Venkatachalam/aging_reg_dataset · Datasets at Hugging Face

CIB1

10519

protein coding

MCF-7

--

Aging

Prevent

Knockdown//SA--gal activity assay

About 65% of KIP knockdown cells were stained intensely for senescence associated ¦Â-galactosidase (SA-¦Â-Gal) activity and exhibited a senescent phenotype at day 15.In contrast, the growth rates of two independent KIP knockdown cells gradually slowed down and almost stopped dividing at ~5 PD.

TRF2

Binding

Pull-down assay

To verify the direct interaction between TRF2 and KIP , we performed GST pulldown experiments. GST-KIP , but not the control GST, bound to FLAG-TRF2 expressed in MCF7 cells, indicating that KIP interacts with TRF2 in vitro.

--

Human

HL

cellular senescence

25,012,820

Gene

0

1

0

1

0

PPIB

5479

protein coding

U251

--

Glioblastoma

Prevent

Knockdown//SA--gal activity assay

As suggested by these findings, although the majority of U251 cells died upon CypB knockdown, surviving cells showed microscopic changes characteristic of senescence, including large and flat morphology, binuclear cells, and massive vacuolization. Furthermore, CypB knockdown in U87 cells markedly increased SA-¦Â-gal staining.Overexpression of oncogenic Ras (HRASV12) in GBM cells evoked similar senescence-associated morphological and molecular changes.

p27

--

Knockdown//Western blot

CypB knockdown or inhibition also increased levels of the cell cycle inhibitor p27/Kip1 (24) and strongly reduced the p27-specific E3 ubiquitin ligase Skp2.

--

Human

HL

cellular senescence

24,272,483

Gene

0

1

0

1

0

NTN4

59277

protein coding

U251MG

--

Glioblastoma

Prevent

SA--gal activity assay

We then detected the number of senescent cells by using senescence-associated beta-galactosidase assay. We observed that silencing of either NTN4 or ITGB4 significantly induced U251MG cell senescence. Next we explored the effects of exogenous recombinant NTN4 on TMZ induced senescence. NTN4 significantly inhibited U251MG cell senescence induced by TMZ.

ITGB4

--

SA-¦Â-gal activity assay

Although NTN4 reduced the senescence rate of wild type or non-targeting control U251MG cells induced by TMZ treatment,NTN4 did not affect ITGB4-silenced U251MG cells.This result indicates that ITGB4 mediates the protective effect of NTN4 on TMZ treated glioblastoma cells.

--

Human

L

cellular senescence

24,265,816

Gene

0

1

0

ITGB4

3691

protein coding

U251MG,U-118 MG,T98G,U87 MG

--

Glioblastoma

Prevent

Knockdown//SA--gal activity assay

We observed that silencing of either NTN4 or ITGB4 significantly induced U251MG cell senescence.Similar results were obtained by silencing the expression of either NTN4 or ITGB4 in U118MG, T98G and U87MG cells.

--

Human

L

cellular senescence

24,265,816

Gene

0

1

0

1

0

SORBS2

8470

protein coding

Primary human keratinocyte

--

Aging

Accelerate

SA--gal activity assay

The highest number of senescent cells was observed for SORBS2-2 fourteen days after transduction.The characteristic perinuclear ¦Â-galactosidase staining pattern of enlarged and flattened cells was observed in case of fibroblasts.In contrast, the morphology of senescing keratinocytes differed in that both enlarged and rounded cells with an overall blue stain were observed. SORBS2-2 and TLR3 could induce senescence in both cell types.

--

Human

HL

cellular senescence

24,165,198

Gene

0

1

0

TLR3

7098

protein coding

Primary human Fibroblast

--

Aging

Accelerate

SA--gal activity assay

The highest number of senescent cells was observed for SORBS2-2 fourteen days after transduction.The characteristic perinuclear ¦Â-galactosidase staining pattern of enlarged and flattened cells was observed in case of fibroblasts.In contrast, the morphology of senescing keratinocytes differed in that both enlarged and rounded cells with an overall blue stain were observed. SORBS2-2 and TLR3 could induce senescence in both cell types.

--

Human

HL

cellular senescence

24,165,198

Gene

0

1

0

HDAC7

51564

protein coding

HeLa,MCF-7

--

Cancer

Prevent

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

HDAC7 knockdown by all the three pairs of oligos significantly decreased the proportion of cells in S phase and increased G0/G1 cell proportion, indicating a G1/S cell cycle arrest. Congruent with the above findings, HDAC7-silenced cells revealed a significant loss of BrdU labeling (16.9%) compared with the control cells (33.2% of BrdU incorporation), suggesting a reduced entry into S phase.HDAC7 silencing markedly elevated the SA-¦Â-Gal activity in HeLa cells (from 0.9% to 16.8%) and MCF-7 cells (from 7.8% to 27.1%).

c-Myc//p21//p27

--//--//--

Western blot

Immunoblot analysis showed that the expression level of c-Myc was remarkably suppressed by HDAC7 silencing, while p21 and p27 levels were drastically upregulated.

--

Human

L

cellular senescence

21,120,446

Gene

0

1

0

1

0

1

0

1

0

IRF5

3663

protein coding

MDAH087-1

--

Aging

Accelerate

SA--gal activity assay

IRF5 or IRF7, induced an additional 5% senescence-associated ¦Â-gal staining over background. The combination of IRF5 with IRF7 was able to induce senescence-associated ¦Â-gal activity in 19.3% of the cells, which is equivalent to that observed in 5-aza-dC-treated immortal MDAH041 cultures. Similar results were observed in IRF5 + IRF7-overexpressing MDAH087-1 cells.

--

Human

HL

cellular senescence

18,505,922

Gene

0

1

0

IRF7

3665

protein coding

MDAH087-1

--

Aging

Accelerate

SA--gal activity assay

IRF5 or IRF7, induced an additional 5% senescence-associated ¦Â-gal staining over background. The combination of IRF5 with IRF7 was able to induce senescence-associated ¦Â-gal activity in 19.3% of the cells, which is equivalent to that observed in 5-aza-dC-treated immortal MDAH041 cultures. Similar results were observed in IRF5 + IRF7-overexpressing MDAH087-1 cells.

--

Human

HL

cellular senescence

18,505,922

Gene

0

1

0

DEK

7913

protein coding

HeLa

--

Aging

Prevent

SA--gal activity assay

Whereas all of the AdE2ts-infected control cells incubated at the permissive temperature exhibited the characteristically senescent morphology, DEK expression resulted in a mixture of cells with senescent as well as nonsenescent features, indicating partial repression of the E2 senescence phenotype.In agreement with these morphological findings, over 70% of the control cell population were positive for the senescence-specific SA¨C¦Â-Gal marker at 32¡ãC, with a reduction to 15% upon DEK overexpression.

--

Human

HL

cellular senescence

16,254,365

Gene

0

1

0

ACACA

31

protein coding

Human primary Fibroblast

--

Aging

Prevent

BrdU assay//SA--gal activity assay

The incorporation of [methyl-3H] thymidine into DNA, normalized to total cell protein, was significantly reduced,as was the percentage of cell nuclei that incorporated BrdU. Besides, a nearly three-fold increase in SA-¦Â-Gal positive cells was observed after silencing of ACC1 gene expression.

p38MAPK

--

Western blot

p38 MAPK phosphorylation was stimulated by ACC1 silencing, while the overall expression of the kinase was not affected . As such, the ratio between the phosphorylated enzyme and total enzyme was more than three-fold higher in ACC1 shRNA treated cells than in the control.

--

Human

L

cellular senescence

27,983,949

Gene

0

1

0

1

0

S100A6

6277

protein coding

HUVEC

--

Aging

Prevent

Knockdown//SA--gal activity assay

Compared with control cells treated with scrambled siRNA,S100A6-depleted cells appeared larger and more "fluffy". Analysis showed that these larger, "fluffy" HUVECs exhibited blue staining characteristic of ¦Â-galactosidase expression, with a higher incidence of blue cells occurring upon depletion of S100A6. Quantification revealed an increase in ¦Â-galactosidase levels of greater than four-fold upon S100A6 depletion.

CDK1

--

Western blot

Depletion of endothelial S100A6 caused a decrease in both CDK1 and phospho-CDK1 levels in subconfluent HUVECs,Importantly, in HUVECs, depletion of the S100A6 levels by S100A6 siRNA#3 caused a decrease of almost 50% in the levels of CDK1 mRNA.

--

Human

HL

cellular senescence

23,095,053

Gene

0

1

0

1

0

CARM1

10498

protein coding

Human bronchial epithelial cell

--

Chronic obstructive pulmonary disease

Prevent

Knockdown//SA--gal activity assay

There was a significant increase in the number of beta-galactosidase positive cells in the airways of NA-treated Carm1+/- mice compared to NA-treated WT animals (31.8 ¡À 3.4% vs 8.2 ¡À 2.8%, p<0.001). Consistent with mouse airway epithelial cells, the siCARM1-transfected 337 human cells showed a significantly higher percentage of beta-galact osidase-positive cells compared with 338 scrambled controls (13.2 ¡À 0.6% vs 3.7 ¡À 0.6%, p<0.001).

SIRT1//p53

--//--

Western blot//qRT-PCR

Moreover, western blot analysis of whole lung homogenates from d14 mice treated with NA revealed a clear reduction in the level of SIRT1 protein in Carm1+/- mice compared to WT controls, with a concomitant increase in p53 levels .Mechanistically, CARM1-silenced cells demonstrated reduced expression of SIRT1, and treating CARM1-silenced cells with the SIRT1 activator resveratrol, reversed the impaired wound healing.

--

Human

HL

cellular senescence

31,461,302

Gene

0

1

0

1

0

KDM3A

55818

protein coding

OVCAR-5,A2780

--

Ovarian cancer

Prevent

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

Interestingly,KDM3A depletion led to G2/M cell cycle arrest, which was accompanied by increased apoptosis,Since we observed that growth arrest was accompanied by the presence of large, flat and vacuolated cells in KDM3A knockdown cells,Indeed,both OVCAR-5/CDDP and A2780/CDDP cells expressing KDM3A shRNAs showed high abundance of senescence-associated ¦Â-galactosidase positive cells as compared with scrambled control cells,indicating that KDM3A prevents the cellular senescence to promote sustained growth of ovarian cancer.

--

Human

HL

cellular senescence

27,694,900

Gene

0

1

0

1

0

1

0

CLU

1191

protein coding

IMR-90

--

Aging

Prevent

Knockdown//SA--gal activity assay

Enhanced senescence was confirmed by significant differences in the SA-¦Â-gal+ cells, where at least two-fold increases were noted compared to untreated pTRIPZ-shCLU IMR-90 cells that expressed sCLU at day 39. Dox-inducible non-silencing shRNA had no affect on sCLU expression, both population doubling times and %SA-¦Â-gal+ cells were not significantly different with or without Dox exposure over the life of the IMR-90 cultures.

ATM

--

Western blot

AT2052 cells expressed significantly lower basal level expression of sCLU compared to wild-type ATM+ IMR-90 cells.

IGF-1R-MAPK-ERK1/2-Egr-1

--

ELISA//Western blot//TUNEL assay

Indeed, IGF-1 expression was approximately two-fold higher in conditioned media from senescent compared to young IMR-90 cells. Analyses of differential signal transduction responses in senescent versus young IMR-90 cells showed that IGF-1 receptor (IGF-1R) levels were activated (elevated P-IGF-1R/tIGF-1R) in senescent cells; antibodies to human IGF-1R are not ideal. Src and Erk-1/2 kinase levels were also elevated in middleaged and senescent compared to young IMR-90 cells. The transcription factor, Egr-1, which binds the hCLU promoter and mediates sCLU expression, was significantly increased during senescence in IMR-90 cells. To further confirm the specificity of IGF-1/IGF-1R/MAPK/ERK-1/2/Egr-1 signaling in sCLU induction during senescence, we treated senescent IMR90 cells with the IGF-1R inhibitor, AG1024, which effectively blocked IR-induced sCLU expression and also suppressed sCLU expression in senescent IMR-90 cells in a dose-dependent manner .

Human

L

cellular senescence

24,937,130

Gene

0

1

0

1

0

ING1

3621

protein coding

Fibroblast

--

Aging

Accelerate

Cell morphological analysis//SA--gal activity assay

Retroviral transduction with p33ING1 resulted in a change to senescence-like cell morphology, as documented by phase-contrast microscopy.We tested for expression of SA-¦Â-Gal activity in p33ING1-infected primary human fibroblasts by use of RasV12 as a control. In accordance with the previous observations, p33ING1 induces SA-¦Â-Gal activity to a extent similar to that seen with oncogenic Ras, suggesting that p33ING1 has potency similar to that of RasV12.

--

Human

L

cellular senescence

15,601,862

Gene

0

1

0

1

0

SFRP1

6422

protein coding

CSC

--

Aging

Accelerate

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

We also confirmed the up-regulation of senescence markers p16, p21, phosphorylated p53, and SA-¦Â-gal in sFRP1-treated CDCs.Importantly, sFRP1-treated CDCs presented SASP, which was characterized by up-regulation of IL-6 and IL-1b, and was consistent with anti-cancer drug-induced cellular senescence.

--

Wnt

Downregulation

Western blot

Different concentrations of recombinant human sFRP1 were administered to CDC cultures for 1 and 48 h. Recombinant sFRP1 treatment caused an increase in phosphorylated b-catenin and a decrease in phosphorylated GSK3b in young patients-derived CDCs,indicating that exogenous sFRP1 inhibited canonical Wnt signaling.

Human

L

cellular senescence

28,435,069

Gene

0

1

0

1

0

1

0

TP53

7157

protein coding

PCI-13

--

Squamous cell carcinoma

Accelerate

Colony formation assay//SA--gal activity assay

Clonogenic assays showed that the 4-Gy surviving fraction in TP53 null or mutant cells was significantly higher than in cells with wtp53. Being that senescence is the primary mechanism of radiation-induced cell death in HNSCC cells, a senescence-associated ¦Â-galactosidase (SA-¦Â-gal) staining was performed and showed that 4 days after treatment with 4-Gy ionizing radiation, cells with wtp53 had a significantly higher SA-¦Â-gal-positive staining compared with the SA-¦Â-galpositive staining in TP53 null and all TP53 mutant cells.

p21

Upregulation

qRT-PCR//Western blot

Among the mutp53 bearing cells, only PCI-13 A161S had minimal, short-lived expression of the p21 gene compared with the enhanced, long-term expression seen in wtp53 cells. All other mutp53 or TP53 null cell lines had no significant elevation in p21 mRNA. Western blot analysis demonstrated long-term expression of the p21 protein in wtp53 cells, with no expression in mutp53 and TP53 null cells, but short-term expression in PCI-13 A161S cells.

--

Human

L

cellular senescence

25,766,317

Gene

0

1

0

1

0

NDRG1

10397

protein coding

HepG2

--

Hepatocellular carcinoma

Prevent

Knockdown//SA--gal activity assay

A large percentage (about 80%) of HepG2 cells transfected with NDRG1-specific siRNA was positive for SA-¦Â-Gal staining.

--

GSK3¦Â-p53/-p16

--

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

The percentage of SA-¦Â-Gal positive cells was also reduced in HepG2 cells co-transfected with NDRG1-specific siRNA and siRNA against p53, p16, p53 & p16, or GSK-3. Western blot results indicated that co-transfection of NDRG1-specific siRNA and siRNA against p53, p16, or p53 & p16 increased p-Rb level which was decreased by NDRG1-specific siRNA . Additionally, GSK-3¦Â siRNA decreased GSK-3¦Â, GSK-3¦Â 9ser, p53, p21 and p16 levels, indicating that GSK-3¦Â can stabilize p53 and p16 levels in HepG2 cells with NDRG1 suppression. Consistently, a decrease in p21 (a p53 downstream target) and increased p-Rb were detected in cells co-treated with NDRG1-siRNA and PFT-¦Á.

Human

HL

cellular senescence

24,302,615

Gene

0

1

0

1

0

RB1

5925

protein coding

MSC

--

Aging

Prevent

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

We observed signs of senescence in shR1 as detected by in situ senescence-associated-beta-galactosidase assay, compared with cells transduced with controls shRNAs.Beta-galactosidasepositive cells showed the characteristic senescent morphology (flat, enlarged cells). Moreover, we detected an increase in the percentage of bi-nucleate cells in shR1 (data not shown), which is another hallmark of senescent cells.

p53//p27//p21

--//--//--

RT-PCR//Western blot

We detected a significant up-regulation (p < 0.05) of p53 protein in shR1 along with an increase in the level of p27kip1 and p21cip1, whereas no modification was observed in p16Ink4a. the increased expression of these genes was also observed at mRNA level.

--

Human

HL

cellular senescence

23,370,776

Gene

0

1

0

1

0

1

0

KIR2DL4

3805

protein coding

NK

--

Aging

Accelerate

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

Primary NK cells had a characteristicflattened and enlarged morphology after activation through CD158d. The average 2D cell area of NK cells activated by CD158d was 19.5 ¡À11 ¦Ìm2(n = 23), up from 6.6 ¡À2.4 ¦Ìm2(n = 22)for cells stimulated with control Ab.There was an increase in senescence-associated ¦Â-galactosidase (SA-¦Â-gal) activity, a widely used senescence biomarker.Furthermore, NK cells activated by CD158d were arrested at the G0/G1 stage of the cell cycle.

--

Human

HL

cellular senescence

23,184,984

Gene

0

1

0

1

0

1

0

NOX4

50507

protein coding

MCF12A

--

Breast cancer

Accelerate

SA--gal activity assay

We performed a SA beta-galactosidase assay on the empty vector and NOX4 overexpressing cells, and determined a higher instance of senescence in the NOX4 overexpressing cell lines when compared to the empty vector controls. An increase in senescence was observed in both breast epithelial and cancer cell lines when NOX4 was overexpressed.

--

Human

HL

cellular senescence

20,523,116

Gene

0

1

0

TNF

7124

protein coding

EPC

--

Aging

Accelerate

SA--gal activity assay

Continuous treatment with TNF-¦Á for 1 wk resulted in a dose-dependent increase in EPC senescence. There was a dose-dependent drop in the EPC population doubling rate with TNF-¦Á treatment that mirrored the observed increase in cell senescence measured by SA-¦Â-Gal staining.

p16

Upregulation

Flow cytometry

We observed a marked up-regulation of p16INK4a expression after the continuous TNF-¦Á treatment, as is shown in a representative flow cytometry histogram.

p38 MAPK

Activation

Western blot

We first demonstrated that the p38 MAPK pathway was activated by TNF-¦Á stimulation, as evidenced by phosphorylation of p38, and that the p38 MAPK inhibitor SB203580 is able to block this activation at a concentration of 10uM.

Human

L

cellular senescence

19,124,561

Gene

0

1

0

MDM2

4193

protein coding

Adipose stromal cell

--

Aging

Prevent

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

Furthermore, as expected, antagonizing MDM2 decreased the expression of HMGA2 significantly supporting the assumption that the p14Arfrepression of HMGA2 is TP53-dependent.Thus, antagonizing MDM2 can induce senescence in mesenchyme-derived stem cells. The reduced levels of HMGA2 expression are also accompanied by an increased number of ¦Â-gal positive cells. For these experiments, canine ADSCs were treated with 30 lM and 50 lM nutlin-3, respectively, for 72 hr. The number of ¦Â-gal positive cells was then determined by counting 500¨C600 cells. With both concentrations,the percentage of positive cells was drastically increased (30 lM: 54.0%; 50 lM: 60.0%) compared with the control cells (9.9%). Also, the increased percentage of positive cells was accompanied by clear morphological changes leading to a majority of flat enlarged cells with an absence of mitoses.

HMGA2

--

qRT-PCR

Furthermore, as expected, antagonizing MDM2 decreased the expression of HMGA2 significantly.

--

Human

L

cellular senescence

21,456,046

Gene

0

1

0

1

0

1

0

CCL2

6347

protein coding

UCB-MSC

--

Aging

Accelerate

Knockdown//SA--gal activity assay

Compared with the untreated control, MCP-1 treatments significantly increased SA-¦Â-gal activity with passaging by up to 8-fold, concomitantly with a considerable delay in cell growth.The use of siRNA to silence MCP-1 in UCB-MSCs significantly blocked both SA-¦Â-gal activity induction and delayed cell growth .

CCR2

--

Flow cytometry//Western blot//SA-¦Â-gal activity assay//Cell growth kinetics//Colony formation assay

MCP-1 is a pro-inflammatory cytokine that exerts its biological effects through its cognate receptor CCR2 (5,45). The expression of CCR2 in UCB-MSCs was lower in the early-phase but it was considerably upregulated in the intermediate-phase and further increased in the terminal senescent phase. Importantly, treatment with CCR2 blocking antibody significantly reduced SA-¦Â-gal expression and enhanced the growth rate and colony-forming capacity of UCB-MSCs in the intermediate-phase.

ROS-p38-MAPK-p53-p21

Activation

Western blot//Knockdown

MCP-1 treatment activated the p53-p21 signaling cascade, increasing the protein level of p53 phosphorylated at Ser392 and total p21.At the molecular level, MCP-1 KD cells showed attenuated activation of the p53-p21 pathway.Moreover, p38-MAPK, a crucial mediator of ROS-induced senescence, was activated from 8 h and peaked at 18 h after MCP-1 stimulation .Importantly, treatment with SB203580, a chemical inhibitor of p38-MAPK, significantly abrogated the increase in phosphorylated p53 and p21 proteins induced by MCP-1 stimulation.

Human

HL

cellular senescence

26,573,462

Gene

0

1

0

1

0

CCR2

729230

protein coding

UCB-MSC

--

Aging

Accelerate

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

The significance of CCR2 was further validated by the fact that CCR2 KD cells had decreased SA-¦Â-gal activity and p53-p21 pathway activation. Furthermore, overexpression of CCR2 in early-phase UCB-MSCs remarkably increased cellular senescence, as indicated by induction of SA-¦Â-gal activity , activation of the p53-p21 signaling cascade, and downregulation of BMI1 expression.

BMI1

Downregulation

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

The significance of CCR2 was further validated by the fact that CCR2 KD cells had decreased SA-¦Â-gal activity and p53-p21 pathway activation. Furthermore, overexpression of CCR2 in early-phase UCB-MSCs remarkably increased cellular senescence, as indicated by induction of SA-¦Â-gal activity, activation of the p53-p21 signaling cascade, and downregulation of BMI1 expression.

p53-p21

Activation

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

The significance of CCR2 was further validated by the fact that CCR2 KD cells had decreased SA-¦Â-gal activity and p53-p21 pathway activation . Furthermore, overexpression of CCR2 in early-phase UCB-MSCs remarkably increased cellular senescence, as indicated by induction of SA-¦Â-gal activity, activation of the p53-p21 signaling cascade, and downregulation of BMI1 expression.

Human

HL

cellular senescence

26,573,462

Gene

0

1

0

1

0

1

0

BMI1

648

protein coding

UCB-MSC

--

Aging

Prevent

Knockdown//SA--gal activity assay

As previously reported, BMI1 KD UCB-MSCs exhibited the typical senescent phenotype, such as an enlarged and flattened morphology and increased SA-¦Â-gal activity.

MCP-1

--

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

As expected, the promoters of MCP-1 in BMI1 KD cells lost the recruitment of BMI1 and the concomitant H2AK119Ub histone modification . The epigenetic change in the MCP-1 locus in BMI1 KD cells coincided with derepression of MCP-1 transcription, which resulted in increased secretion of MCP-1 protein. The enhanced secretion of MCP-1 protein in BMI1 KD cells triggered cellular senescence, as judged by the increase in SA-¦Â-gal activity and the activation of the p53-p21 signaling pathway. Most importantly, the senescent phenotypes shown in BMI1 KD cells were significantly rescued after silencing of MCP-1.

p53-p21

--

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

The enhanced secretion of MCP-1 protein in BMI1 KD cells triggered cellular senescence, as judged by the increase in SA-¦Â-gal activity and the activation of the p53-p21 signaling pathway .

Human

HL

cellular senescence

26,573,462

Gene

0

1

0

1

0

RBP2

5948

protein coding

AGS,HeLa,SiHa,HGC-27,BGC-823

--

Gastric cancer

Prevent

BrdU assay//SA--gal activity assay

Significant fractions of the cells treated with RBP2 siRNA became positive for SA-¦Â-Gal staining. Consistent with positive SA-¦Â-Gal staining, these RBP2-depleted cells exhibited significantly lower BrdU labeling than did control cells.

p16//p21//p27

--//--//--

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

p16ink4a, p21CIP1, and p27kip1 promoter activity significantly increased following RBP2 depletion in AGS and BGC cells. In contrast, cotransfection with the RBP2 expression vector led to a substantially declined activity of all 3 promoters.

--

Human

L

cellular senescence

19,850,045

Gene

0

1

0

1

0

PIM1

5292

protein coding

2BS

--

Aging

Accelerate

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

Additionally, Pim-1 overexpression increased levels of the senescence-associated protein p16 and decreased levels of the DNA methyltransferase DNMT1, which is known to negatively correlate with senescence.Conversely, HBP1 knockdown attenuated the effect of Pim-1 on the expression of these two genes. HBP1 knockdown also attenuated Pim-1-induced senescence as indicated by SA-¦Â-gal staining and growth arrest in 2BS cells, suggesting that HBP1 also participates in Pim-1-induced premature senescence in normal cells.

HBP1

Binding

Western blot

Exogenous Pim-1 increased HBP1 protein levels but had no effect on HBP1 mRNA expression. Moreover, Pim-1 knockdown decreased HBP1 protein levels but had no effect on HBP1 mRNA.

--

Human

HL

cellular senescence

28,348,080

Gene

0

1

0

1

0

1

0

1

0

SIRT1

23411

protein coding

PAEC

--

Atherosclerosis

Prevent

Crystal violet assay//SA--gal activity assay

Compared to cells transfected with pcDNA vector, the number of senescent cells was reduced significantly in PAECs overexpressing hSIRT1 (by ~42%) or S47A (by ~56%), and the proliferation was enhanced significantly by augmenting either of these two proteins.

CDK5

Binding

Co-IP//Western blot

Direct protein-protein interaction between SIRT1 and endogenous CDK5 was confirmed by co-immunoprecipitation. Using the M2 anti-FLAG agarose beads, CDK5 was detected in the immune-complexes precipitated from PAECs transiently transfected with Flag-hSIRT1-WT but not pcDNA vector. The in vitro phosphorylation assay further validated the ability of CDK5 complexes to phosphorylate SIRT1 at S47.

--

Human

HL

cellular senescence

22,753,194

Gene

0

1

0

1

0

OTX2

5015

protein coding

Daoy ,MED8A

--

Medulloblastoma

Accelerate

Cell morphological analysis//SA--gal activity assay

Furthermore,sustained induction of OTX2 in MED8A or DAOY cells resulted in clear morphologic changes. Cells became flattened and showed an increase in the amount of cytoplasm .¦Â-Galactosidase activity was detected in cells withOTX2expression, but not in control cells.

IL-6//IGFBP7

Upregulation//Upregulation

Western blot

Indeed,the expression of senescence-associated secretory factors IL-6 (27) and IGFBP7 (28) was strongly increased after OTX2 induction .

p53

Activation

Luciferase reporter assay

To confirm P53 activation after OTX2 induction, we transfected wild-type and mutant reporter constructs for P53 activity into MED8A-OTX2 and MED8A-control cells. The results plotted show an up to 4-fold increase in luciferase activity after OTX2 induction in MED8A-OTX2 cells, but not in MED8A-control cells or in MED8A-OTX2 cells transfected with a mutated reporter construct.

Human

HL

cellular senescence

21,047,732

Gene

0

1

0

1

0

ZEB1

6935

protein coding

EPC1-hTERT

--

Endometrial cancer

Prevent

SA--gal activity assay//Western blot

Stable knockdown of either ZEB1 or ZEB2 resulted in upregulation of p15INK4B and p16INK4A, accompanied by transcriptional activation of the respective promoters, and senescence in a subset of the cells, indicating the possibility that ZEB may mitigate EGFR-induced senescence.

p15INK4b//p16

Upregulation//Upregulation

Western blot

ZEB1 or ZEB2 resulted in upregulation of p15INK4B and p16INK4A, accompanied by transcriptional activation of the respective promoters.

--

Human

L

cellular senescence

20,424,117

Gene

0

1

0

1

0

ZEB2

9839

protein coding

EPC2-hTERT

--

Endometrial cancer

Prevent

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

Stable knockdown of either ZEB1 or ZEB2 resulted in upregulation of p15INK4B and p16INK4A, accompanied by transcriptional activation of the respective promoters , and senescence in a subset of the cells, indicating the possibility that ZEB may mitigate EGFR-induced senescence.

p15INK4b//p16

Upregulation//Upregulation

Western blot

ZEB1 or ZEB2 resulted in upregulation of p15INK4B and p16INK4A, accompanied by transcriptional activation of the respective promoters.

--

Human

L

cellular senescence

20,424,117

Gene

0

1

0

1

0

1

0

EGFR

1956

protein coding

EPC2-hTERT

--

Endometrial cancer

Accelerate

SA--gal activity assay//Western blot

We suspected that EGFR overexpression may trigger senescence. In fact, senescence was observed in 30¨C40% of EPC2-hTERT cells shortly after drug selection upon retrovirus-mediated transduction of EGFR, but not a control empty vector.

--

Human

L

cellular senescence

20,424,117

Gene

0

1

0

1

0

PPARGC1A

10891

protein coding

HBMVEC

--

Aging

Prevent

Cell counting//Cell migration assay//Knockdown//SA--gal activity assay//Tube formation assay

PGC1¦Á downregulation also induced cellular senescence-associated phenotypes, such as decreased tube formation, cell proliferation, and cell migration and increased SA-¦Â-gal activity,suggesting that PGC1¦Á contributes to endothelial cell senescence by maintaining mitochondrial function.and the overexpression of PGC1¦Á in old cells increased cell proliferation and partially reduced the senescent phenotype as shown by SA-¦Â-gal staining.

HDAC1//SIRT1

--//--

Knockdown//Western blot

Reduction of HDAC1 by transfection of HDAC1 siRNA into cells increased PGC1¦Á acetylation.Sirt1 knockdown also induced PGC1¦Á acetylation.

--

Human

L

cellular senescence

30,016,403

Gene

0

1

0

1

0

1

0

1

0

1

0

CDKN2A

1029

protein coding

iCSCL-10A

--

Cancer

Accelerate

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

Cell cycle analysis demonstrated a significantly increased G1 population of p16INK4a-transduced cells compared with the control vector-transduced cells .Cells transduced with p16INK4aexhibited an enlarged, flattened and irregular shape, and flow cytometric analysis revealed this effect in terms of forward scatter and side scatter profiles.These cells also positively stained with senescence-associated ¦Â-galactosidase, whereas no such cells were observed among the control vector-transduced population.

--

Human

HL

cellular senescence

23,318,426

Gene

0

1

0

1

0

1

0

1

0

MCRS1

10445

protein coding

IMR-90,Hs68

--

Aging

Accelerate

Cell morphological analysis//PI staining//SA--gal activity assay

To confirm that MSP58-overexpressing cells entered senescence, we analyzed DNA content using flow cytometry with propidium iodide staining. HT1080 MSP58 clone 11 and 14 cells showed decreased fractions in the G1phase resulting from cells accumulating in the S and G2/M phases and elevated forward and side scatter relative to control cells, respectively reflecting increased cell size and granularity.Notably, stable HT1080 and NIH3T3 transfectants expressing MSP58 showed elevated SA-¦Â-gal activity, a classical biomarker of senescence.After selection, MSP58-infected cells displayed a large size with a flattened shape and significantly increased SA-¦Â-gal activity compared with control cells.

BRG1

Binding

Yeast Two-Hybrid assay//Co-IP//Western blot

MSP58 selectively bound to the N-terminal domain (amino acids 1 750) of BRG1.Overexpressed MSP58 and BRG1 proteins were reciprocally coimmunoprecipitated in COS-1 cells.

p53-p21

Activation

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

Elevated levels of p53 and p21 proteins were also detected in MSP58-overexpressing IMR90, Hs68, and H184B5F5/M10 cells;Cells transfected with the truncated constructs showed substantially reduced responses as the p21-dm construct was not induced, indicating that MSP58 regulates p21 promoter activity through the p53 protein.Furthermore, depletion of ATM or p21 using shRNA transfection significantly reduced the incidence of MSP58-induced senescent phenotypes as shown by the representative stable clones HT1080 MSP58 clone 11-ATMsi 9 and MSP58 clone 11-p21si6.

Human

L

cellular senescence

22,563,078

Gene

0

1

0

1

0

1

0

EID3

493861

protein coding

MCF-7

--

Breast cancer

Accelerate

SA--gal activity assay

Induction of EID3 expression with doxycycline augmented the effects of IR on the cells, resulting in a significant increase in the induction of SA-¦Â-gal positive senescent cells compared to the cells exposed to IR alone without the induction of EID3 by doxycycline.

p21//p57

Upregulation//Upregulation

qRT-PCR//Western blot

The increase in p21 and p57 expression was augmented by the induction of EID3 with doxycycline.

--

Human

HL

cellular senescence

30,114,644

Gene

0

1

0

CHD5

26038

protein coding

HepG2,Hep3B

--

Hepatocellular carcinoma

Accelerate

SA--gal activity assay//WST-8 assay

Similarly, the proliferation of Hep3B-CHD5 was lower than Hep3B-empty vector. Consistent with the lack of proliferative capacity, HepG2-CHD5 and Hep3B-CHD5 expressed senescence-associated ¦Â-galac-tosidase (SA-¦Â-gal) activity, a marker of senescent cells. However, very few senescent cells were detected in HepG2-empty vector and Hep3B-empty vector cells.

--

Human

HL

cellular senescence

24,529,164

Gene

0

1

0

1

0

BCL11B

64919

protein coding

U87,U251

--

Glioma

Prevent

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

The histochemical assay for SA-¦Â-Gal activity showed that SA-¦Â-Gal activity was strongly produced in U87-KD1 and U87-KD2 cells with a flat, enlarged cell shape . The extensive SA-¦Â-Gal activity was also detected in U251-KD1 and U251-KD2 cells compared to that of U251-Mock cells. We noted that the activity of SA-¦Â-Gal was weak in either U87- or U251-Mock cells.The percentage of U87-KD1 and U87-KD2 cells at the G0/G1 phase was increased when compared to that observed in U87-Mock cells. In addition, the reduction of Bcl11b expression caused an increase in the arrest of U251 cells at the G0/G1 (KD1) or S (KD2) phase, along with a reduction in the amount of cells at the G2/M phase.

p21//p53//SOX2//Bmi-1

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

qPCR//Western blot

Similar to p21, the production of p53 mRNA and proteins was increased in C6 and U87 cells after Bcl11b gene knockdown.In addition, the transcripts of the stemness genes, Sox-2 and Bmi-1, were downregulated in U87 and U251 after Bcl11b knockdown, as well as their protein levels.Similar to p21, the production of p53 mRNA and proteins was increased in C6 and U87 cells after Bcl11b gene knockdown.

--

Human

L

cellular senescence

26,096,706

Gene

0

1

0

1

0

1

0

1

0

SIRT6

51548

protein coding

NHAC-kn

--

Aging

Prevent

Knockdown//SA--gal activity assay

Furthermore, the SA-¦Â-Gal assay showed that the percentage of SA-¦Â-Gal-positive cells was significantly higher in the SIRT6 siRNA group (24.3 ¡À 4.2 %) than in the control group (11.3 ¡À 3.0 %), indicating that the depletion of SIRT6 induced premature senescence.

¦ÃH2AX//TIFs//p16//p21

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

Immunofluorescence microscopic analysis//Western blot

Immunofluorescence microscopic analyses showed that ¦ÃH2AX foci and TIFs were increased in the SIRT6-depleted chondrocytes than in controls.The p16 protein level was higher and p21 protein level was lower in the SIRT6-depleted chondrocytes than those in control chondrocytes.

--

Human

L

cellular senescence

25,819,580

Gene

0

1

0

1

0

CREB1

1385

protein coding

WI-38

--

Aging

Prevent

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

Permanent CREB reduction in WI-38 fibroblasts highly influenced their viability and proliferation capacity.Moreover, an increased number of cells with senescence-associated ¦Â-galactosidase activity and flatten cell morphology could be observed.

--

Human

HL

cellular senescence

23,991,634

Gene

0

1

0

1

0

1

0

CDCA3

83461

protein coding

HBEC3,HBEC4,HBEC5,H2228,H460,SKMES-1,EBC-1,HTB-182,CRL-5889,A549

--

Lung cancer

Prevent

Cell morphological analysis//SA--gal activity assay

NSCLC cells depleted of CDCA3 were larger than the control cells with MFI values ~4 fold greater than control cells. An enlarged cellular morphology is consistent with cells that have entered senescence.CDCA3 depletion resulted in an increased proportion of cells staining positive for SA-¦Â-gal.

p21

--

Immunofluorescence

Depletion of CDCA3 induced an increase in nuclear immunofluorescent staining of p21 in all cell lines with the exception of the three HBEC cell lines. Quantification of the immunofluorescence with high content microscopy demonstrated a >3 fold increase in nuclear p21 in all NSCLC cell lines with the exception of A549 cells which demonstrated a ~2 fold increase in p21 protein.

--

Human

HL

cellular senescence

28,487,093

Gene

0

1

0

1

0

DNMT1

1786

protein coding

HDF

--

Aging

Prevent

Knockdown//SA--gal activity assay

Moreover, siRNA-mediated knockdown of DNMT1 alone was sufficient to induce HDF senescence.

UHRF1//WNT5A

--//--

Knockdown//qRT-PCR//Western blot//Flow cytometry

Although UHRF1 suppression significantly down-regulated both mRNA and protein expression of DNMT1, DNMT1 knockdown did not alter UHRF1 expression, indicating the role of UHRF1 as an upstream regulator of DNMT1 transcription. However, DNMT1 promoter activity was significantly decreased by siRNA-mediated UHRF1 suppression , further supporting regulation of DNMT1 transcriptional activity by decreased UHRF1 expression. Knockdown of either DNMT1 or UHRF1 clearly induced WNT5A protein expression, whereas HELLS knockdown showed a minor effect.

--

Human

HL

cellular senescence

28,100,769

Gene

0

1

0

1

0

UHRF1

29128

protein coding

HDF

--

Aging

Prevent

Knockdown//SA--gal activity assay

Compared with DNMT1 knockdown, UHRF1 knockdown more effectively led to the gain of senescent phenotypes.

DNMT1//WNT5A

--//--

Knockdown//qRT-PCR//Western blot//Flow cytometry

Although UHRF1 suppression significantly down-regulated both mRNA and protein expression of DNMT1, DNMT1 knockdown did not alter UHRF1 expression, indicating the role of UHRF1 as an upstream regulator of DNMT1 transcription. However, DNMT1 promoter activity was significantly decreased by siRNA-mediated UHRF1 suppression, further supporting regulation of DNMT1 transcriptional activity by decreased UHRF1 expression. Knockdown of either DNMT1 or UHRF1 clearly induced WNT5A protein expression, whereas HELLS knockdown showed a minor effect.

--

Human

HL

cellular senescence

28,100,769

Gene

0

1

0

1

0

XPG

17295032

protein coding

Embryonic Fibroblast

--

Aging

Prevent

Knockdown//MTT assay

With continuous culture, cells from -/- embryos ceased growing by 4 to 5 weeks after the start of in vitro culture, while cells from the +/+ or +/- embryos kept their growth capability 3 to 4 weeks longer, indicating that -/- cells underwent premature replication senescence,These results indicate that cells from xpg-deficient mice have a short replication life span and readily gain immortality and malignant phenotypes, suggesting that the -/- cells are genetically unstable.

--

Human

L

cellular senescence

10,022,922

Gene

0

1

0

1

0

ZNF148

7707

protein coding

NCI-H460

--

Lung cancer

Prevent

Knockdown//SA--gal activity assay

Knockdown of endogenous ZBP-89 promoted NCI-H460 cell senescence, a 1.5-fold increase in SA-¦Â-gal activity was seen after 7 days of ZBP-89i transfection, whereas overexpression of ZBP- 89 led to a reverse effect. In addition, cells transfected with ZBP-89i exhibited phenotypic changes that are typical of cells undergoing replicative senescence.

p16

Downregulation

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

ZBP-89 restrained senescence of NCI-H460 cells through p16 repression. Representative photomicrographs of the SA-¦Â-gal staining at day 7 after ZBP-89i transfection, or ZBP-89i plus p16i transfection. An irrelevant siRNA vector was used as the control.Western blotting demon- strated that p16 protein expression was decreased on ZBP-89 ectopic expression, whereas it was enhanced¡£Western blotting demonstrated that p16 protein expression was decreased on ZBP-89 ectopic expression, whereas it was enhanced by knockdown of the endogenous ZBP-89 in NCI- H460 cells by knockdown of the endogenous ZBP-89 in NCI- H460 cells .Overexpression of ZBP-89 greatly inhibited p16 promoter activity¡£The p16 mRNA level was decreased on ectopic expression of ZBP-89, but increased by knockdown of endoge- nous ZBP-89.

--

Human

L

cellular senescence

19,583,777

Gene

0

1

0

1

0

HDAC1

3065

protein coding

WI-38

--

Aging

Prevent

SA--gal activity assay

Young WI-38 cells cultured in the presence of either HDAC inhibitor cells. Untreated proliferating WI-38 cells propagated in parallel had no SA-¦Â-gal activity acquired the perinuclear staining for SA-¦Â-gal activity normally associated with senescen£¬treatment with butyrate or TSA for 24 hours induced the expression of p21WAF1 in proliferating WI-38 cells. Distinct morphological changes also occurred when WI-38 cells enter replicative senescence. Senescent cells became larger and assumed irregular shapes, while proliferating WI-38 cells formed long£¬and striated parallel array.

p21

--

Western blot

Ttreatment with HDAC inhibitors butyrate or TSA induced p21WAF1 expression in proliferating WI-38 cells.

--

Human

L

cellular senescence

16,250,917

Gene

0

1

0

CD59

966

protein coding

ECA-109

--

Aging

Prevent

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

The results showed that significant cell cycle arrest at the G2/M phase occurred at 12, 24, and 48 h post-irradiation in CD59-deficient cells compared with that in CD59- sufficient cells .We next detected the effect of CD59 expression on the occurrence of senescence at day 6 after ionizing radiation by SA-¦Â-gal staining. The results revealed that CD59 deficiency significantly increased the percentage of senescent Eca109 cells upon irradiation compared with that observed in CD59-sufficient Eca109 cells.The expression levels of the associated signaling molecules p21 and p16 significantly decreased at 72 h after irradiation in both CD59-sufficient and -deficient cells compared with the levels in the corresponding untreated cells, in which CD59-sufficient cells showed a greater decrease in p21 and p16 expression than CD59-deficient cells .

--

Human

L

cellular senescence

30,166,523

Gene

0

1

0

1

0

1

0

PTTG1

9232

protein coding

HCT116

--

Aging

Prevent

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

With similar cell numbers plated, HCT116 WT and PTTG1-/- cells exhibited similar absorbance (OD 0.4) at 0 hour. After 48 hours, HCT116 WT cells showed an absorbance of 1.8 and PTTG1/- cells 1.3, suggesting that HCT116 PTTG1-/- cell number were less than WT. Reintroduction of PTTG1 into HCT116 PTTG1/- cells (PTTG1-/- +PTTG1) increased cell number after 48 hours , and these cells exhibited an OD value of 2.1 (1.6 fold increase compared to PTTG1-/- expressing an empty vector).After doxorubicin (0.005 uM) treatment, 56% of HCT116 PTTG1-/- cells exhibited enhanced ¦Â¨CGal staining, compared to 5% of WT cells£¬we reintroduced PTTG1 into HCT116 PTTG1-/- cells£¬¦Â¨CGal staining indicated fewer senescent cells when PTTG1 was re-introduced (6%) than in control HCT116 PTTG1-/- cells .

p21

--

Western blot

To test the requirement of p21 for enhanced senescence observed in PTTG1-/- cells, we knocked down p21 and then assessed effects of cell treatments. PTTG1-/- HCT116 cells exhibited 21% senescent cells when p21 was suppressed, compared to 58% in control siRNA treated cells, indicating the requirement for p21 in PTTG1 mediated senescence. Moreover, HCT116 PTTG1-/- cells possess higher p21 levels.

--

Human

L

cellular senescence

21,858,218

Gene

0

1

0

1

0

1

0

GAPDH

2597

protein coding

A549

--

Lung cancer

Prevent

Knockdown//SA--gal activity assay

Knockdown of GAPDH by siGAPDH caused cell proliferation arrest, in contrast to control A549 cells transfected with scrambled siRNA. Cells treated with siGAPDH manifested proliferation arrest, revealed enlarged morphology, and distinct blue staining characteristic for SA-¦Â-galactosidase activation.

AMPK//p53

--//--

Western blot//Knockdown

Following inhibition of glycolysis via GAPDH depletion,the decrease of ATP level resulted in AMPK phosphorylation, and p53 stabilization. In line with these findings, we detected accumulation of p53 phosphorylated at Ser15 in GAPDH knockdown cells.

--

Human

HL

cellular senescence

21,749,859

Gene

0

1

0

1

0

ABI3

51225

protein coding

ARO,WRO

--

Aging

Accelerate

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

Although we observed a trend toward increased apoptosis in WRO cells expressing ABI3 when compared to control cells, the growth attenuation was, however, not accompanied by a corresponding increase in apoptotic cells.The ABI3-induced growth suppression in was further studied by flow cytometric analysis, which revealed a decrease in cell viability and cell cycle arrest at the G0/G1 phase.The number of ¦Â-Gal positive cells was higher in ARO and WRO cells expressing ABI3 at days 3 and 5 postseeding.

p21//E2F1

Upregulation//Downregulation

RT-PCR

Stable expression of ABI3 in WRO cells induced p21WAF1 while reduced E2F1 expression at days 3 and 5 post-seeding.

--

Human

L

cellular senescence

21,223,585

Gene

0

1

0

1

0

1

0

1

0

EHF

26298

protein coding

MEF

--

Prostate cancer

Prevent

Knockdown//SA--gal activity assay

Treatment of MEFs with the EHF-targeting siRNA resulted in decreased EHF, enlargement and flattening of the cells, and positive SA-¦Â-Gal activity£¬EHF overexpression effectively rescued siEHF-induced senescence and the corresponding SA-¦Â-Gal positivity.

p16//p27

--//--

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

EHF-knockdown cells had increased levels of p16 and p27 expression.The presence of sip27 prevented accumulation of p27 protein and reduced the SA-¦Â-gal ¨Cpositive population in MEFs and PC-3 cells, compared with controls transfected with siEHF alone.

--

Human

L

cellular senescence

17,172,423

Gene

0

1

0

1

0

LMNA

4000

protein coding

VSMC,HUVEC

--

Hutchinson-Gilford progeria syndrome

Accelerate

Cell morphological analysis

Introduction of progerin into VSMCs strongly reduced cell growth and shortened the replicative lifespan,In contrast, introduction of progerin had no effect on the growth and lifespan of HUVECs.

p53

--

Knockdown

We introduced siRNA targeting p53 into progerin-infected VSMCs and found that this siRNA counteracted the anti-proliferative effect of progerin on VSMC growth.

--

Human

HL

cellular senescence

28,423,660

Gene

0

1

0

RHOA

387

protein coding

HeLa

--

Aging

Prevent

Flow cytometry//SA--gal activity assay

Cell cycle analysis (by flow cytometry using PI) of irradiated cells suggested that, after exposure to 15 Gy of ¦Ã- radiation, cells with constitutively high levels of activated RhoA (HeLa-RhoA-V14) remain arrested in S and G2/M, whereas HeLa cells expressing the dominant negative RhoAN19 remain predominantly in the G1 and S phases of the cell cycle. As expected, we observed a marked arrest of HeLa cells in the G2/M phase of the cell cycle after treatment with high (15 Gy) dose of ¦Ã-radiation .Cells expressing HeLa-RhoAN19, expected to be deficient in RhoA activity, display higher senescence levels at lower doses of 0.5 and 5 Gy of ¦Ã-radiation, which correlates with their preferential arrest at G1 and S phases.These cells also showed increased apoptosis 48 and 72 h after exposure to the highest (15 Gy) dose of ¦Ã-radiation.

--

Human

L

cellular senescence

26,649,141

Gene

0

1

0

1

0

BRG1

834543

protein coding

IMR-90

--

Aging

Accelerate

Knockdown//SA--gal activity assay

We ectopically expressed wild type BRG1 in IMR90 normal human fibroblasts. Indeed, there was a significant increase in SAHF formation in cells ectopically expressing BRG1 compared with controls. Consistently,other markers of SAHF such as formation of H3K9Me2 and mH2A1.2 foci were also induced by the ectopically expressed wild type BRG1.Expression of SA-¦Â-gal activity, a hallmark of cellular senescence, was also induced by wild type BRG1 expression.

BRCA1//p16//p21

--//Upregulation//Upregulation

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

Compared with shBRCA1 alone cells, expression of wild type BRG1 but not mutant BRG1 induced a more pronounced senescence phenotype as indicated by a significant increase in SAHF formation and expression of SA-¦Â-gal activity.This correlated with an increased induction of p16INK4a and P21cip1.

--

Human

L

cellular senescence

23,438,604

Gene

0

1

0

1

0

ING1

3621

protein coding

Fibroblast

--

Aging

Accelerate

SA--gal activity assay

ING1a induced the expression of both p16 and Rb when ectopically expressed in young fibroblasts. ING1a also induced senescence-associated ¦Â-galactosidase staining and cell cycle arrest at the G0/G1phase of the cell cycle after about 48 h of ectopic expression.

ITSN2

Upregulation

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

We measured the expression of ITSN2 in senescent cells after knocking down ING1a using siRNA. We found that knockdown of ING1a in senescent cells led to significant down-regulation of ITSN2 mRNA levels, further suggesting a role for ING1a in regulating ITSN2 expression .When ITSN2 was knocked down in ING1a-expressing cells, we found that EGFR internalization kinetics were similar to control GFP-expressing cells and were significantly restored when compared to the ING1a- expressing A431 cells. We next checked if loss of ITSN2 in ING1a-expressing cells affected the senescence phenotype. We found that senescence-associated ¦Â-gal staining was reduced significantly in these cells, and they showed increased proliferation when assayed using the BrdU incorporation assay. High levels of p16 were no longer induced by ING1a , suggesting that ITSN2 was indeed a direct mediator of ING1a-induced senescence signal. Since we previously noted higher levels of both ING1a and ITSN2 in senescent versus low passage fibroblasts.

Rb-E2F

--

Western blot

Since hypophosphorylated Rb is the Activate form that binds and inhibits E2F, we next asked whether the Rb in ING1a-expressing cells physically associated with E2F. Western blot analysis of immunoprecipitated E2F1 in these samples confirmed that E2F bound Rb, avidly in the presence of ING1a compared to the GFP-expressing cells.

Human

HL

cellular senescence

23,472,054

Gene

0

1

0

SIRT1

23411

protein coding

HepG2

--

Hepatocellular carcinoma

Prevent

Colony formation assay//FACS analysis//Knockdown//SA--gal activity assay

The morphology of cells infected with shSIRT1 viruses was transformed into a larger, flattened shape compared to shScr and non-infected controls. Morphological changes in HepG2 cells were associated with a decrease in proliferation, measured by colony formation and alamarBlue? assays,more cells arrested in G1 and less in S phase of the cell cycle £¬Their complete lack of growth was accompanied with an induction of cellular senescence as shown by enlarged cell size and expression of pH-dependent beta-galactosidase activity.

--

Human

L

cellular senescence

23,339,189

Gene

0

1

0

1

0

1

0

1

0

XRCC4

7518

protein coding

Fibroblast

--

Aging

Prevent

EdU assay//SA--gal activity assay

Overexpression of both XRCC4 and Lig4 suppresses the onset of SIPS.

--

Human

L

cellular senescence

27,391,797

Gene

0

1

0

1

0

LIG4

3981

protein coding

Fibroblast

--

Aging

Prevent

EdU assay//SA--gal activity assay

Overexpression of both XRCC4 and Lig4 suppresses the onset of SIPS.

--

Human

L

cellular senescence

27,391,797

Gene

0

1

0

1

0

TACC3

10460

protein coding

MCF-7

--

Aging

Prevent

Knockdown//SA--gal activity assay

shRNA-mediated depletion of TACC3 resulted in a strong inhibition of MCF-7 cell proliferation, as evidenced by the progressive accumulation of cells arrested in G1and a decline in bromodeoxyuridine incorporation as a marker for DNA synthesis£¬Consistently, these cells developed several signs of a senescent phenotype, including a flattened and enlarged morphology and increased expression of senescence-associated ¦Âgal activity.

--

Human

L

cellular senescence

20,729,911

Gene

0

1

0

1

0

PTEN

5728

protein coding

--

Prostate

Prostate cancer

Prevent

SA--gal activity assay

Ptenpc?/? mutants showed a strong senescence response as determined by SA-¦Â-gal activity.

--

Human

HL

cellular senescence

19,690,330

Gene

0

1

0

AKT1

207

protein coding

Human endothelial cell

--

Aging

Accelerate

SA--gal activity assay

Long-term culture studies showed that constitutive activation of Akt significantly shortened the lifespan of the endothelial cells, whereas inhibition of Akt activity delayed senescence compared with mock-infected cells ,AktCA-transduced endothelial cells were flattened and enlarged, while mock- or AktDN-infected endothelial cells exhibited normal morphology and growth.Senescence-associated b-galactosidase activity was also increased in AktCA-transduced cells.

--

p53-p21

Upregulation

Western blot//Luciferase reporter assay

Expression of p53 and p21Waf1/Cip1, but not p16Ink4a, was elevated, while the level of phosphorylated Rb was decreased in AktCA-infected cells compared with mock infected cells, suggesting that Akt may induce growth arrest by upregulating p53 and p21£¬Aktinduced cell growth arrest was restored in p21-deficient MEF, suggesting that p21 is essential for Aktinduced growth arrest of these cells.Introduction of AktCA induced p53 promoter-driven luciferase activity compared with mock infection, but not luciferase activity driven by a promoter containing 15 copies of a similar sequence with mutation at critical positions (MG15),Ablation of p53 also lessened the decrease in the lifespan of AktCA-infected cells.

Human

L

cellular senescence

14,713,953

Gene

0

1

0

TAZ

6901

protein coding

LN-229

--

Glioblastoma

Prevent

Knockdown//SA--gal activity assay

TAZ knockdown reduces the expression of CTGF and Cyr61 in these cells. Under this circumstance, more LN229 cells undergo senescence after irradiation. Notably, reduction of TAZ expression per se is not sufficient to mimic radiation in inducing senescence, although a modestly increased senescence frequency was observed. Therefore, inhibition of TAZ could facilitate tumor cells to undergo senescence after irradiation.

--

Wnt

Downregulation

Western blot

To test if radiation-induced TAZ degradation is through inhibiting the Wnt signaling, we first examined the expression of ¦Âcatenin. Similar to TAZ, the protein level of ¦Â-catenin was also reduced progressively in response to irradiation in LN229 cells.

Human

HL

cellular senescence

30,542,117

Gene

0

1

0

1

0

NNT

23530

protein coding

MSC

--

Aging

Prevent

SA--gal activity assay

Replicative senescence in MSCs was clearly reduced by overexpression of NNT or NMNAT3 as determined by SA-¦Â-gal staining.The proportions of SA-¦Â-gal1 cells markedly decreased by 52.0% and 75.8% following overexpression of NNT and NMNAT3, respectively, compared with old control cells.

--

Human

L

cellular senescence

27,428,041

Gene

0

1

0

NMNAT3

349565

protein coding

MSC

--

Aging

Prevent

SA--gal activity assay

Replicative senescence in MSCs was clearly reduced by overexpression of NNT or NMNAT3 as determined by SA-¦Â-gal staining£¬ The proportions of SA-¦Â-gal1 cells markedly decreased by 52.0% and 75.8% following overexpression of NNT and NMNAT3, respectively, compared with old control cells.

--

Human

L

cellular senescence

27,428,041

Gene

0

1

0

MUS81

80198

protein coding

SKOV3,A2780

--

Ovarian cancer

Prevent

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

The stable transduced shMUS81 SKOV3 and A2780 cells were arrested in G0/G1 phase ,and the percentage of S phase cells decreased obviously.Knockdown of MUS81 in SKOV3 and A2780 cells increased the emergence of premature senescence cells.

--

Human

L

cellular senescence

27,255,997

Gene

0

1

0

1

0

1

0

ATRAID

51374

protein coding

ARPE?19

--

Aging

Accelerate

LDH activity assay//SA--gal activity assay

SA ¦Â-gal staining demonstrated that ~62.7% of cells overexpressing Apr3 were positive for SA ¦Â?gal, compared with 17.3% of the parent cells.Cell viability was then determined using an LDH activity assay and it was demonstrated cells overexpressing Apr3 exhibited decreased cell viability compared with the parent cells .Apr3 overexpression moderately inhibited cell proliferation by 34.5% compared with the parent cells as determined by a 3 [H]-thymidine incorporation assay.

--

Human

L

cellular senescence

26,934,949

Gene

0

1

0

1

0

CDKN2A

1029

protein coding

MSC

--

Systemic Lupus Erythematosus

Accelerate

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

There were less SA-¦Â-gal-positive cells when p16INK4A expression was knocked down in MSCs from SLE patients. The cell proliferation assay showed that the proliferation rate of p16INK4A knockdown MSCs from SLE patients was restored to that of the normal MSCs. Cell-cycle analysis revealed that G1 phase arrest was reversed in p16INK4A-knockdown MSCs from SLE patients .

--

Human

L

cellular senescence

22,820,504

Gene

0

1

0

1

0

1

0

1

0

CDKN2A

1029

protein coding

Human WMM1175 melanoma cell

--

Melanoma

Accelerate

SA--gal activity assay

At 3 days post-induction the cells induced for p16INK4a expression had adopted characteristics of senescent cells, appearing enlarged and flattened. These cells were negative for the proliferation marker Ki67 and acquired SA-¦Â-gal activity.

CDK4//CDK6

Downregulation//Downregulation

SA-¦Â-gal activity assay

In the presence of ectopic CDK4 or CDK6 expression, p16INK4a did not promote cell enlargement, heterochromatic foci or SA-¦Â-gal activity .These data confirm that the inhibition of both CDK4 and CDK6 kinase activity is required for p16INK4a-mediated cell cycle arrest and senescence.

--

Human

L

cellular senescence

18,843,795

Gene

0

1

0

DNMT1

1786

protein coding

HDF

--

Aging

Prevent

SA--gal activity assay//Western blot

Upregulation of DNMT1 was found to partially reverse the observed UVA-induced increase in SA¦Â-gal positive cells.DNMT1 expression attenuated the observed increase in p53, p21, and p16 expression following UVA irradiation.

ZEB1

Binding

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

Compared with the control vector, ZEB1 over-expression caused a significant increase in luciferase activity in the cells that were cotransfected with the WT and Mut2-4 reporter vectors. Conversely, cotransfection of ZEB1-expressing vector with the Mut1 reporter vector gave a level of luciferase activity that was equivalent to the control.ZEB1 up-regulation partially reversed the UVA-induced increase in SA-¦Â-gal positive cells and upregulation of p53, p21, and p16 expression, while ZEB1 down-regulation caused the opposite effects to be observed.

--

Human

HL

cellular senescence

29,466,247

Gene

0

1

0

1

0

FOXA1

3169

protein coding

HEC-1B

--

Endometrial cancer

Accelerate

MTT assay//SA--gal activity assay

Restoring FOXA1 expression induced morphological features of senescence, including accumulation of granular cytoplasmic inclusions and elevated ¦Â-galactosidase activity.MTT and plate colony formation assays revealed that HEC-1B cells transiently transfected with a FOXA1 expression plasmid showed significant growth inhibition and limited colony formation compared with the cells transfected with the negative control vector.

p16

Activation

qRT-PCR

Following treatment with LY 294002, expression of the senescence-associated genes p16INK4a and p27kip1 was markedly increased, while PTEN gene expression was decreased.

Akt

Downregulation

Western blot

Western blot analysis of the protein levels of p(Ser473)-AKT and AKT revealed almost complete inhibition of p-AKT expression in cells treated with LY 294002 at 1, 10 and 20 ¦ÌM, with optimal inhibition achieved at 10 ¦ÌM.

Human

L

cellular senescence

27,349,269

Gene

0

1

0

1

0

RAC3

5881

protein coding

HEK293

--

Aging

Prevent

SA--gal activity assay

RAC3 overexpression significantly reduced the number of cells having large and heterochromatic nuclei and the positive SA ¦Â-Gal-stained cells induced by H2O2 or rapamycin.

--

Human

L

cellular senescence

26,469,953

Gene

0

1

0

TP53

7157

protein coding

HepG2

--

Hepatocellular carcinoma

Accelerate

MTT assay//SA--gal activity assay

Both colony formation and cell survival were significantly reduced in the TP53+/¦¤40 clones compared to the RI clones.The percentage of senescence-associated ¦Âgalactosidase (SA-¦Â-gal)-positive cells was significantly higher in the TP53+/¦¤40 clones compared to the RI clones.

--

Human

L

cellular senescence

27,980,070

Gene

0

1

0

1

0

ARHGAP18

93663

protein coding

HUVEC

--

Aging

Accelerate

Cell morphological analysis//SA--gal activity assay

After 48 h, ARHGAP18 overexpression resulted in a significant increase in large cells, defined as senescent based on size and flattened appearance,expression of SA-¦Â-galactosidase (SA-¦Â-gal) and of p21 expression.

AP1

--

Western blot

ARHGAP18-senescent cells also prevented the phosphorylation of c-Jun, an essential component of the transcription factor AP-1, which has also been shown to regulate components of the SASP and for adhesion molecule expression on EC.

NF-¦ÊB

Upregulation

Western blot

In the absence of TNFa stimulation, both EV and ARHGAP18-senescent cells showed basal levels of NF-jB expression. Following TNFa stimulation, EV cells showed increased expression of the RelA/p65 subunit of the NF-kB complex , whereas the ARHGAP18-senescent cells did not show this same upregulation.

Human

L

cellular senescence

25,407,919

Gene

0

1

0

1

0

PPARD

5467

protein coding

HCAEC

--

Aging

Prevent

SA--gal activity assay

The increase in SA-¦Â-gal activity was significantly reduced in the presence of GW501561, a PPAR¦Äspecific ligand, demonstrating the involvement of PPAR¦Äin the inhibition of Ang II-induced premature senescence.

SIRT1

Upregulation

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

When cells were exposed to 100 nM GW501516, the increase in SIRT1 mRNA was significant at 1 h and reached a maximum at 3 h.Whereas knockdown of SIRT1 by siRNA significantly reversed the effect of GW501516 on both ROS generation and cellular senescence in HCAECs following exposure to Ang II.

--

Human

L

cellular senescence

23,000,914

Gene

0

1

0

SIRT1

23411

protein coding

HDF

--

Aging

Prevent

Flow cytometry//SA--gal activity assay

SIRT1 adenovirus resulted in the marked upregulation of SIRT1 protein. Furthermore, cells overexpressing SIRT1 showed attenuated UVB-induced cell senescence, In addition, SIRT1 overexpression significantly upregulated cell growth and rescued cells from G1 cell cycle arrest.

FOXO3¦Á//p53

--//--

Western blot

Total acetylation of p53 and acetylation of p53 at Lys382 were increased.SIRT1 overexpression decreased the UVB-induced acetylation of p53. These results suggest that overexpressed SIRT1 deacetylates p53 and FOXO3¦Á to elicit its protective action against the UVB-induced senescence.

--

Human

L

cellular senescence

25,165,029

Gene

0

1

0

1

0

CDKN2A

1029

protein coding

CPC

--

Aging

Accelerate

Knockdown//SA--gal activity assay

Interestingly, SA¦ÂG staining showed decreased numbers of senescent cells in the experimental hCPCs infected with lentivirus expressing shRNA against p16INK4A.

--

NF-¦ÊB

Upregulation

Western blot//RT-PCR

RT-PCR and Western Blot with cytokines and NF¦ÊB related genes was performed and showed upregulation in NFKB1, TBK1, RELA, IKBKB, and TICAM2. NFKB2 and HMOX1 showed 3~4-fold increased mRNA expression, while TLR3 showed almost 5.5-fold increased mRNA expression in the sh-p16INK4A infected aging hCPCs compared to the control hCPCs.

Human

L

cellular senescence

29,675,777

Gene

0

1

0

1

0

STIM1

6786

protein coding

PC-3,DU 145

--

Prostate cancer

Accelerate

Flow cytometry//Growth curve assay//SA--gal activity assay

The percentages of ¦Â-Gal staining (+) cells (senescent cells) were significantly higher in DU145 and PC3 cells overexpressing STIM1-YFP, ORAI1 and ORAI1-STIM1-YFP than in the corresponding controls.We observed a low growth rate in DU145-ORAI1, DU145-STIM-YFP and DU145-Orai-STIM1-YFP cells, indicating that STIM1 and ORAI1 have inhibitory effects on cell proliferation.We found that DU145 and PC3 cells overexpressing STIM1-YFP, ORAI1 and ORAI1-STIM1-YFP were enriched in the G0/G1 phase and that the percentage of these cells in the G2/M phase was significantly reduced.

--

Human

HL

cellular senescence

26,257,076

Gene

0

1

0

1

0

1

0

RTN4

57142

protein coding

DU 145

--

Prostate cancer

Accelerate

Flow cytometry//SA--gal activity assay

RTN4 overexpressed cells were in G2/M phase but only 22.4% of control cells were in this stage, suggesting that cell cycle was blocked in G2/M phase (p < 0.05).Blue perinuclear staining in a small subset of RTN4 overexpressed DU145 cells was observed while the control group cells showed no visible staining.

--

Human

L

cellular senescence

30,480,803

Gene

0

1

0

1

0

YY1

7528

protein coding

H460

--

Aging

Prevent

Knockdown//SA--gal activity assay

Cells transfected with YY1i exhibited phenotypic changes that resembled those observed in the cells undergoing replicative senescence.These changes include increased SA-¦Â-gal staining, flattened cell morphology, and enlarged cell size.

p16

Downregulation

Knockdown//Western blot//CHIP

We used a p16siRNA vector (p16i) to knockdown the endogenous p16 expression [30]. Apparently, compared with the cells transfected with YY1i alone, co-transfection of cells with YY1i and p16i vectors failed to induce cell senescence.Western blotting assays demonstrated that the p16 protein expression was decreased upon overexpression of YY1, while it was enhanced by knockdown of the endogenous YY1 in NCI-H460 cells.The ChIP data revealed that YY1 was enriched at the p16 promoter (P3) upon the overexpression of YY1.

--

Human

L

cellular senescence

18,558,095

Gene

0

1

0

1

0

PIM1

5292

protein coding

2BS

--

Aging

Accelerate

SA--gal activity assay

PIM1 induces cell senescence. SA-¦Â-gal activity staining experiments showed strong staining compared to the empty vector control and mPIM1K67M groups.

CBX8

Downregulation

CHIP//CCK-8 assay//EdU assay//Colony formation assay//SA-¦Â-gal activity assay

To further verify whether endogenous CBX8 and PIM1 interact with each other, co-IP experiments were performed in extracts of senescent cells induced by RasV12 as PIM1 expression was upregulated in RasV12-induced senescent cells [9,26]. CBX8 was observed to co-immunoprecipitate with PIM1. In reciprocal immunoprecipitations, PIM1 was observed presenting in CBX8 immunoprecipitates PIM1 overexpression inhibited the growth of 2BS cells, while ectopic expression of CBX8 could obviously mitigate such growth inhibition by PIM1 as indicated in CCK8, EdU incorporation and colony formation assays. Furthermore, SA ¦Â-gal activity staining experiment showed that overexpressing CBX8 could make the percentage of stained cells decreasing as compared with that of cells with PIM1 overexpressing alone, which suggests that overexpression of CBX8 may effectively counteract PIM1-induced senescence.

--

Human

L

cellular senescence

29,763,603

Gene

0

1

0

GLIS2

84662

protein coding

--

Kidney

Aging

Prevent

SA--gal activity assay

Concordant with H3K9me3 content, senescence-associated lysosomal ¦Â-galactosidase activity was virtually absent in Ksp-creKif3af/-;Glis2+/mut kidneys but was clearly detected in Ksp-creKif3af/-;Glis2mut/mut double mutants and more intensely in Glis2mut/mut kidneys.

--

Human

L

cellular senescence

27,181,777

Gene

0

1

0

HTRA1

5654

protein coding

ARPE-19

--

Aging

Accelerate

SA--gal activity assay

There were more SA-¦Â-galactosidase-positive cells among HtrA1-expressing ARPE-19 cells (55%) than among control cells (44.3%) after H2O2 treatment.

--

p38 MAPK

Activation

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

p38 MAPK is activated simultaneously with the HtrA1-enhanced cell senescence in MEFs.To confirm that HtrA1 activates cell senescence through the p38 MAPK cascade, we examined the effects of a p38 MAPK inhibitor, SB203580, on the induction of cell senescence by recombinant HtrA1,SB203580 completely abolished the HtrA1- stimulated expression of p21CIP1/WAF1 and p16INK4a. Similarly, induction of SA-¦Â-galactosidase activity by recombinant HtrA1 was inhibited by SB203580,showing that HtrA1 enhances cell senescence through the p38 MAKP cascade.

Human

L

cellular senescence

23,623,979

Gene

0

1

0

MAML1

9794

protein coding

B16

--

Melanoma

Prevent

SA--gal activity assay

ShMaml1-B16 cells exhibited a large, flat morphology indicative of senescence, which was confirmed by enhanced SA-¦Â-gal staining.

STAT1

--

Western blot

Western blot analysis showed an increase in both the total and phosphorylated levels of STAT1 in the shMaml1-B16 cells.

--

Human

L

cellular senescence

22,864,395

Gene

0

1

0

MCL1

4170

protein coding

Human glioma cell

--

Glioma

Prevent

Knockdown//SA--gal activity assay

The MCL1-siRNA group showed decreased cell density, enlarged body of some cells with flat triangle, and an intense sense of grain, and vacuolation, which were the characteristics of cell senescence, and the positive expression rate of ¦Â-galactosidase was (14.26 2.1)% .

--

PI3K-Akt

Downregulation

Western blot

Compared with the blank and NC groups, the protein levels of MCL1, Bcl-2, PI3K, Akt, Bmi-1 and extents of PI3K and Akt phosphorylation increased remarkably in the IGF-1 group, and the protein levels of Bax and PTEN decreased significantly ; while the protein levels of MCL1, PI3K, Akt, Bcl-2, Bmi-1 and extents of PI3K and Akt phosphorylation decreased and that of Bax and PTEN elevated significantly in the IGF-1 + MCL1-siRNA group.

Human

L

cellular senescence

30,296,359

Gene

0

1

0

1

0

PINK1

65018

protein coding

NPC

--

Aging

Prevent

Knockdown//SA--gal activity assay

These results indicated that PINK1/Parkin mediated mitophagy was inhibited in NPCs by down-regulation of PINK1. Furthermore, SA-¦Â-Gal staining results showed that NPCs suffered PINK1-shRNA transfection accompanying with H2O2 treatment had the highest percentage of SA-¦Â-Gal-positive cells (blue), and staining intensity.However, down-regulating PINK1 alone didn't increase the percentage of SA ¦Â-Gal-positive cells and staining intensity comparing with the control group.

Parkin

--

Western blot

The expression of LC-3 II/I decreased and Parkin, P62 increased markedly in NPCs with PINK1-shRNA transfection.

--

Human

L

cellular senescence

29,890,141

Gene

0

1

0

1

0

CABLES1

91768

protein coding

HUVEC

--

Aging

Accelerate

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

The EdU results showed that the Cables1 transfection reduced cell proliferation. Similar results were also observed by flow cytometry. The results suggested that HUVECs had a lower rate of proliferation (S: 3.00 ¡À 0.20%) in the Cables1 group than in the control group (S: 10.78 ¡À 0.9%). Cellular senescence was evaluated after Ang II treatment, and SA-Gal activity was found to be significantly increased in Cables1-transfected HUVECs compared with the control cells.

--

p21

Upregulation

Western blot

p21 activation was analyzed by Western blotting. The results showed that Cables1 significantly increased p21 expression compared with the control group.

Human

L

cellular senescence

28,118,639

Gene

0

1

0

1

0

1

0

SIRT1

23411

protein coding

CEP

--

Disc degenerative disease

Prevent

SA--gal activity assay

The overexpression of SIRT1 also decreased the population of G1 phase cells (p< 0.05), the percentege of SA-¦Â-Gal-positive cells (blue), and staining intensity of the CEP cells.

--

p53-p21

Downregulation

Western blot

The overexpression of SIRT1 and inhibition of endogenous SIRT1 activity significantly influenced the p53/p21 pathway and, thereby, CEP cell senescence.

Human

L

cellular senescence

26,940,203

Gene

0

1

0

COX5B

1329

protein coding

MDA-MB-231,MDA-MB-468,MCF-7

--

Breast cancer

Prevent

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

The efficiency of knockdown was confirmed with mRNA and protein level analyses. We then explored the effects of COX5B depletion on cell proliferation and migration. The result showed that loss of COX5B inhibited both cell proliferation and migration in the three cell lines. In the process, cells with sh-COX5B became flattened and elongated after three passages, which were morphological transformation of cell senescence. Thus, a ¦Â-galactosidase staining assay was used to determine whether the cells undergo cell senescence. We found that more COX5B-depleted cells (25~45%) stained positively than control cells (7~16%), suggesting that loss of COX5B induced senescence.

--

Human

L

cellular senescence

26,506,233

Gene

0

1

0

1

0

1

0

1

0

1

0

ARID1A

8289

protein coding

HPNE

--

Pancreatic cancer

Accelerate

Knockdown//SA--gal activity assay

ARID1A knockdown had no effect on cell apoptosis as demonstrated by Annexin V/Propidium Iodide apoptosis assay, but accelerated cell cycle progression in HPNE/KRASG12D cells. Second, ARID1A knockdown significantly reduced the percentage of senescence-associated bgal staining-positive cells. Third, accelerated cell cycle progression and inhibited senescence were also observed in the stable HPNE/KRASG12D/shARID1A cells compared with HPNE/KRASG12D/shNC control cells.

p16

Upregulation

Western blot

The results showed that restored ARID1A expression led to increased expression of p16, promotion of cell senescence.

--

Human

L

cellular senescence

28,942,143

Gene

0

1

0

1

0

EZH2

2146

protein coding

MKN28

--

Gastric cancer

Prevent

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

To further elucidate reasons why inhibiting EZH2 promoted DOX-induced cell proliferation inhibition, wecounted cells positive for SA-¦Â-gal expression, and observed SAHF formation again. In MKN28 cells carrying mutant p53, siRNA and EGCG caused marked increase in positive level of SA-¦Â-gal staining and SAHF and there was increase in percentage of G0/G1 phase cells.These results suggested that EZH2 depletion promoted DOX-induced senescence in MKN28 cells.

p53

--

Western blot

EZH2 depletion increased accumulation of p53 protein and protein of its target gene p21, in AGS cells under co-treatment.

--

Human

L

cellular senescence

24,738,879

Gene

0

1

0

1

0

1

0

SREBF1

6720

protein coding

HDF

--

Aging

Accelerate

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

SREBP-1 knockdown effectively attenuated all senescence phenotypes induced by H2O2.Similar and more remarkable results by overexpressing SREBP-1 on gain of senescence phenotypes, including SA-¦Â-galactosidase, granularity (SSC), and delayed cell growth in addition to induction of lipogenic enzymes were obtained in young HDFs.

--

Human

L

cellular senescence

20,615,871

Gene

0

1

0

1

0

1

0

GRPR

2925

protein coding

A172

--

Glioblastoma

Prevent

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

GRPR knockdown reduces GBM cell proliferation.Silencing of GRPR increases significantly the percentage of senescent cells based on cell count using ImageJ.

EGFR//p38//p16//p21

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

Western blot

We also observed a reduction of p38 and an increase in activation of EGFR as assessed by the content of pEGFR in GRPRi cells.GRPRi cells showed increased expression levels of p53, as well as p21 and p16,suggesting that senescence was enhanced in these cells through the activation of the p53 pathway.

p53

--

Western blot//Knockdown

GRPRi cells showed increased expression levels of p53, as well as p21 and p16,suggesting that senescence was enhanced in these cells through the activation of the p53 pathway .

Human

L

cellular senescence

26,780,458

Gene

0

1

0

1

0

1

0

CPEB1

64506

protein coding

U87,U251

--

Glioma

Prevent

Knockdown//SA--gal activity assay

U251 and U87 cell growth was inhibited by CPEB1 knockdown. Senescent cells can be detected with an assay that detects senescence-associated ¦Â-gal activity.

miR-101//p53

--//--

Luciferase reporter assay//qPCR

When the cells were individually cotransfected with miR-101 and pMIR-CPEB1-30-UTR-WT, there was a significant reduction in luciferase activity compared with cells transfected with the negative control, thereby confirming that miR-101 directly interacted with the 30-UTR of CPEB1 in glioma cells. It was gratifying to observe that miR101 could inhibit the expression of CPEB1, and that the suppression of miR-101 could enhance the expression of CPEB1.After the knockdown of CPEB1, the level of expression of p53 was decreased in the U251 cells but not changed in the U87 cells. Furthermore, it was discovered that the knockdown of CPEB1 induced the overexpression of p21, which is downstream of p53.

--

Human

L

cellular senescence

23,788,032

Gene

0

1

0

1

0

NRSN2

80023

protein coding

SMMC-7721

--

Hepatocellular carcinoma

Accelerate

Cell viability assay//SA--gal activity assay

We found that when NRSN2 was overexpressed, the cellular viability of SMMC-7721 decreased and the senescent cells increased after doxorubicin treatment for 48 h .

Bcl-2

--

Western blot//qRT-PCR

Considering the increased cell viabilities and the anti-senescence/apoptosis effect after NRSN2 silencing, we examined the change of PI3K/Akt pathway, which plays an important role in tumor growth [11] and Bcl-2 family proteins, which function as a critical modulator in cell apoptosis .Meanwhile, the cleaved PARP and pro-apoptotic protein Bax was decreased, while the anti-apoptotic protein Bcl-2 was significantly increased after NRSN2 silencing.

PI3K-Akt-mTOR//p53-p21

--//--

Western blot

Knockdown of NRSN2 led to increased (473)p-Akt levels in MHCC-LM3 cells. After silencing NRSN2 in Hep3B, we treated these transfected cells with doxorubicin, a chemotherapeutic agent which is commonly used to induce cell senescence , for 2 days and we surprisingly found that compared to the control, the levels of p53 and p21 were decreased.

Human

L

cellular senescence

26,055,238

Gene

0

1

0

1

0

SIRT1

23411

protein coding

iPSC-Ecs

--

Aging

Prevent

SA--gal activity assay

Overexpression of SIRT1 led to a significant reduction of cells entering senescence when compared to control groups. Blocking SIRT1 with the inhibitor Ex-527 led to a higher percentage of senescent cells in all groups, suggesting a contribution of endogenous SIRT1.

--

Human

L

cellular senescence

26,413,932

Gene

0

1

0

HRH4

59340

protein coding

MDA-MB-231,MCF-7

--

Breast cancer

Accelerate

SA--gal activity assay

Present results indicate that the negative effect on proliferation exerted by histamine in MDA-MB-231 cells was elated to a 2-fold increase in cell senescence. In addition, this effect was reversed by JNJ7777120 treatment and H4R agonists mimicked histamine effect, suggesting the involvement of H4R in histamine-induced cell senescence .In the same way, histamine was able to augment the percentage of MCF-7 senescent cells (15.7¡À1.3 vs. 7.8¡À0.4 in untreated, P<0.001), which was partially blocked by the treatment with JNJ7777120. Moreover, H4R agonists elicited a similar response, increasing cell senescence.

--

Human

L

cellular senescence

21,622,113

Gene

0

1

0

CDKN1A

1026

protein coding

HUVEC

--

Aging

Accelerate

SA--gal activity assay

When compared with the results of empty vector control, HUVECs transfected with p16 exhibited a rapid increase in the proportion of senescent cells, as assessed by SA-¦Â-Gal staining on days 3 and 5. Cells transfected by p21 also showed accelerated senescence, but this occurred only after 5 days of incubation and was milder than the effect of p16 transfection.

--

Human

L

cellular senescence

16,243,918

Gene

0

1

0

CDKN2A

1029

protein coding

HUVEC

--

Aging

Accelerate

SA--gal activity assay

When compared with the results of empty vector control, HUVECs transfected with p16 exhibited a rapid increase in the proportion of senescent cells, as assessed by SA-¦Â-Gal staining on days 3 and 5. Cells transfected by p21 also showed accelerated senescence, but this occurred only after 5 days of incubation and was milder than the effect of p16 transfection.

--

Human

L

cellular senescence

16,243,918

Gene

0

1

0