--//Downregulation//--

Western blot

Whereas pretreatment with HGF, but not VEGF, prevented Ang II¨Cinduced rac1 binding to GTP at 60 minutes. HGF also prevented Ang II¨Cinduced rac1 translocation from the cytosol to the plasma membrane.we examined the PIP3 production, and, finally, we found that pretreatment with HGF significantly inhibited Ang II¨Cinduced PIP3 production, whereas VEGF did not inhibit it.Consistently, pretreatment with HGF,but not VEGF, significantly inhibited Ang II¨Cinduced phosphorylation of Akt in EPCs.

--

Human

L

cellular senescence

19,047,582

Gene

0

1

0

1

0

CBS

875

protein coding

HUVEC,HAEC,HDF

--

Aging

Prevent

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

CBS depletion led to decreased cell numbers in HUVEC but not HDF; it also significantly reduced the rate of cell proliferation, measured by BrdU incorporation studies, but had no effect on the rate of apoptotic cell death. CBS knockdown also reduced the proliferative capacity of human aortic endothelial cells (HAEC), included as an additional control, included as an additional control.Both in HUVEC and HAEC, CBS knockdown led to a premature accumulation of cells staining positive for senescence associated ?-galactosidase (SA-?-gal).

--

Human

L

cellular senescence

23,117,410

Gene

0

1

0

1

0

1

0

HMGA2

8091

protein coding

ULM

--

Uterine leiomyomas

Prevent

Knockdown//SA--gal activity assay

Cells treated with HMGA2 siRNA had significantly increased levels of ¦Â-Gal staining, indicative of cellular senescence than the cells treated with control siRNA.

p16//p21

--//--

Western blot

In addition, we observed that silencing of HMGA2 in ULM led to upregulation of both p16 and p21.

Akt

--

Western blot//Knockdown

A dose-dependent upregulation of pAKT was observed by Western blot analysis along with an increase in HMGA2 levels. Next, using HMGA2 ULM, HMGA2 was silenced using siRNA. With decreased HMGA2 levels, decreased pAKT levels were observed .

Human

HL

cellular senescence

29,790,226

Gene

0

1

0

1

0

GNAO1

2775

protein coding

GY-7703,SMMC-7721

--

Hepatocellular carcinoma

Accelerate

CCK-8 assay//Knockdown//SA--gal activity assay//Western blot

In the 96 h observation, we found that after 72 h of transfection, the cell proliferation was comparably faster in cells transfected with siGNAO1-1 and siGNAO1-2, in which the protein expression of GNAO1 was remarkably inhibited, than that in cells transfected with NC(P < 0.05).In ¦Â-gal staining assay, after 72 h of transfection, a lower proportion of senescent cells was observed in the group transfected with GNAO1 siRNA compared with that in the group of NC and NTC, indicating that down-regulation of GNAO1 inhibitedthe senescence of HCC cells.

--

Human

L

cellular senescence

23,984,917

Gene

0

1

0

1

0

1

0

1

0

BRG1

834543

protein coding

SW 13

--

Retinoblastoma

Accelerate

SA--gal activity assay

16.8% of the cells transfected with BRG1 were ¦Â-galactosidase positive compared to only 1.4% with the control vector.

p21/WAF1/SDI

Upregulation

Western blot

Western blotting showed that the expression of p21 was significantly down-regulated by inhibition of the BAF47 protein, while the expression level of p16 was not significantly changed in HeLa cells.

--

Human

L

cellular senescence

14,729,964

Gene

0

1

0

FOXO1

2308

protein coding

T98G,LN-18

--

Glioblastoma

Accelerate

Flow cytometry//Knockdown//MTT assay

The results revealed that the overexpression of FOXO1 arrested the cell cycle in the G0/G1 phase, suppressing cells from entering the S phase. The depletion of FOXO1 increased the colony forming ability in cell proliferation of the LN18 and T98G cells.

SIRT1//p16

Downregulation//Upregulation

CHIP//Western blot//qRT-PCR

A ChIP assay was performed with FOXO1 antibody, which revealed that the SIRT1 promoter region interacted with FOXO1.In LN18 cells overexpressing FOXO1, the expression of SIRT1 was suppressed.The opposite results were obtained in the FOXO1-depleted cells, which showed SIRT1 was increased and p16INK4a was decreased .The mRNA levels of SIRT1 and p16INK4a were also regulated by FOXO1, which suggested that FOXO1 regulated SIRT1 and p16INK4a at the transcriptional level.

--

Human

L

cellular senescence

29,207,098

Gene

0

1

0

1

0

1

0

FOXO4

4303

protein coding

COLO 829

--

Melanoma

Accelerate

SA--gal activity assay

Ectopic FOXO4 expression rendered Colo829 cells positive for SA-¦Â-GAL activity.

BRAFV600E//p21

--

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

We therefore wondered whether BRAFV600E could signal through JNK toward FOXO4 to promote senescence. To fully address this question, we first determined all possible JNK sites of in vitro phosphorylated FOXO4 by liquid chromatography¨Ctandem mass spectrometry analysis.In vitro phosphorylation by JNK significantly increased detection of wild-type FOXO4 by these respective antisera, especially the newly discovered Thr223 and Ser226, whereas FOXO4-4A in which these residues are mutated to Ala was not detected. Together, these results indicate that BRAFV600E promotes JNK-mediated phosphorylation of FOXO4.BRAFV600Ecooperated with FOXO4 to induce p21cip1 expression, and in correlation with the induction of senescence, FOXO4 expression increased p21cip1 expression in Colo829 cells. Similar effects were observed on p21cip1mRNA expression determined by quantitative real-time PCR. Moreover, BRAFV600Eand FOXO4 expression resulted in a synergistic activation of a luciferase-reporter gene driven by the p21cip1promoter.

--

Human

L

cellular senescence

20,959,475

Gene

0

1

0

BMI1

648

protein coding

GIST882

--

Gastrointestinal stromal tumor

Prevent

CCK-8 assay//Colony formation assay//Knockdown//SA--gal activity assay

The ¦Â-Gal activity assay was applied in order to detect cell senescence . In comparison with the control group, the percentage of senescent cells involved with the Bmi-1 shRNA group significantly increased (26.7% ¡À 3.9% vs. 17.2% ¡À 2.7%; P < 0.05).The results of the soft agar colony formation assay indicated that the number of colonies contained in the control group was closer to that in the NC group (9.5 ¡À 3.1 vs. 10.1 ¡À 2.2; P > 0.05). In comparison with the control group, the number of colonies in the Bmi-1 shRNA group were reduced significantly (9.5 ¡À 3.1 vs. 3.2 ¡À 1.5; P < 0.05).

p16//p14

Downregulation//Downregulation

qRT-PCR//Western blot//Knockdown

The results of the RT-qPCR method for detection of the mRNA expressions of Bmi-1, p16Ink4A, p14ARF, cyclinD 1, and CDK4 in the GIST882 cells among the three groups are shown . These results showed no remarkable differences of Bmi-1 mRNA expression between the control and NC groups (P > 0.05). In comparison with the control group, the Bmi-1 mRNA expression in the Bmi-1 shRNA group was reduced significantly (P < 0.05). In comparison to the control group, both p16Ink4A and p14ARF mRNA expressions in the Bmi-1 shRNA group were markedly increased (both P < 0.05).. Bmi-1 protein expression in the Bmi-1 shRNA group was shown to be evidently lower than that found in the control group(P < 0.05). In comparison with the control group, both p16Ink4A and p14ARF protein expressions were markedly increased, while both cyclinD 1 and CDK4 protein expressions were significantly reduced in the Bmi-1 shRNA group (all P < 0.05).

--

Human

L

cellular senescence

29,042,141

Gene

0

1

0

1

0

1

0

1

0

MEOX1

4222

protein coding

HUVEC

--

Aging

Accelerate

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

Likewise, we observed a decrease in the proportion of S phase cells when MEOX1 or MEOX2 were expressed in HUVECs. MEOX1 had the greatest effect on cellular proliferation and MEOX2Q235E decreased the proportion of S phase cells to the same extent as wild-type MEOX2. This corresponds to an increase from 4.5% senescence to 9.8% and 10.9% senescence, when comparing untransduced cells to cells ectopically expressing MEOX1 and MEOX2, respectively.

p21/WAF1//p16

Activation//Activation

Luciferase reporter assay//Western blot

MEOX1 and MEOX2 induced greater than two fold activation of the luciferase reporter from the 2272 bp p21CIP1/WAF1 promoter, when compared to the empty vector control .Interestingly, the DNA binding deficient MEOX1Q220E and MEOX2Q235E proteins were able to activate this luciferase reporter to a level comparable to wild-type proteins .Compared to the EGFP control, we observed a three-fold increase in p21CIP1/WAF1 mRNA levels and more than a two-fold increase in p21CIP1/WAF1 protein levels 48 hours after adenoviral delivery of p53. Likewise, expression of MEOX1 or MEOX2 resulted in significantly increased p21CIP1/WAF1 mRNA expression.Comparable to MEOX2, MEOX1 activated the expression of a luciferase reporter from the 2272 bp p21CIP1/WAF1 promoter in HEK293 cells.

--

Human

L

cellular senescence

22,206,000

Gene

0

1

0

1

0

1

0

MEOX2

4223

protein coding

HUVEC

--

Aging

Accelerate

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

Likewise, we observed a decrease in the proportion of S phase cells when MEOX1 or MEOX2 were expressed in HUVECs. MEOX1 had the greatest effect on cellular proliferation and MEOX2Q235E decreased the proportion of S phase cells to the same extent as wild-type MEOX2. This corresponds to an increase from 4.5% senescence to 9.8% and 10.9% senescence, when comparing untransduced cells to cells ectopically expressing MEOX1 and MEOX3, respectively.

p21/WAF1//p16INK5a

Activation//Activation

Luciferase reporter assay//Western blot

MEOX1 and MEOX2 induced greater than two fold activation of the luciferase reporter from the 2272 bp p21CIP1/WAF1 promoter, when compared to the empty vector control .Interestingly, the DNA binding deficient MEOX1Q220E and MEOX2Q235E proteins were able to activate this luciferase reporter to a level comparable to wild-type proteins .Compared to the EGFP control, we observed a three-fold increase in p21CIP1/WAF1 mRNA levels and more than a two-fold increase in p21CIP1/WAF1 protein levels 48 hours after adenoviral delivery of p53. Likewise, expression of MEOX1 or MEOX2 resulted in significantly increased p21CIP1/WAF1 mRNA expression.Comparable to MEOX2, MEOX1 activated the expression of a luciferase reporter from the 2272 bp p21CIP1/WAF1 promoter in HEK293 cells.

--

Human

L

cellular senescence

22,206,000

Gene

0

1

0

1

0

1

0

ATM

472

protein coding

HUVEC

--

Aging

Accelerate

Knockdown//SA--gal activity assay

Furthermore, knockdown of ATM by siRNA suppressed increase in SA-¦Â-gal-positive cells induced by H2O2.

--

ATM-Akt-p53-p21

Activation

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

To assess whether ATM phosphorylation was caused by H2O2-induced redox imbalance. Western blot analysis showed that pretreatment of endothelial cells with NAC blocked the stimulatory effects of H2O2 on the expression of ATM-S1981, Akt-S473, p53-S15 and p21 .Cells transfected with ATM siRNA showed reduced ATM expression with inhibition of Akt and p53 phosphorylation in addition to down-regulation of p21 expression .Moreover, down-regulation of Akt, p53, or p21 by siRNA also suppressed an increase in SA-¦Â-gal-positive cells induced by H2O2.

Human

L

cellular senescence

20,639,198

Gene

0

1

0

1

0

ADIPOQ

9370

protein coding

--

Liver

Tumor

Accelerate

Knockdown//SA--gal activity assay

The number of SA-¦Â-gal positive cells in liver tumor tissue from APN KO mice was markedly less compared with that from WT mice, indicating that adiponectin promotes cell senescence in tumor tissues.

CXCL1

Upregulation

qRT-PCR

qPCR analysis further confirmed that CXCL1 was highly up-regulated in the adiponectin-treated SL4 cancer cells.

p53//p16

Activation//Activation

Immunohistochemistry//Immunofluorescence

p53- and p16-positive cells was detected in adiponectin treated co-cultured cells but not without adiponectin treatment, no expression was detected for p19 and p21 moleculars. Moreover, immunostaining of cocultured cells with PDGFRa (a marker of fibroblasts) confirmed the p53- and p16-positive cells are fibroblasts. Intervention experiments also showed p53 and p16 were involved in angiogenesis and tumor growth.

Human

HL

cellular senescence

27,092,462

Gene

0

1

0

1

0

ATF3

467

protein coding

HKC,SCC

--

Skin cancer

Prevent

SA--gal activity assay

In vivo, ectopic ATF3 promoted tumorigenicity of H-rasV12-expressing HKCs and SCC cells, producing aggressive tumours with reduced senescence as those caused by calcineurin/NFAT inhibitors. Conversely, Atf3 knockdown overcame the tumour-promoting effects of these compounds and restored senescence.

p53

Downregulation

Western blot//qRT-PCR

Functionally, ectopic ATF3 expression, at levels comparable to those occurring in SCCs from CsA-treated patients ,blocked expression of p53 and senescence-associated genes.

--

Human

HL

cellular senescence

20,485,437

Gene

0

1

0

RCC1

1104

protein coding

hTERT-RPE1

--

Aging

Prevent

Immunoblotting//SA--gal activity assay

In contrast, SABG-negative and proliferating cells gradually prevailed in the hTERT-RPE1RCC1-V5 cultures, concomitant with increased interphase and mitotic markers and the decline in cyclin D1 expression.

--

Human

L

cellular senescence

26,864,624

Gene

0

1

0

1

0

PSMB5

5693

protein coding

B-MSC

--

Aging

Prevent

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

The number of BrdU+ cells was significantly increased by 14% following PSMB5 overexpression (p < 0.01 vs. GFP control),Meanwhile, overexpressing PSMB5 attenuated the reduction of Cyclin D1 and CDK4 proteins, raising their expressions by 56% and 23%, respectively.The SA-¦Â-gal assay was applied to identify the aged hBMSCs by staining them blue. Enhanced proteasomal activity by PSMB5 overexpression significantly reduced the percentage of blue cells from 55 ¡À 3% to 40 ¡À 4%.

--

Human

L

cellular senescence

24,393,841

Gene

0

1

0

1

0

1

0

TBX2

6909

protein coding

MCF-7

--

Breast cancer

Prevent

Clonogenic assay//Knockdown//SA--gal activity assay

We performed TBX2 siRNA knockdown in three breast cancer cell lines and after confirming knockdown we assessed effects on cell growth using clonogenic assays. We observed dramatic growth inhibition in all three cell lines.Finally, using senescence-associated ¦Â-galactosidase staining we could also show that knockdown of either TBX2 or EGR1 by transfection of NDRG1 into MCF7 cells that resulted in increased expression of Dec1, indicating that NDRG1 may contribute to the cell senescence observed after TBX2 inhibition.

NDRG1

Downregulation

Western blot//RT-PCR//Knockdown

The most consistently TBX2-regulated gene was NDRG1, which showed consistent upregulation after TBX2 siRNA in several breast cancer cell lines. We also observed NDRG1 upregulation both at the mRNA and protein level after induction of DN-TBX2 in MCF7 cells . Together, these data show that TBX2 represses multiple target genes with NDRG1 consistently upregulated after TBX2 inhibition.

--

Human

HL

cellular senescence

20,348,948

Gene

0

1

0

1

0

1

0

ZEB1

6935

protein coding

SW480

--

Colorectal cancer

Prevent

Knockdown//SA--gal activity assay

We found that ZEB1 knockdown increased the number of SA ¦Â-gal-positive cells and that Wnt3a inhibited basal SA ¦Â-gal activity in SW480-shCtl cells but not in SW480-shZEB1-A cells.

DDK1//mutant p53//MDM2//CtBP//macroH2A1

Activation//Activation//Activation//Activation//Downregulation

Knockdown//Western blot//CHIP//qRT-PCR

Accordingly, the transcriptional activity of serial deletions of the DKK1 promoter increased when the site at?490 bp was included.The ability of endogenous ZEB1 to directly bind to the DKK1 promoter was tested for the site at ?490 bp by ChIP assay.Furthermore, mutation of the CtBP binding sites in ZEB1 (ZEB1-CIDmut) hampered ZEB1¡¯s repression of H2AFY,We found that blocking of CtBP activity in SW480 cells by MTOB induced senescence and upregulated H2AFY.We found that overexpression of DKK1 or addition of rhDKK1, but not rhDKK3, in mutant SW480 cells upregulated mutant TP53 and MDM2 expression in these cells . In turn, DKK1 knockdown downregulated both mutant TP53 and MDM2 and this downregulation was reverted by addition of rhDKK1, but not of rhDKK3. The downstream effect of DKK1 via mutant p53 was corroborated by the knockdown of the latter with a specific siRNA (siTP53). siTP53, but not siCtl, downregulated mRNA expression levels of MDM2 and CTBP while increasing those of H2AFY .

--

Human

HL

cellular senescence

27,965,283

Gene

0

1

0

1

0

THRB

7068

protein coding

ARO,MCF-7,COS-7

--

Aging

Accelerate

SA--gal activity assay

We examined SA-¦Â-gal activity and found that the number of positively stained cells was significantly higher in the irradiated cultures overexpressing wtTHRB than in those treated by any singular agent or by any other combination. Effect of wtTHRB-Ad on the accumulation of SA-¦Â-gal-positive cells was more than additive in irradiated MCF-7 and COS7 cultures, and additive in ARO, Infection with mutTHRB-Ad significantly decreased the number of SA-¦Â-gal-positive cells in irradiated ARO and COS7 cultures.

p21//p16//Rb

Upregulation//Upregulation//Downregulation

Western blot

One hundred-twenty hours after irradiation, i.e. when growth inhibition and senescence-like changes have already taken place in the cultures, cultures infected with wtTHRBAd consistently displayed the decreased phosphorylation of Rb, suggestive of cell accumulation in the G1 phase. Concordantly, these cells also had elevated p21.

--

Human

L

cellular senescence

17,965,546

Gene

0

1

0

H1-3

3007

protein coding

WI-38

--

Aging

Accelerate

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

Concomitant with the decreases in histone H1 protein levels, the expression of N fusions caused severe growth defects in young WI-38 cells as revealed by the growth curves and the BrdU incorporation assay. N fusions also induced a large, flat morphology and increased the percentage of SA¨C¦Â-gal¨Cpositive cells, suggesting that the ectopic expression of N fusions induced premature senescence in WI-38 cells.

p53//p21//Rb

--//Upregulation//--

Western blot//Cell transfection

Cells expressing N fusions showed decreased levels of phosphorylated Rb and cyclin A and increased levels of p21 and phosphorylated p53.

--

Human

L

cellular senescence

17,158,953

Gene

0

1

0

1

0

1

0

TGFB1

7040

protein coding

MEF

--

Aging

Accelerate

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

Compared to physioxia, hyperoxia promoted senescence and triggered activation of TGF-¦Â signaling.Activation of TGF-¦Â signaling by oxidative stress was accompanied by increased miR-29 accumulation and accelerated reduction in the abundance of total H4K20me3.Inhibition of TGF-¦Â signaling by inhibitor treatment and Smad4 depletion diminished miR-29 expression, attenuated the reduction in Suv4-20h1 and Suv4-20h2 expression and produced partial recovery of H4K20me3. Moreover, alterations in TGF-¦Â signaling changed the progression of senescence and specifically altered H4K20me3 without affecting other histone modifications. Disruption of TGF-¦Â signaling by E-616452 restored H4K20me3 levels,attenuated SA-¦Â-gal staining and enhanced the Mki67 signals in Suv4-20h-depleted cells, suggesting that H4K20me3 is a downstream effector of TGF-¦Â signaling. In addition, after miR-29 was destroyed by miRNA inhibitors, activation of TGF-¦Â signaling did not down-regulate Suv4-20h and H4K20me3.

miR-29//H4K20me3

Upregulation//Downregulation

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

We performed histone modifications scanning in senescent cells. The results showed that H4K20me1, -me2, and -me3 exhibited prominent down-regulation in senescent mouse embryonic fibroblasts (MEFs). Accordingly, the expression levels of Suv4-20h1 and Suv4-20h2, the two major methyltransferases mediating H4K20me3, were also decreased during senescence. Furthermore, depletion of Suv4-20h1, Suv4-20h2 or both (designated as Suv4-20h) by shRNAs and treatment with selective Suv4-20h inhibitor A-19636 led to reduced H4K20me3 protein abundance and premature senescence.Meanwhile, disruption of the TGF-¦Â pathway by E-616452 treatment mitigated the increase in miR-29 abundance and progression of senescence in cells with ectopic miR-29.

--

Human

L

cellular senescence

29,967,491

Gene

0

1

0

1

0

1

0

DGCR8

54487

protein coding

IMR-90

--

Colorectal cancer

Prevent

Knockdown//SA--gal activity assay

shDGCR8 IMR90 cells also showed a significant increase in the number of cells with senescent©\associated beta©\galactosidase (SA©\Beta Gal) activity as well as nuclei with SAHFs (senescence©\associated heterochromatin foci), regions of facultative heterochromatin characteristic of senescent cells.

p21

--

BrdU assay

Inactivation of DGCR8 in human and murine fibroblasts caused a modest, but reproducible increase in the protein levels of the cell©\cycle inhibitor p21CIP1. Also, a 3©\fold increase in p21CIP1 transcript and increased number of p21CIP1©\positive cells by immunofluorescence was observed in shDGCR8 human fibroblasts.

--

Human

HL

cellular senescence

23,773,483

Gene

0

1

0

1

0

HOPX

84525

protein coding

HBEC,Y-BE

--

Lung cancer

Accelerate

Cell morphological analysis//SA--gal activity assay

SA-¦Â-Gal staining showed that HOPX transfectant cells displayed cellular enlargement and flattening as well as positivity for SA-¦Â-Gal staining. Additionally, cells exhibited senescence-associated nuclear properties, including elevation of SAHF and accumulation of p-H2AX (gamma-H2AX, Ser139). Forced expression of HOPX in immortalized human bronchial epithelial cells Y-BE led to increased SA-¦Â-Gal staining activity, while knockdown of HOPX in HBECs resulted in decreased SA-¦Â-Gal positive staining, indicating that HOPX-mediated senescence is not restricted to malignant cells.

--

Ras-MAPK//Akt

Activation//Downregulation

Western blot

We found that ectopic expression of HOPX resulted in activation of Ras in both HOPX-positive transfectants HOPX-3 and HOPX-V5-7, as revealed by Ras GTPase assay, which in turn led to activation/phosphorylation of ERK and p38, two major players of the MAPK pathway.Overexpression of HOPX led to inactivation of Akt and down-regulation of MDM2, a negative regulator of p53, as well as the p53 downstream target p21.Decreased expression of HOPX led to reversed effects on the Ras/MAPK pathway and the Akt pathway, with reduced phosphorylated levels of ERK and p38, as well as increased phosphorylated levels of Akt, MDM2, and down-regulation of p21.

Human

HL

cellular senescence

25,345,926

Gene

0

1

0

1

0

BHLHE40

8553

protein coding

HEK293,EC9706

--

Endometrial cancer

Accelerate

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

We transfected DEC1 into EC9706 and confirmed that overexpression of DEC1 induces senescence by SA-¦Â-Gal staining assay and the typically enlarged and flattened cell shape. We found DEC1 overexpression significantly suppressed the growth of EC9706 and HEK293, as measured by growth rate and colony formation assay.

--

Human

HL

cellular senescence

22,844,531

Gene

0

1

0

1

0

1

0

1

0

SIRT6

51548

protein coding

HBEC

--

Lung cancer

Prevent

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

Overexpression of SIRT6 significantly suppressed HBEC senescence after CSE exposure.Intriguingly, compared with control vector, a slight decrease in HBEC senescence was observed by SIRT6 overexpression even in the absence of CSE, especially when measured by means of SA-¦Â-gal staining. Conversely, SIRT6 knockdown increased the percentage of senescent cells, indicating that intrinsic SIRT6 is not sufficient to completely inhibit senescence but has the ability to antagonize CSE-induced cellular senescence in HBECs.

--

IGF-Akt-mTOR

Downregulation

Knockdown//Western blot

SIRT6 knockdown and mutant SIRT6 H133Y overexpression enhanced phosphorylation of IGF-1R accompanied by a modest increase in IGF-1R expression. Next, we examined phosphorylation of Akt and S6K. Consistent with IGF-1R phosphorylation, overexpression of SIRT6 clearly diminished Akt and S6K phosphorylation regardless of presence or absence of CSE. In contrast, SIRT6 knockdown and mutant SIRT6 H133Y overexpression induced Akt and S6K phosphorylation .

Human

L

cellular senescence

24,367,027

Gene

0

1

0

1

0

1

0

GADD45G

10912

protein coding

SK-Hep-1,Hep3B,SMMC-7721

--

Hepatocellular carcinoma

Accelerate

RT-PCR//SA--gal activity assay

Approximately 50%©\70% of these cells ectopically expressing GADD45G were stained positively for senescence©\associated marker ¦Â©\galactosidase . Furthermore, mRNA levels of the senescence©\associated secretory cytokine, interleukin (IL)©\8, were also significantly elevated.

--

JAK-STAT3

Downregulation

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

We found that the constitutive phosphorylation of Stat3 (Tyr705) was substantially inhibited by GADD45G expression in the human liver cancer cell lines, Sk©\Hep1, SMMC©\7721, and Hep3B.In Sk©\Hep1 cells, we found that levels of Tyr705©\phosphorylated Stat3 rapidly decreased, starting within 2 hours after GADD45G induction; coincidently, levels of phosphorylated Jak2 and tyrosine kinase 2 (Tyk2), the upstream activators of Stat3, were also severely down©\regulated.We extended our observation to two subclones (numbers 1 and 2) of mouse LPC©\H©\RasV12 cells, wherein GADD45G could significantly induce cell senescence.Western blot levels of phosphorylated Stat3, Jak2, and Tyk2 were also substantially decreased in these cells .

Human

HL

cellular senescence

23,897,841

Gene

0

1

0

1

0

PRKN

5071

protein coding

Airway epithelial cell

Lung

Chronic obstructive pulmonary disease

Prevent

ELISA//Immunohistochemical staining//SA--gal activity assay//Western blot

Senescence-associated-GLB1/¦Â-galactosidase staining showed clear positive staining of airway epithelial cells in CS-exposed prkn KO mice but few positive staining cells were detected in CS-exposed wild-type mice.Immunohistochemical staining and western blotting of CDKN1A/p21 and CDKN2A/p16 (senescence-associated cyclin dependent kinase inhibitors) were performed. Both airway epithelial cells and alveolar epithelial cells in CS-exposed prkn KO mice demonstrated clear positive staining in both CDKN1A and CDKN2A . Consistent with the degree of cellular senescence, CXCL1 and IL1B protein levels were significantly increased in CS-exposed prkn KO mice compared to CS-exposed wild-type mice .

--

Human

L

cellular senescence

30,290,714

Gene

0

1

0

1

0

1

0

1

0

CD40L

100286596

protein coding

A549,H460

--

Lung cancer

Prevent

CCK-8 assay//Flow cytometry//SA--gal activity assay//Western blot

We used pcDNA3.1+, pcDNA3.1+-CD40L-WT and pcDNA3.1+-CD40L-M to transfect CD40-positive NSCLC cell lines. CD40L expression was increased in the A549, A549/TR, A549/DDP and H460 cells 48 h after transfection.In addition, the culture cell size was enlarged in the A549/TR, A549/DDP and H460 cells, a trait associated with senescence. These data were confirmed with SA-¦Â-gal staining, yet SA-¦Â-gal staining was not observed in the A549 cells.CCK-8 assay data showed that CD40L-M expression significantly reduced cell proliferation.Flow cytometry data showed that the percentage of S phase cells was decreased and the percentage of G0/G1 phase cells was increased in the CD40L-M-expressed cell group compared with these percentages noted in the controls or negative cells,indicating that CD40L-M expression blocked cell cycle progression.

--

ATM-Chk2

--

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

We assessed p-Chk2 and p-Chk1 in CD40L-induced senescent A549/TR cells. Data showed that only p-Chk2 was significantly induced 48 h after transfection, indicating that the ATM/Chk2 pathway was activated in response to CD40L-M-induced senescence.We treated CD40L-M-transfected A549/TR cells with an inhibitor of ATM kinase, KU-55933. KU-55933 inhibited phosphorylation of the ATM target protein Chk2 and decreased SA-¦Â-gal activity , suggesting that ATM/Chk2 mediated CD40L-M-induced senescence.

Human

L

cellular senescence

29,565,449

Gene

0

1

0

1

0

1

0

1

0

FOXO3

2309

protein coding

MEF

--

Aging

Accelerate

Knockdown//SA--gal activity assay

We delivered the siRNA into MEFs and confirmed that transfection with this siRNA silenced circ-Foxo3 levels. We also delivered an siRNA targeting the linear mRNA of Foxo3 into the cells. The cells transfected with circ-Foxo3 siRNA showed less ¦Â-gal staining than the cells transfected with Foxo3 siRNA or the control oligo. Primary cardiomyocytes transfected with the siRNA also displayed lower levels of beta-Gal staining and circ-Foxo3 expression.

--

Human

L

cellular senescence

26,873,092

Gene

0

1

0

1

0

KNDC1

85442

protein coding

HUVEC

--

Aging

Accelerate

Flow cytometry//SA--gal activity assay

The levels of KNDC1 mRNA and protein in HUVECs at P4 were downregulated by gene silencing with KNDC1-siRNA1/2. Following repression of KNDC1 levels in HUVECs at P4, the size of the cells was similar to the size of P1 cells and the activity of SA-¦Â-gal was decreased compared with that in NT-siRNA-transfected cells.The number of cells in G1 phase was decreased in cells transfected with KNDC1-siRNA.

--

p53-p21-p16

--

Western blot

The phosphorylation of p53 and the expression of p21 and p16 decreased significantly compared with NT-siRNA-transfected control cells.

Human

L

cellular senescence

24,788,352

Gene

0

1

0

1

0

HDAC2

3066

protein coding

MCF-7

--

Aging

Prevent

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

We found that knockdown of HDAC2, but not HDAC1, resulted in a G1 arrest.Consistent with the arrest in G1, cellular proliferation and colony formation were inhibited upon knockdown of HDAC2. We also found that HDAC2 affected colony formation in a dose-dependent manner because more effective knockdown of HDAC2 resulted in more marked growth inhibition .In contrast, knockdown of HDAC1 or tetracycline had no effect on cellular proliferation or colony formation. Furthermore, we found that knockdown of HDAC2 resulted in cellular senescence.

--

Human

HL

cellular senescence

17,409,421

Gene

0

1

0

1

0

1

0

1

0

DAO

1610

protein coding

U2OS,HepG2

--

Aging

Accelerate

Colony formation assay//EdU assay//SA--gal activity assay//Western blot

We observed that the percentage of SA-¦Â-gal¨Cpositive cells was increased after the treatment with etoposide both in U2OS and HepG2 cells, knockdown of DAO partially restored etoposide-induced loss of proliferative capacity.we next tested the impact of inhibiting DAO activity on senescence using 6-chlorobenzo[d]isoxazol-3-ol (CBIO), previously characterized for the ability to inhibit enzymatic activity of DAO .CBIO suppressed the SA-¦Â-gal activation and loss of proliferative capacity following etoposide treatment in both U2OS and HepG2 cells.CBIO was also effective in suppressing senescence of Hs68 cells, as judged by SA-¦Â-gal, EdU incorporation proliferation assays, and immunoblot analysis, We observed that ectopic expression of DAO enhanced etoposide-induced senescence, although DAO overexpression in the absence of etoposide had no significant effect, indicating that DAO expression alone is not sufficient to induce senescence but accelerates senescence under stress conditions.

--

Human

L

cellular senescence

30,659,069

Gene

0

1

0

1

0

1

0

1

0

SIRT7

51547

protein coding

WI-38

--

Aging

Prevent

Knockdown//SA--gal activity assay

We found that shRNA depletion of SIRT7 from WI38 primary human fibroblasts led to substantial accumulation of senescent cells within days after SIRT7 depletion. Levels of the senescence marker p16 were also increased in the SIRT7-deficient cell cultures.

--

Human

L

cellular senescence

29,728,458

Gene

0

1

0

1

0

DPY30

84661

protein coding

IMR-90

--

Aging

Prevent

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

ShDPY30 IMR90 cells also contained DAPI-stained SAHFs, another characteristic for senescent human fibroblasts (Narita et al, 2003). Additionally, the overall morphology of the DPY30 knockdown IMR90 cells changed, towards a more rounded and flattened phenotype with enlarged nuclei. SA-¦Â-galactosidase assays showed that >90% of the shDPY30 IMR90 cells expressed Activate SA-¦Â-galactosidase. Further, in proliferation assays, growth curves, and colony formation assays, shDPY30 IMR90 cells arrested and stopped proliferating.

ID

--

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

In ID1 and ID3 overexpressing cells, DPY30 knockdown reduced mRNA levels, but not proteins levels, of ID1 and ID3.the shDPY30 cells with ID1 or ID3 overexpression regained their fibroblast features and began to proliferate again, albeit not to the same degree as the IMR90 control cells, while the shDPY30 cells remained in a senescence-like state.?A reduced SA-¦Â-galactosidase staining was observed in shDPY30 cells overexpressing either ID1 or ID3 (from 82 to 55 or 28%, respectively).

--

Human

HL

cellular senescence

23,872,946

Gene

0

1

0

1

0

1

0

1

0

1

0

HRAS

3265

protein coding

BJ

--

Aging

Accelerate

Immunofluorescence//SA--gal activity assay

Doxycycline-induced H-RASV12?expression induces cellular senescence in normal BJ human fibroblasts.Few cells stained positive for (SA-¦Â-GAL) activity during the proliferative phase at day 4 but SA-¦Â-GAL positivity increased at day 12 in DOX-treated but not in untreated cultures, coinciding with the onset of growth arrest. By day 16 around 65 % of DOX-treated cells were SA-¦Â-GAL positive compared to 15% of the control cells.?DOX stimulation increased the percentage of cells that showed intense H3K9me3 staining (90 % compared to 30 % in the control), appearing as SAHF or a more widespread nuclear staining .

p53

--

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

RAS-induced SA-¦Â-GAL activity was almost completely abolished in p53 knockdown cells, which is in good agreement with their continuous proliferation. Further, only a modest increase in the expression of p16 and H3K9me3 was observed in DOX-treated shp53 cells compared with shCtrl cells, suggesting that p53 depletion inhibited senescence induced by RAS.

--

Human

L

cellular senescence

30,526,305

Gene

0

1

0

1

0

Hsp27

39078

protein coding

MRC-5

--

Aging

Prevent

Knockdown//SA--gal activity assay//qPCR

After 3 days of HSP27 knockdown, we observed significant increases in cells positive for senescence-associated ¦Â-gal (SA-¦Â-gal) and significant up-regulation of pro-inflammatory cytokines IL-1¦Á and IL-8.

E2F4//p130

--//--

qPCR//Western blot//Immunofluorescence

By using qPCR, we found that the expression of E2F-4 was significantly increased upon HSP27 knockdown, HSP27 knockdown increased the expression of p130.We also confirmed strong increases of E2F-4 and p130 proteins by HSP27 knockdown using immunoblot analysis and immunocytochemistry.

--

Human

L

cellular senescence

30,166,342

Gene

0

1

0

1

0

1

0

XAF1

54739

protein coding

HMVEC

--

Aging

Accelerate

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

Decreased cell proliferation in Doxo- or IR-treated cells was partially recovered by XAF1 downregulation. Repression of XAF1 levels in senescent cells caused morphological changes similar to young cells and a decrease in SA-¦Â-gal activity. Additionally, XAF1 downregulation in premature senescent cells decreased the population of cells in the G1 phase and increased the population of cells in the S and G2/M phases.

--

p53

--

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

The Doxo- and IR-induced increase in p53 expression and activation was reduced in the XAF1 knockdown cells .By measuring the SA-¦Â-gal activity, we demonstrated that p53 knockdown inhibited XAF1-induced cellular senescence but p16 knockdown did not.

Human

L

cellular senescence

26,802,028

Gene

0

1

0

1

0

1

0

IGFBP4

3487

protein coding

MSC

--

Aging

Accelerate

Annexin V binding assay//Cell apoptosis assay//Cell proliferation assay

Treatment with anti-IGFBP4 and/or anti-IGFBP7 blocked the pro-senescence activity of CM-P10 even at the lowest concentration tested .It is to be noted that the simultaneous treatment with both anti-IGFBP4 and anti-IGFBP7 provided an enhanced reduction of senescence induced by CM-P10 on P1 MSC.treatment of CM-P10 with both anti-IGFBP4 and anti-IGFBP7 showed a significant reduction of the apoptosis level. CM-P10 treatment with anti-IGFBP4 or anti-IGFBP7 showed a significant increase of cell proliferation rates with respect to P1 MSC cultured with untreated CM-P10 at 72?h.the simultaneous addition of both rIGFBP4 and rIGFBP7 increased MSC senescence and induced apoptosis at the highest concentration assayed .

--

Human

L

cellular senescence

24,201,810

Gene

0

1

0

1

0

1

0

IGFBP7

3490

protein coding

MSC

--

Aging

Accelerate

Annexin V binding assay//Cell apoptosis assay//Cell proliferation assay

Treatment with anti-IGFBP4 and/or anti-IGFBP7 blocked the pro-senescence activity of CM-P10 even at the lowest concentration tested.It is to be noted that the simultaneous treatment with both anti-IGFBP4 and anti-IGFBP7 provided an enhanced reduction of senescence induced by CM-P10 on P1 MSC.treatment of CM-P10 with both anti-IGFBP4 and anti-IGFBP7 showed a significant reduction of the apoptosis level.CM-P10 treatment with anti-IGFBP4 or anti-IGFBP7 showed a significant increase of cell proliferation rates with respect to P1 MSC cultured with untreated CM-P10 at 72?h.the simultaneous addition of both rIGFBP4 and rIGFBP7 increased MSC senescence and induced apoptosis at the highest concentration assayed .

--

Human

L

cellular senescence

24,201,810

Gene

0

1

0

1

0

1

0

ABI3BP

25890

protein coding

ARO

--

Aging

Accelerate

Flow cytometry//SA--gal activity assay

We first observed that SA-¦Â-Gal positive cells,indicated by blue staining, were increased when ABI3BP is re-expressed.Secondly, immunofluorescence of HP1¦Ã showed an expression pattern concentrated in DNA foci of cells expressing ABI3BP compared to controls which suggested that the re-expression of ABI3BP was associated with the nuclear changes that occur during senescence. Conversely in vector-transfected cells, the staining for HP1¦Ã gave a weak signal and it was disperse through the nucleoplasm .Cell cycle distribution was analyzed by flow cytometry. G0/G1 arrest occurred in ARO cells expressing ABI3BP as indicated by an increase in the percentage number of cells at this phase.

--

p21

Upregulation

qPCR

P21 mRNA levels were 3-fold increased in ARO cells following ABI3BP re-expression . G0/G1 arrest occurred in ARO cells expressing ABI3BP as indicated by an increase in the percentage number of cells at this phase. Although cell cycle was not investigated at day 5, when senescence phenotype was marked observed, the cell proliferation marker Ki67 was down-regulated and the cell cycle inhibitor p21 was up-regulated at day 5.

Human

L

cellular senescence

18,559,958

Gene

0

1

0

1

0

PIM1

5292

protein coding

22Rv1

--

Aging

Accelerate

Cell morphological analysis//SA--gal activity assay

By the final passage (numbers 20¨C25), PIM1-expressing cells barely divided. The final-passage cells were found to have a flat and spread morphology and to be ¦Â-galactosidase (¦Â-Gal) positive, consistent with the suggestion that they had undergone senescence.

--

p53-p21

Activation

SA-¦Â-gal activity assay//qRT-PCR//MTT assay//Western blot

A marked change in the level of phosphorylation of these proteins occurs in late-passage PIM1-containing 22Rv1 cells and mirrors increases in the levels of the p21 protein . To examine the role of p21 in these cells, lentiviral vectors were used to transduce a Tet/ON-inducible shRNA directed at p21 (sh-p21) or a control sequence (sh-c) into 22Rv1 cells constitutively expressing PIM1 or a control vector. This shRNA was successful in decreasing p21 levels in control and PIM1-containing cells, but had no effect on the growth of 22Rv1 cells. In contrast, the growth of PIM1-containing 22Rv1 cells was markedly stimulated by decreasing the p21 levels. In these cells, decreased p21 levels correlated with a markedly lower level of ¦Ã-H2AX, staining of these late-passage PIM1¨Coverexpressing cells with ¦Â-Gal shows that blocking p53 activity also prevents the induction of this marker of cell senescence by PIM1.

Human

L

cellular senescence

20,647,331

Gene

0

1

0

1

0

HEPACAM

220296

protein coding

MCF-7

--

Aging

Accelerate

Cell morphological analysis//SA--gal activity assay

About 54.2% ( P ?<?0.001) of MCF7 cells expressing hepaCAM were found to have flat enlarged morphology, a typical characteristic of senescent cells. 40% ( P ?<?0.001) of MCF7 cells expressing hepaCAM were stained blue representing ¦Â-galactosidase activity. Conversely, 12.2 and 15.6% of cells expressing vector or hCAM-tailless showed ¦Â-galactosidase staining, respectively.

--

p53-p21

Upregulation

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

Higher protein levels of p53, p21 and p27, but lower levels of cyclin B1 and cdc2, were detected in cells expressing wild-type hepaCAM when compared with cells expressing vector and hCAM-tailless.MCF7 cells expressing hepaCAM were either untransfected or transfected with either control or p53 siRNA, and the protein levels of p21 and p27 were evaluated. In comparison with either the untransfected cells or cells transfected with control siRNA, knockdown of p53 by p53 siRNA lowered the expression of p21 but did not significantly change p27, indicating that, in the presence of hepaCAM, the increase of p21 expression is p53 dependent. In addition, downregulation of p53 in cells expressing hepaCAM resulted in decreased ¦Â-galactosidase staining, reduced cell size and increased cell growth, suggesting that suppression of p53 alleviates senescence induced by hepaCAM.

Human

L

cellular senescence

18,845,560

Gene

0

1

0

1

0

LSD1

827786

protein coding

PC-3

--

Aging

Prevent

Knockdown//RT-qPCR//SA--gal activity assay

LSD1-depleted cells underwent cellular senescence as manifested by increased expression of senescence markers, including positivity of pronounced foci for the heterochromatin marker H3K9 trimethylation (H3K9me3)6,19, senescence-associated ¦Â-galactosidase (SA-¦Â-gal) activity and upregulation of MMP3 gene encoding a senescent cell secretory protein20.Analogous to the effect of gene knockdown, the LSD1 inhibitor pargyline11,21 also induced senescence in both LNCaP and PC-3 cells .

H3K9me2

--

CHIP//Immunocytochemistry//SA-¦Â-gal activity assay

LSD1 knockdown alone induced robust cellular senescence, including increased SA-¦Â-gal activity and positivity of pronounced foci for the heterochromatin marker H3K9me3 .Chromatin immunoprecipitation (ChIP) assay showed that the level of the transcription-repressive mark H3K9me2 in SKP2 and CDC25A promoters was significantly increased after LSD1 knockdown, and the effect was almost completely reversed by restored expression of LSD1.

--

Human

HL

cellular senescence

28,991,226

Gene

0

1

0

1

0

1

0

DICER1

23405

protein coding

WI-38,IMR-90

--

Aging

Prevent

Knockdown//SA--gal activity assay

SiRNA©\mediated downregulation of DICER1 levels increased cellular senescence and blocked the ability of metformin to lower the number of SA©\¦Â©\gal©\positive cells in both WI©\38 and IMR©\90 cells. Conversely, overexpression of DICER1 decreased cellular senescence.

--

Human

HL

cellular senescence

26,990,999

Gene

0

1

0

1

0

HDAC9

9734

protein coding

MRC-5

--

Aging

Prevent

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

We show that sodium butyrate (NaB) and panobinostat (LBH589), two broad-spectrum HDAC inhibitors up-regulate hsa-miR-31 (miR-31).The results of the Western blot analysis showed that NaB and LBH589 induced p53, p21, and p16, and reduced comparative levels of phosphorylated pRb in control cells.The results indicated that NaB and LBH589 induced a robust senescent phenotype in control cells .The results indicated that NaB and LBH589 increased ¦ÃH2AX foci formation in control cells.

miR-31

--

SA-¦Â-gal activity assay//EdU assay//Western blot//¦ÃH2AX staining

The results of the Western blot analysis showed that NaB and LBH589 induced p53, p21, and p16, and reduced comparative levels of phosphorylated pRb in control but not in miR-31 inhibitor expressing cells. The data also indicated that the miR-31 inhibitor overcomes the proliferation inhibitory effect of NaB and LBH589 in MRC5 cells, and that the miR-31 inhibitor promotes cell proliferation. The results indicated that NaB and LBH589 induced a robust senescent phenotype in control but not in miR-31 inhibitor expressing cells as indicated by an increase in SA-¦Â-gal- and decrease in EdU-positive cells.The results indicated that NaB and LBH589 increased ¦ÃH2AX foci formation in control but not in miR-31 inhibitor expressing cells.

--

Human

HL

Others

25,737,447

Gene

0

1

0

1

0

1

0

PPARD

5467

protein coding

VSMC

--

Aging

Prevent

SA--gal activity assay//Western blot

Activation of PPAR¦Ä by GW501516, a specific ligand for PPAR¦Ä, significantly attenuated Ang II-induced generation of superoxides and suppressed senescence of VSMCs.When VSMCs were treated with Ang II, the senescence-associated ¦Â-galactosidase (SA ¦Â-gal) activity, a biomarker for cellular senescence, was significantly increased in Ang II-treated VSMCs.This increase was significantly suppressed in the presence of GW501516, suggesting the involvement of PPAR¦Ä in the inhibition of Ang II-induced premature senescence . The siRNA-mediated down-regulation of PPAR¦Ä significantly suppressed the GW501516-mediated inhibition of premature senescence of VSMCs induced by Ang II. Similar results were obtained when the cells were analyzed with another PPAR¦Ä siRNA_2.

PTEN

Upregulation

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

Treatment with GW501516 significantly increased the level of PTEN transcript in a time-dependent manner. The up-regulation of PTEN by GW501516 was suppressed in the presence of siRNA against PPAR¦Ä, suggesting the causal role of PPAR¦Ä in the up-regulation of PTEN.PTEN siRNA reversed the GW501516-mediated suppression of superoxide generation in Ang II-treated cells. The number of SA ¦Â-gal-positive cells reduced by GW501516 was also recovered by transfection with PTEN siRNA.

PI3K-Akt

--

Western blot

The activation of Akt by Ang II was markedly reduced in the presence of GW501516 and almost completely abolished in the presence of both GW501516 and LY294002. Down-regulation of PPAR¦Ä by siRNA reversed the GW501516-induced decrease in phosphorylated Akt in cells treated with Ang II. Similar results were obtained with another PPAR¦Ä siRNA_2.The short time pretreatment (30 min) did not affect the level of phosphorylated Akt, whereas the long time pretreatment (24 h) significantly suppressed the Ang II-induced increase in phosphorylated Akt.

Human

L

cellular senescence

22,072,715

Gene

0

1

0

1

0

GLO1

2739

protein coding

RPTEC

--

Aging

Prevent

SA--gal activity assay//Western blot

Empty vector-transfected cells treated with etoposide showed a significant increase in SABG-positive cells, by 2.3 ¡À 1.0-fold, which was significantly suppressed by GLO1 overexpression.When GLO1-knocked down cells were treated with etoposide, the number of cells positive for SABG staining was significantly increased, compared with control siRNA-transfected cells . Other senescence markers, such as transcript expression level of p53, p21WAF1/CIP1, and p16INK4A or protein expression level of p53 in whole cellular lysate, were also markedly augmented by knockdown of GLO1.

--

Human

L

cellular senescence

22,001,178

Gene

0

1

0

1

0

HDAC1

3065

protein coding

UCD-Mel-J

--

Aging

Accelerate

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

The Tet-regulated UCD-derivative (UCD-HDAC1) cell line expressed HDAC1 when treated with doxycycline (Dox), a potent tetracycline analog. An MTT-cell proliferation assay demonstrated that 10¨C14 days after HDAC1 induction, UCD-HDAC1 cells became growth arrested,whereas uninduced UCD-HDAC1 cells, or UCD cells harboring an empty vector in the presence or absence of Dox, displayed normal exponential growth.After 3 weeks of sustained HDAC1 levels the cells became irreversibly arrested.These cells did not resume growth upon Dox removal, and continued to display typical markers of normal melanocyte senescence (Bennett & Medrano, 2002) including expression of the senescent-associated ¦Â-galactosidase (SA-¦Â-gal) marker, a flat morphology, and aberrant cytokinesis.

--

Human

L

cellular senescence

17,578,512

Gene

0

1

0

1

0

1

0

RAD21

5885

protein coding

MDA-MB-231,HCT116,LNCaP,DU 145

--

Aging

Prevent

BrdU assay//Immunofluorescence//SA--gal activity assay

The more effective siRNA #2 was used in the subsequent experiments, which showed that RAD21 suppression significantly inhibited MDA-MB-231 cell proliferation , decreased bromodeoxyuridine (BrdU) incorporation, and elevated the activity of the senescence marker SA-¦Â-gal.Heterochromatin foci are a typical signature of human cells undergoing senescence.Heterochromatin foci were readily visible in senescent cells transfected with RAD21 siRNA after DAPI staining, with 45% of nuclei displaying a punctate DAPI staining pattern.We also found that RAD21 silencing induced senescence in HCT116 colon cancer cells, as well as LNCap and DU145 prostate cancer cells .

c-Myc

--

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

c-Myc overexpression in cells transfected with RAD21 siRNA and the pcDNA3.1-c-Myc expression plasmid did not result in reduction of c-Myc protein, whereas decrease in c-Myc protein was induced in cells cotransfected with RAD21 siRNA and the control plasmid. Furthermore, c-Myc overexpression could also lead to functional inactivation of RB1 by increasing its phosphorylation in cells transfected with RAD21 siRNA. As expected,co-expression of RAD21 siRNA and c-Myc protein dramatically reduced the number of senescent cells.

Rb-E2F

--

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

RAD21-depleted MDA-MB-231 cells displayed lower levels of phospho-RB1 .all phenotypic markers of cellular senescence tested were significantly inhibited when either p21 or RB1 was silenced with RAD21.RAD21 siRNA-induced senescence was characterized by RB1 hypophosphorylation, downregulation of the E2F-dependent genes proliferating cell nuclear antigen (PCNA), cyclinA, and cyclinD1, as well as downregulation of c-Myc, a key regulator of the RB1/E2F pathway.c-Myc overexpression could also lead to functional inactivation of RB1 by increasing its phosphorylation in cells transfected with RAD21 siRNA.

Human

L

cellular senescence

26,529,363

Gene

0

1

0

1

0

1

0

XRCC5

7520

protein coding

HUVEC

--

Aging

Prevent

SA--gal activity assay//Western blot

The number of SA-¦Â-gal+ (positive) cells was significantly reduced, indicating that pcDNA3.1-Ku86 significantly inhibited the increase in the number of SA-¦Â-gal+ cells when co-incubated with HUVECs (p?<?0.01). The SA-¦Â-gal+ cell population was significantly increased by Ku86 knockdown (p?<?0.05). Additionally, the expression of senescence-related proteins in HUVECs lacking Ku86 showed the opposite trend, compared with that observed in Ku86-overexpressing cells (p?<?0.01).

--

Human

L

cellular senescence

30,616,437

Gene

0

1

0

1

0

PTGES2

10728

protein coding

HUVEC

--

Aging

Accelerate

Immunofluorescence//SA--gal activity assay

Starting from 2 days after the beginning of the treatment, PGE2 had a marked effect on growth inhibition, only in the presence of a high concentration of glucose.In accordance, we observed a statistically significant increase in the percentage of cells expressing the cell-cycle inhibitor p16, only in the presence of a high concentration of glucose. PGE2 also induced an increase in the number of SA-¦Â-Galpositive cells, again only in the presence of a high concentration of glucose .It is worth to note that the growth, the p16 expression, and the SA-¦Â-Gal staining were not affected by the supplementation in glucose perse.

EP3

--

SA-¦Â-gal activity assay

The EP3 antagonist led to a statistically significant decrease of SA-¦Â-Gal activity.We treated NHDFs with an agonist of EP3 (sulprostone) and found that it increased the number of SA-¦Â-Gal-positive cells at the same level of that induced by PGE2.

--

Human

L

cellular senescence

24,046,862

Gene

0

1

0

1

0

SIRT1

23411

protein coding

BOEC

Peripheral Blood

Chronic obstructive pulmonary disease

Prevent

SA--gal activity assay

We next confirmed the protective role of SIRT1 against senescence in BOEC, as inhibition of SIRT1 increased SA-¦Â-gal activity in SIRT1-deficient BOEC compared to control siRNA-treated cells, both in baseline and oxidant conditions.There was a significant negative correlation between both SIRT1 protein levels and activity and SA-¦Â-gal staining in all samples.

p53

--

Western blot

Inhibition of SIRT1 expression in BOEC, which caused increased senescence, also resulted in increased acetylation of p53 at Lys-382, suggesting that the protective effect of SIRT1 against senescence in BOEC may be in part mediated by deacetylation of p53.

--

Human

L

cellular senescence

23,897,750

Gene

0

1

0

ATM

472

protein coding

HUVEC

--

Chronic obstructive pulmonary disease

Accelerate

SA--gal activity assay//Western blot

Inhibition of ATM caused a significant increase in SIRT1 mRNA and protein levels and a reduction in SA-¦Â-gal activity compared to control cells. Inhibition of ATM, in H2O2-treated HUVEC induced a significant increase in SIRT1 protein levels and reduction in senescence compared to control siRNA-treated cells.

SIRT1

--

Western blot

Inhibition of ATM, in H2O2-treated HUVEC induced a significant increase in SIRT1 protein levels and reduction in senescence compared to control siRNA-treated cells.

--

Human

L

cellular senescence

23,897,750

Gene

0

1

0

1

0

CKB

1152

protein coding

HBEC

--

Aging

Prevent

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

CKB knockdown concomitant with CSE exposure further increased the percentage of SA-¦Â-gal¨Cpositive cells. We also investigated the effects of CSE exposure and CKB knockdown on p21/waf-1, a senescence-associated cyclin-dependent kinase inhibitor, by Western blotting.Both CSE and CKB knockdown induced the expression of p21/waf-1, respectively, and CKB knockdown, concomitant with CSE exposure, further induced p21/waf-1 expression.

--

Human

L

cellular senescence

21,980,054

Gene

0

1

0

1

0

1

0

MKRN1

23608

protein coding

AGS,SNU601,MEF

--

Gastric cancer

Prevent

Knockdown//SA--gal activity assay

Upon ablation of MKRN1, both cells exhibited growth retardation and senescence acceleration detected using SA-¦Â-galactosidase staining. The results indicated that 53% of MKRN1?/? MEFs that were counted displayed premature senescence, whereas only 7.3% of wild-type MEFs went through senescence at passage 6 (MKRN1+/+ vs MKRN1?/?, mean percentage = 7.3% vs 53%, difference = 45.7%, 95% CI = 39.07% to 52.33%, P =0.005).

p14

--

Western blot

MKRN1 induced degradation of exogenous p14ARF in H1299 cells in a concentration-dependent manner. Experiments employing cycloheximide (CHX) treatment to measure the protein half-life showed that approximately half of the exogenous p14ARF protein disappeared in less than 2h in the presence of MKRN1, compared with almost 5h without MKRN1.

--

Human

HL

cellular senescence

23,104,211

Gene

0

1

0

1

0

CBX7

23492

protein coding

SGC-7901

--

Gastric cancer

Prevent

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

Western blot analysis of CBX7 indicted that CBX7 siRNA efficiently knockdowned CBX7 expression.Stable expression of CBX7 siRNA in SGC-7901 cells led to an increase of senescence and a decrease in colony formation in soft agar.Compared to control, the rate of senescent cells was higher in CBX7 knockdown SGC-7901 cells , and the soft agar colonies in CBX7 knockdown SGC-7901 cells were less in frequency and also smaller in size.

p16

--

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

Cell lines with low or no p16(INK4a) overexpressing CBX7 suggested a negative correlation between the expression of CBX7 and p16(INK4a).We found that knockdown of CBX7 resulted in increased p16(INK4a) expression.

--

Human

L

cellular senescence

20,723,236

Gene

0

1

0

1

0

1

0

1

0

SYK

6850

protein coding

A375

--

Melanoma

Accelerate

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

We observed that a major population of cells arrested by Syk displayed a flat, enlarged morphology with increased granularity that is characteristic of senescent cells .A blue staining indicative of acidic ¦Â-gal activity was observed in the growth-arrested A375 infected with Ad-Syk. Notably, ¡«40% of cells were ¦Â-gal positive in Syk-expressing populations, whereas <2% of A375 cells were positive in vector-infected cultures. Immunofluorescence analysis for HP1¦Â, a marker of SAHF, revealed punctuated regions of DNA corresponding to heterochromatic foci in Syk-expressing cells, but not in control Syk-negative cells.

--

p53-p21//pRb

Upregulation//Upregulation

Western blot//immunofluorescence

Increased levels of activating phosphorylation of p53 at Ser15 and induction of the cdk inhibitor p21 were observed in Syk-expressing A375 cells compared with controls . We also noticed that Syk expression resulted in modest, but consistent, increase in the levels of total p53. In addition, Syk-expressing cells harbored dephosphorylation of pRb and lost cyclin A expression. Consistent with changes in p53 regulation, immunofluorescence of individual cells showed nuclear p53 accumulation in Syk-positive cells .

Human

L

cellular senescence

19,293,188

Gene

0

1

0

1

0

1

0

FBXO31

79791

protein coding

MCF-7

--

Breast cancer

Accelerate

SA--gal activity assay

The MCF-7 cell line has a spontaneous background of senescent cells usually evident as occasional blue staining cells.Following several weeks of growth, 35% to 40% of colonies ectopically expressing FBXO31 showed senescence compared with 5% to 7% of senescent colonies with vector alone. In subsequent experiments, MCF-7 cells ectopically expressing an EGFP-tagged FBXO31 showed that the presence of cell senescence was correlated to the expression of FBXO31.

--

Human

L

cellular senescence

16,357,137

Gene

0

1

0

PRC2

3647044

protein coding

TIG-3

--

Aging

Prevent

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

Therefore, to formally establish that a decreasing pool of the PRC2 complex directly leads to senescence and displacement of BMI1, we targeted the expression of EZH2 and SUZ12 by short hairpin interference.The loss of either protein results in the disruption of the PRC2 complex (Pasini et al. 2004b) and leads to the decrease of H3K27me3 and displacement of both BMI1 and CBX8 from the INK4A promoter. Significantly, cells depleted of EZH2 or SUZ12, similar to cells depleted of BMI1, activate INK4A transcription and become senescent .

INK4a

--

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

We targeted the expression of EZH2 and SUZ12 by short hairpin interference. The loss of either protein results in the disruption of the PRC2 complex (Pasini et al. 2004b).cells depleted of EZH2 or SUZ12, similar to cells depleted of BMI1, activate INK4A transcription and become senescent.

--

Human

HL

cellular senescence

17,344,414

Gene

0

1

0

1

0

1

0

1

0

TNF

7124

protein coding

HMVEC

--

Aging

Accelerate

Cell morphological analysis//SA--gal activity assay

Staining with SA-¦Â-gal, pH 6.0 revealed a significant increase (N52%) of SA-¦Â-gal positive cells in young HMVEC-Ls treated with 10 and 20 ng/ml TNF-¦Á for 15 consecutive treatments over a period of 15 days when compared to the young, untreated cells. In parallel with an increase in the SA-¦Â-gal expression, young HMVEC-Ls exposed to chronic TNF-¦Á treatment became enlarged and flattened, which is a typical senescent phenotypic morphology, similar to those of the RS cells.There were 5.5 ¡À 1.7% SA¦Â-gal positive in untreated young cells whereas both RS and chronic TNF-¦Á-treated young untreated cells demonstrated significantly higher SA-¦Â-gal positive cells, which were ~49.4 ¡À 4.6% and 57.8 ¡À 10.0%, respectively.The TNF-¦Á-treated cell and RS cell cultures contained significantly higher percentages of G0/G1 cell population i.e. ~67% and ~71%, respectively, compared to the untreated young cell culture (~54%).

Hsa-miR-20b

Upregulation

qRT-PCR

qRT-PCR results corroborated with microarray expression, whereby hsa-miR-17 and -20a were downregulated in RS and TNF-¦Á-treated cells when compared to young untreated cells .However, hsa-miR-20b was instead found upregulated with qRT-PCR analysis and this discrepancy could be due to the inherent differences between the two methods in detecting miRNAs.

--

Human

HL

cellular senescence

28,595,801

Gene

0

1

0

1

0

DIP2C

22982

protein coding

RKO

--

Colorectal cancer

Prevent

Knockdown//RT-qPCR//SA--gal activity assay

We performed qPCR and found p14 ARF and p16 INK4a upregulated to the same extent in DIP2C ?/? cells . As p16 INK4a is a marker of cellular senescence we then stained cells for ¦Â-galactosidase activity at pH 6, which is another characteristic of senescent cells, detecting staining of up to 1.9% of DIP2C ?/? cells, compared to 0.2% of parental RKO cells.Cell enlargement is another sign of senescence and imaging of live cells revealed that DIP2C knock-out cells appeared stretched-out relative to parental RKO cells .

--

Human

HL

cellular senescence

28,716,088

Gene

0

1

0

1

0

1

0

ASF1A

25842

protein coding

HepG2,LNCaP

--

Aging

Prevent

Knockdown//SA--gal activity assay

We, thus, performed ¦Â-gal staining for senescence evaluation. Blue ¦Â-gal staining was readily seen in siASF1a-treated cells, while hardly detected in control cells, implying that knockdown of ASF1a led to senescence of HepG2 and LNCaP cells.

--

p53-p21

--

Western blot//Knockdown

Both p21cip1 and p53 were up-regulated in HCT116-Cas9 cells after ASF1a knockdown.

Human

L

cellular senescence

30,692,519

Gene

0

1

0

1

0

OPTN

10133

protein coding

RTEC

Kidney

Aging

Prevent

SA--gal activity assay//Western blot

Overexpression of OPTN increased mitophagosome formation under HG stimulation , while prevented features of cellular senescence .

--

Human

L

cellular senescence

29,367,621

Gene

0

1

0

1

0

POLB

5423

protein coding

MEF

--

Aging

Prevent

SA--gal activity assay//Western blot

In the absence of HU or other DNA damaging agents, we find robust SA-¦Â-Gal staining in the Polbcells, a much greater senescence response due to loss of Polb than we had anticipated .In addition to significant increase in SA-¦Â-gal positive cells induced by loss of Polb, p16 was also significantly upregulated in the Polbcells, further supporting that senescence is induced when Polb is absent.

--

Human

L

cellular senescence

29,968,395

Gene

0

1

0

1

0

CARF

79800

protein coding

Fibroblast

--

Aging

Accelerate

Autofluorescence//SA--gal activity assay

We examined senescence specific markers in control and CARF-overexpressing cells and found that consistent with senescence-like morphology and loss of proliferation, the CARF-overexpressing cells showed autofluorescence, senescence-specific -gal staining and enhanced levels of p53 protein and its downstream effector, p21WAF1.

--

p53

Activation

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

We have found that CARF is up-regulated in senescent fibroblasts and showed a strong correlation with ¦Â-gal activity as well as increase in p53 activity and p21WAF1 levels, established molecular markers of senescence .

Human

L

cellular senescence

19,001,376

Gene

0

1

0

1

0

CD40LG

959

protein coding

A549,H460

--

Lung cancer

Prevent

EdU assay//SA--gal activity assay

Results showed a greater number of positively stained CD40L-WT/CD40L-M cells than control cells in A549/TR, A549/DDP, and H460 cells.We then explored the effects of CD40L-WT/CD40L-M overexpression on DNA synthesis and cell proliferation, which are characteristics of cell senescence. The results showed that overexpression of CD40L-WT/CD40L-M inhibited DNA synthesis and cell proliferation.

--

NF-¦ÊB

--

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

CD40L-WT or CD40L-M-overexpressed cells showed nuclear translocation of NF-¦ÊB p65.To address the functional significanof NF-¦ÊB in the senescence program of CD40L-M-overexpressing NSCLC cells, transfected cells were treated with the NF-¦ÊB inhibitor Bay11-7082 . Results of this experiment showed a decrease in the expression of p53. Further, NF-¦ÊB signaling was inhibited via p65 siRNA, and the protein levels of p53 and p21 were attenuated. SA-¦Â-gal staining was also detected.

Human

L

cellular senescence

30,078,020

Gene

0

1

0

1

0

NSUN2

54888

protein coding

HUVEC

--

Aging

Accelerate

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

Exposure of HUVECs to high glucose increased the levels of SHC, TP53, p16, and p-p38, the levels of cellular ROS, the percentage of G1 cells, and SA-¦Â-gal activity, while knockdown of NSUN2 mitigated all of these effects.

SHC

Upregulation

Western blot//Knockdown

Overexpression of NSUN2 increased the levels of SHC proteins p66SHC, p52SHC, and p46SHC (~3.9-, ~5.6-, and ~3.8-fold, respectively), while NSUN2 knockdown decreased the same (by ~70%, ~80%, and ~80%, respectively).

--

Human

HL

cellular senescence

26,992,231

Gene

0

1

0

1

0

1

0

MEF2A

4205

protein coding

VSMC

--

Aging

Accelerate

Knockdown//SA--gal activity assay

Our results showed that over-expression of MEF2A significantly enhanced senescence compared with the cells in the EGFP group. Consistently, MEF2A knockdown inhibited VSMC senescence.

miR-143

Upregulation

Knockdown//qRT-PCR

After viral transfection for 24 h, we performed RT-qPCR analysis to confirm MEF2A overexpression in VSMCs with a corresponding increase in miR-143 expression compared to cells transduced with EGFP. Consistently, MEF2A knockdown in VSMCs resulted in down-regulated miR-143.

--

Human

L

cellular senescence

25,655,189

Gene

0

1

0

1

0

CCN2

1490

protein coding

Human airway epithelial cell

--

Aging

Accelerate

Cell morphological analysis//SA--gal activity assay//qRT-PCR

CTGF overexpression markedly reduced cell growth, and this arrest in growth was associated with the cells, showing an enlarged morphology and an increase in (SA)-¦Â-gal activity, a marker of senescence.

--

Human

L

cellular senescence

28,026,993

Gene

0

1

0

1

0

1

0

NR0B2

8431

protein coding

CNE-1,CNE-2

--

Nasopharyngeal carcinoma

Prevent

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

Morphologically, CNE-2 and CNE-2-empty vector cells were vacuolated, flattened and much larger in size compared with CNE-2 SHP-1 overexpression cells.¦Â-galactosidase staining revealed higher senescence in CNE-1 SHP-1 shRNA cells compared with CNE-1-scramble shRNA cells (23.6?¡À?3.4 % vs. 11.4?¡À?1.8 %, P?<?0.001), and lower senescence in CNE-2 SHP-1 overexpression cells compared with CNE-2-empty vector cells (3.6?¡À?2.7 % vs. 13.2?¡À?3.3 %, P?=?0.001.Compared with CNE-2-empty vector cells, CNE-2 SHP-1 overexpression cells had lower proportions of cells in G1 (55.7?¡À?2.6 % vs. 71.8?¡À?2.9 %, P?<?0.001) and G2/M (4.7?¡À?0.8 % vs. 8.18?¡À?1.3 %, P <?0.001) phases, and a higher proportion of cells in S phase (39.7?¡À?2.2 % vs. 20.1?¡À?2.9 %, P?=?0.001).

p16//pRb

Downregulation//Upregulation

Western blot

On the other hand, compared with CNE-2-empty vector cells, CNE-2 SHP-1 overexpression cells showed decreased expression of p16 (?95 %, P?<?0.001), and increased expressions of Rb (+358 %, P?<?0.001) and pRb (+248 %, P?<?0.001).

--

Human

L

cellular senescence

26,215,037

Gene

0

1

0

1

0

1

0

WNT9A

7483

protein coding

Mouse renal tubular cell

Kidney

Aging

Accelerate

Immunostaining//SA--gal activity assay

Immunostaining results indicated that in vivo expression of exogenous Wnt9a significantly aggravated the IRI-induced increase in p16INK4A and TGF-¦Â1 expression and SA¨C¦Â-gal activity in tubules.In primary cultured mouse renal tubular cells, incubation with recombinant Wnt9a triggered the upregulation of SA¨C¦Â-gal activity.

--

Human

L

cellular senescence

29,440,280

Gene

0

1

0

1

0

IFNG

3458

protein coding

HeLa

--

Aging

Accelerate

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

The number of DDR foci gradually increased during the treatment, accompanied by activation of the DDR kinase Chk2 (phosphorylated on threonine 68) and serine15-phosphorylation of p53, an established target of DDR kinases such as ATM. Cell proliferation was impaired, as judged from diminished numbers of BrdU-incorporating cells, with concomitant development of premature senescence-like phenotype including cell spreading, nuclear enlargement, SA-¦Â-gal and elevated cdk inhibitors p15INK4B (p15) and p21Cip1. Enhanced cyclin A, decreased PLK-1 and cyclin B at day 14 indicated a predominant G2-phase delay/arrest. Senescence-associated promyelocytic leukemia (PML) nuclear bodies were also increased, consistent with the recognized role of IFNs in stimulation of PML expression .

NOX4//ANT2

--//Downregulation

Western blot//Flow cytometry

Indeed, a 24 h treatment of HeLa cells with IFN¦Ã (100 U/ml) transiently activated STAT1 (not shown)and induced TGF¦Â secretion, SMAD2 phosphorylation, Nox1 and Nox4 expression and induction, ROS production, and the DDR and inhibited cell proliferation, all detected at day 6 after a single dose of IFN¦Ã. This multifaceted esponse indicated that even a short-term exposure of cells to IFN¦Ã is sufficient to trigger a cascade of events leading to cellular senescence. Downregulation of ANT2 in IFN¦Ã-treated cells led to increased proportion of apoptotic cells compared with IFN¦Ã treatment alone, indicating the contribution of ANT2 suppression to increased genotoxic stress.

TGF¦Â-SMAD

Activation

Western blot

TGF¦Â signaling pathways detected as phosphorylated SMAD2/SMAD3 were activated by IFN¦Ã.

Human

L

cellular senescence

25,982,278

Gene

0

1

0

1

0

1

0

1

0

RNF114

55905

protein coding

MEF,IMR-90,MCF-7

--

Aging

Prevent

SA--gal activity assay//Western blot

Intriguingly, we found that ZNF313 depletion triggers cellular senescence in various human cancer cells . Moreover, ZNF313 expression was substantially lower in senescent versus young cells and its level correlated inversely with the replicative senescence of mouse embryonic fibroblast (MEF) and human fetal lung fibroblast (IMR90). It was also observed that adriamycin-induced senescence of MCF7 cells is accompanied with ZNF313 reduction and that basal senescence is suppressed and stimulated by elevation and depletion of ZNF313, respectively . Similarly, adriamycin- or ¦Ã-IR-induced senescence of HCT116 cells was down- and upregulated by WT-ZNF313 and siZNF313, respectively. ZNF313 inhibited basal or adriamycin-induced senescence in the presence or absence of XAF1 expression.

p21

--

Western blot

ZNF313 depletion resulted in a marked increase in p21WAF1 protein stability. ZNF313 depletion attenuated p21WAF1 ubiquitination.

--

Human

L

cellular senescence

23,645,206

Gene

0

1

0

1

0

CSNK2A1

1457

protein coding

HCT116

--

Aging

Prevent

Knockdown//SA--gal activity assay

Transfection of CKII a siRNA markedly increased SA- ¦Â-gal staining, whereas rapamycin, vitamin C, and vitamin E treatment suppressed SA- ¦Â-gal staining .

--

PI3K-Akt-mTOR-ROS

--

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

We found that triciribine reduced the rate of CKII inhibition induced senescence.Consistent with this, we observed that triciribine completely abolished CKII downregulation induced overexpression of p53 and p21 Cip1/WAF1.In addition, triciribine reduced phosphorylation of mTOR (S2448) and AKT (S473), suggesting that AKT acts as a positive regulator of mTOR in cells made senescent by CKII inhibition. Furthermore, overexpression of CKII a clearly suppressed phosphorylat ion of AKT (473) as well as mTOR (2448) . Finally, incubation with triciribine reduced production of hydrogen peroxide and superoxide anion in CKII a-downregulated cells. co-treatment of cells with wortmannin dramaticall y reduced the rate of SA- ¦Â-gal staining . Whereas p53 and p21 Cip1/WAF1 expression levels were elevated in DRB-treated cells compared with to control cells, wortmannin apparentl y downregulated the expression of these proteins.Consistent with this, wortmannin almost completely blocked activation of p53 and p21 Cip1/WAF1 in cells transfected with CKIIa siRNA wortmannin attenuated phosphorylation of mTOR (S2448), p70S6K, and AKT (S473), suggesting that PI3K acted as a positive regulator of mTOR in cells made senescent by CKII inhibition. CKII a knockdown significantly increased hydrogen peroxide and superoxide anion levels relative to control cells, as indicated by green and red fluorescence.incubation with wortmannin apparently prevented green and red fluorescence in CKII adownregulated cells.wortmannin had no effect on the catalytic activity of CKII in cells, indicating that CKII is an upstream regulator of PI3K .Western blot analysis revealed that overexpression of CKII a reduced phosphorylation of both mTOR (2448) and p70S6K by 60% compared to control cells.DRB significantly increased hydrogen peroxide and superoxide anion levels relative to untreated control cells, as indicated by green and red fluorescence. However, incubation with rapamycin, vitamin C, and vitamin E apparently prevented both fluorescent signals in DRB-treated cells. Vitamin C and vitamin E are well known ROS scavenger s, whereas rapamyci n is an mTOR inhibitor.

Human

L

cellular senescence

23,523,798

Gene

0

1

0

1

0

IFNB1

3456

protein coding

LXSN,K16,K38,ME-180,Caski,HeLa,SiHa

--

Aging

Accelerate

SA--gal activity assay

Increasingly high percentages of senescent cells were observed exclusively in K38 cells transformed by E6 and E7 proteins of cutaneous HPV genotype, starting from 4 days of IFN-¦Â treatment, compared to control keratinocytes (LXSN), K16 cells and mucosal high risk HPV-positive cell lines.

PML//p21//p53

Upregulation//Upregulation//Activation

Western blot//qRT-PCR

The expression of HPV38 E6 and E7 in human keratinocytes induces the stabilization of p53, as shown by WB analysis of p53 in K38 cells compared with control keratinocytes (LXSN), K16 cells and high risk HPV-positive cell lines SiHA and ME-180. Three different siRNAs were used for PML, p53 and p21 genes. Upon IFN-¦Â treatment, PML was up-regulated as well as p21. PML seems to be an essential component of senescence response in K38 cells, since, when PML expression is inhibited by specific siRNAs, IFN-¦Â-induced senescence is strongly reduced. p21 silencing partially affects IFN-¦Â-induced senescence, while p53 silencing appears much more effective, IFN-¦Â modulates p53 phosphorylation status at different phosphorylation sites while acetylation is mainly downregulated in Lys-320.In fact, when PML expression is silenced, DNp73 protein levels are not downregulated by IFN-¦Â. The DNp73 protein expression appears to be reduced upon IFN-¦Â treatment, probably as a result of the p53 post-translational modifications induced by IFN-¦Â.In fact, when PML expression is silenced, DNp73 protein levels are not downregulated by IFN-¦Â.PML depletion reduces IFN-¦Â induction of Bax and Pig3 in K-38 cells, indicating the role of PML in the ability of IFN-¦Â to recover p53 transactivation activity of specific target genes.

--

Human

HL

cellular senescence

22,615,843

Gene

0

1

0

POU5F1

5460

protein coding

PA-1

--

Embryonal carcinoma

Accelerate

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

OCT4A-silenced cells underwent a profound G2M arrest with apoptosis being greatly reduced. The cells displayed the large, flattened morphology and Sa-¦Â-gal staining associated with senescence on day 5, identical to that previously reported when silencing p53. Prolonged loss of OCT4A lead to the polyploidisation of these senescent cells and a reduction in clonal recovery.

p21

Downregulation

Western blot//Knockdown

Efficient (80¨C95%) knock-down of OCT4A was confirmed with 3 different siRNA, in addition to a 3 siRNA pool and the molecular response to ETO was then examined by immunoblotting for key regulators of DNA damage, (pCHK2, p53, p21Cip1, and RAD51).These experiments revealed p53, RAD51 and pCHK2 were all upregulated from day 1 independently of OCT4A expression. p21Cip1 expression rose gradually from day 1 throughout the course of experiment as seen in previous experiments. Silencing of OCT4A resulted in a marked increase in p21Cip1 expression from day 1.

--

Human

L

cellular senescence

26,102,294

Gene

0

1

0

1

0

1

0

KRAS

3845

protein coding

--

Gills,bone,skin,gut,liver,heart,brain

Aging

Accelerate

Immunofluorescence//SA--gal activity assay

The increase in cell proliferation in the kidney and liver of CS-like fish leads to a net expansion in organ size. We also detected an increased number of SA-¦Âgal-positive cells in the brain, heart, liver and bone of CS-like fish. In CS-like fish, but not in wild-type controls, distinct nuclear foci containing ¦ÃH2AX and pAtm were detected in various tissues. Strikingly, we noticed that the nuclei that stained for ¦ÃH2AX and pAtm in the brain and heart of CS-like fish are localized in specific regions and throughout the heart ventricle, corresponding to areas enriched with adult proliferating cells (Adolf et al., 2006; Grandel et al., 2006; Poss, 2007). There was a clear inverse correlation between the decrease in BrdU+ cells and the increase of SA-¦Âgal and DDR markers in the brain and heart of CS-like fish .

tp53

--

RT-PCR

We found an increase in the expression of the tp53 target gene cdkn1a/p21 in RNA extracted from whole CS-like fish and from specific tissues, and tissue sections processed for in situ hybridization, from CS-like fish.

--

Human

L

cellular senescence

19,132,118

Gene

0

1

0

1

0

PPARD

5467

protein coding

Human primary keratinocyte

--

Aging

Prevent

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

This increase, however, was significantly suppressed in the presence of GW501516, suggesting the involvement of PPAR¦Ä in the inhibition of UVB-induced premature senescence .A marked increase in the levels of p53 and p21 was observed in keratinocytes exposed to UVB, whereas pre-treatment with either GW501516 or NAC diminished the effect of UVB on these proteins.The level of PPAR¦Ä in keratinocytes was markedly reduced upon transfection with PPAR¦Ä siRNA in the presence of GW501516 and/or UVB radiation, whereas control siRNA, consisting of a pool of non-specific sequences, had no effect on PPAR¦Ä levels. As expected, the siRNA-mediated down-regulation of PPAR¦Ä significantly suppressed the GW501516-mediated inhibition of premature senescence and ¦Ã-H2A.X foci of keratinocytes induced by UVB radiation.

PTEN

Upregulation

Western blot//Fluorescence microscopy

Treatment with GW501516 markedly increased the levels of both PTEN transcript and protein in a time-dependent manner. The high levels of PTEN mRNA and protein observed 24 h after treatment with GW501516 are consistent with the observation that pre-treatment with GW501516 for 24 h, but not for 30 min, significantly suppressed the UVB-induced phosphorylation of Akt. The up-regulation of PTEN by GW501516 was suppressed in the presence of siRNA against PPAR¦Ä, suggesting that PPAR¦Ä has a causal role in the up-regulation of PTEN.Although UVB radiation significantly increased ROS generation, pre-treatment with GW501516 for 24 h significantly suppressed the effect of UVB radiation . The reduction in ROS generation in response to GW501516 was recovered in cells transfected with PPAR¦Ä siRNA, suggesting a PPAR¦Ä-dependent effect of GW501516 on ROS production.

PI3K-Akt-Rac1

Downregulation

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

UVB-induced ROS production was significantly reduced in the presence of LY294002, a PI3K (phosphatidylinositol 3-kinase) inhibitor, but not in the presence of GF109203X, a PKC (protein kinase C) inhibitor. The effect of LY294002 was further augmented in cells incubated with GW501516. Prior incubation with DPI, a non-specific inhibitor of NADPH oxidase, significantly attenuated UVB-induced ROS production in keratinocytes. In addition, LY294002 reduced the percentage of SA ¦Â-gal-positive cells following UVB irradiation to the same extent as did GW501516. Markedly reduced in the presence of GW501516 and almost completely abolished in the presence of both GW501516 and LY294002 . Down-regulation of PPAR¦Ä by siRNA reversed the GW501516-induced decrease in Akt phosphorylation in cells exposed to UVB.Although UVB irradiation caused the rapid translocation of Rac1 to the cell membrane, the addition of GW501516 and/or LY294002 almost completely abolished the UVB-mediated translocation of Rac1. Consistent with these results, immunoblot analysis using Rac1-specific antibody revealed that Rac1 accumulated in the membrane fraction following UVB irradiation and inhibition of UVB-induced membrane translocation by both GW501516 and LY294002.UVB irradiation activated Rac1, whereas treatment with either GW501516 or LY294002 suppressed UVB-induced activation of Rac1.

Human

L

cellular senescence

22,335,598

Gene

0

1

0

1

0

1

0

MYC

4609

protein coding

--

Pancreas

Aging

Accelerate

Immunohistochemistry//RT-qPCR

We confirmed that the expression of SASP factors in i4F;Arfnull pancreas was increased to similar levels as those observed in i4F pancreas upon OSKM activation.

INK4a//p21//p53//IL-6

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

SA-¦Â-gal activity assay//Immunohistochemistry

In parallel to this, i4F;Ink4a-null pancreas presented limited senescence after OSKM activation, and little expression of senescence-associated genes like Arf, p53, and p21 as well as Il6 and other factors involved in the SASP, such as Tnf, Il1a, Il1b, Il1rn, Mmp3, and Pai1. We also observed an increased accumulation of reprogrammed (NANOG+) and senescent (SA¦ÂG+) cells in the pancreas of i4F;p21-null mice compared to i4F controls. In agreement, the levels of SASP factors, such as Il6, Tnf, and Pai1, presented the same tendency, that is, i4F<i4F;p21-null. We next evaluated the accumulation of senescent cells and observed that it was decreased i4F;p53-null;Ink4a/Arf-null pancreas compared to i4F;p53-null pancreas, reinforcing the importance of Ink4a in the induction of OSKM-driven senescence (Mosteiro et al., 2016).We observed that after 7 days of doxycycline treatment (0.2 mg/ml), the pancreatic dysplasia in i4F;Il6mutant mice was much reduced compared to their i4F counterparts. In this genetic background and at the time point analyzed, the number of NANOG+ cells was too low to assess differences between genotypes , but we observed a suggestive reduction in the levels of senescent cells in the pancreas together with a residual induction of Ink4a, Arf, and several SASP factors, such as Tnf, Il1a.

--

Human

L

cellular senescence

29,280,266

Gene

0

1

0

1

0

HRAS

3265

protein coding

IMR-90

--

Aging

Accelerate

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

HRasV12 expressing IMR90 cells display the typical hallmarks of a senescent cell, including persistent cell growth arrest, increased activity of senescence-associated ¦Â-galactosidase (SA-¦Â-gal) (4.7% in control vs 79.6% in Ras-treated group, P<0.05 ), expression of cell-cycle inhibitors, and levels of the senescence marker H3K9me3, as well as persistent DDR. OIS-induced senescence also significantly increased the mRNA levels of SASP-associated genes by at least 19.7-fold (Illustrated by the case of CXCL6. P<0.05) compared with the control group .

--

Human

HL

cellular senescence

28,247,536

Gene

0

1

0

1

0

1

0

1

0

G6PD

2539

protein coding

HFF-3,HFF-1

--

Aging

Prevent

SA--gal activity assay

HFF1 but not HFF3 cells showed intense staining for SA-¦Â-Gal at PDL 50.There was a significant increase in the percentage of SA-¦Â-Gal-positive HFF1 cells upon serial passage. In comparison, such increase was modest for HFF3 cells .Here we showed that the intracellular NADPH/NADP+ ratio of HFF1 cells decreased by around 50% as they were passaged from PDL 12 to 50.In contrast, the NADPH/ NADP+ ratio of HFF3 cells, which was about 50% higher than that of HFF1 at PDL 12, diminished only slightly. The change in NADPH/NADP+ ratio paralleled that of intracellular G6PD activity.The G6PD activity in HFF1 cells decreased significantly with the increasing PDL, whereas that of HFF3 cells was only slightly reduced. The level of 8-OHdG, an oxidative DNA damage marker, in HFF1 cells increased significantly with PDL.

--

Human

L

cellular senescence

14,980,702

Gene

0

1

0

PML

5371

protein coding

MEF

--

Aging

Accelerate

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

The frequency of SA-¦Â-gal+ cells was markedly lower in Ras-expressing PMLMEFs than in controls.Morphologically, they became flat and enlarged and were strongly positive for SA-¦Â-gal. Although PML and Ras induced equivalent levels of p53 and p21CIP1, PML did not influence p16 expression.

p53

Upregulation

Western blot

Expression of oncogenic Ras or PML induced a modest, yet reproducible, increase in acetylation of p53 at K382, but not K320 (data not shown).

--

Human

L

cellular senescence

10,910,364

Gene

0

1

0

1

0

1

0

TP53BP2

7159

protein coding

MEF

--

Aging

Accelerate

SA--gal activity assay

Senescence associated-¦Â-galactosidase (SA-¦Â-gal) activity was detected in around 30% or 41% of cells infected with retroviruses expressing ASPP2 (1¨C1,128) or ASPP2(1¨C360), respectively, whereas SA¦Â-gal activity was not detected in ASPP2(123¨C1,128)-infected cells. Moreover, rapamycin induced long-lived protein degradation under all conditions, but the most profound increase of about 17-fold was observed in HRAS V12-expressing ASPP2.

--

Human

L

cellular senescence

22,847,423

Gene

0

1

0

STMN1

3925

protein coding

IMR-90

--

Aging

Prevent

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

Microscopic observation of the stathmin-depleted IMR-90 cells revealed enlarged and flattened cells, suggesting that they might be undergoing senescence. Compared to the control-shRNA treated cells, stathmin-shRNA treated and hydrogen peroxide-treated cells showed blue staining with SA-¦Â-gal. Over 80% percent of stathmin-depleted IMR-90 cells stained positive, whereas only 6% of hydrogen peroxide-treated cells were stained .Levels of Ser 15 phosphorylated as well as total p53 were much higher in stathmin-depleted IMR-90 cells when compared with the control shRNA-treated cells . Phosphorylated Rb at Ser 780 was barely detected in stathmin-depleted IMR-90 cells, while it was easily detected in control shRNA-treated cells. The amount of total Rb was similar in positive control, control and stathmin shRNA-treated cells.

--

Human

L

cellular senescence

28,885,720

Gene

0

1

0

1

0

1

0

1

0

IL1RN

3557

protein coding

MEF,Primary MEF,LNCaP

--

Cancer

Prevent

SA--gal activity assay

Remarkably, treatment with IL-1RA decreased both SA-¦Â-gal stainingand levels of the tumour suppressor protein p53 in Pten2/2 cells.Importantly, IL-1RA also blocked docetaxel-induced senescence in human prostate cancer cells.

--

Human

HL

cellular senescence

25,156,255

Gene

0

1

0

PGR

5241

protein coding

ES©\2

--

Ovarian cancer

Accelerate

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

PH 6.49 GFP-PR cells exposed to R5020 for 4 d significantly induced SA¦ÂGal (40%) relative to minimal SA¦ÂGal (10%) in empty vector and vehicle-treated cellsc;ell cycle analysis of GFP-PR cells by propidium iodide staining for DNA content demonstrated a significant increase in the percentage of cells in the G0/G1?phase accompanied with a decrease in the percentage of cells in the S phase of the cell cycle following 4 d of ligand exposure relative to vehicle controls.

p21//FOXO1

Upregulation//--

qRT-PCR//Western blot//Knockdown//CHIP

Notably, in the presence of progestin, p21 exhibited significantly increased mRNA and protein expression;p21 mRNA levels were greatly diminished in cells expressing sh-FOXO1 relative to sh-controls in both vehicle- and R5020-treated conditions.

--

Human

HL

cellular senescence

23,574,718

Gene

0

1

0

1

0

1

0

YAP1

10413

protein coding

PDLSC

--

Aging

Prevent

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

EdU result showed that the proliferation rate of shYAP reduced markedly after transfection compared with NC group (P<0.01)£»The percentage of early and late apoptotic cells in shYAP group were significantly increased after YAP knockdown compared with NC group (p<0.001)£»compared with NC group, the proportion of cells in G0/G1 phase increased evidently (P < 0.05), while that in S and G2/M phase decreased (P < 0.05) in shYAP group£»shYAP group showed higher senescence cells rate compared with NC group (P<0.01).

--

ERK//Bcl-2

Upregulation//Downregulation

Knockdown//Western blot

The expression of p-C-Raf338 and p-MEK, which take part in the phosphorylation of Erk, was inhibited when YAP was knocked down; Bcl-2 family members (Bak, Bax, Bad, Bid and Bik), their expression levels increased separately after knocking down YAP.

Human

L

cellular senescence

29,104,479

Gene

0

1

0

1

0

1

0

1

0

1

0