Venkatachalam/aging_reg_dataset · Datasets at Fast360

SPHK2

56848

protein coding

--

Spleen,Skin,Testes tissue

Lung cancer

Prevent

Knockdown//SA--gal activity assay

SphK2-/-mice exhibited accelerated senescence and aging phenotype measured by increased ¦Â-gal expression in their paws (37) at generation 6, and decreased weight of testes atgeneration4 or 5 compared with WT and SphK1-/-mice (generation matched controls). Examination of spleen,skin, and testes tissues at generation 6 using H&E staining also confirmed increased senescence and aging phenotype in SphK2-/-compared with WT mice.

--

p16

--

Immunofluorescence//Western blot

ShRNA-mediated knockdown of SPHK2 induced caspase-3, and ectopic expression of p16 attenuated this process compared with Scr-shRNA¨C expressing A549 cells.

Human

HL

apoptosis

29,748,384

Gene

0

1

0

1

0

GKN1

56287

protein coding

AGS,MKN1,AGSGKN1,MKN1GKN1

--

Gastric cancer

Accelerate

SA--gal activity assay//qPCR

Average telomere length and telomerase activity decreased significantly in GKN1-transfected AGS and MKN1 cells, compared to those in mock-transfected cells. Furthermore, unlike mock-transfected cells, AGSGKN1 and MKN1GKN1 cells that stably expressed GKN1 demonstrated shortened telomeres and diminished telomerase activity with additional passages.These stable transfectants from passage 5 showed significant shortening of telomeres, as well as decreased telomerase activity and hTERT mRNA expression in a time-dependent manner. The mean percentage of SA-¦Â-gal-positive AGSGKN1 and MKN1GKN1 stable cells increased by 13% and 5.5% at 24 hr and by 24% and 19% 48 hr (P = 0.0134 and P = 0.0247), respectively.

c-Myc//hTERT//TRF1

Downregulation//Downregulation//Upregulation

qPCR//Western blot

GKN1 directly bound to the c-myc protein in the immunoprecipitation assay and down-regulated its expression, as well as inhibited its binding activity to TRF1.Interestingly, c-myc mRNA expression was positively correlated with hTERT expression, whereas GKN1 expression was inversely correlated with c-myc and hTERT mRNA expression in 35 gastric cancer tissues. GKN1 protein expression was positively and inversely correlated with TRF1 and c-myc protein expression.

--

Human

L

apoptosis

25,344,918

Gene

0

1

0

1

0

PAPSS2

9060

protein coding

MCF-7

Tumor tissue

Breast cancer

Prevent

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

MCF7 human breast cancer cells treated with PAPSS2 Si were fewer in number and showed poor colony-forming ability compared with control siRNA (Con Si)-transfected cells. PAPSS2-depleted cells accumulated in the G1 phase with no increase in the proportion of cells either in the subG1 phase or that were positive for propidium iodide staining. PAPSS2-depleted cells displayed hallmarks of senescent cells such as enlarged and flattened morphology and SA-¦Â-Gal staining.As PAPSS2 was depleted, the cells showed a loss of phosphorylated pRb with changes of cell cycle regulatory protein levels and accumulation of p53 and p21.

--

FGFR-AKT-p53-p21

--

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

PAPSS2-mediated premature senescence, including a loss of phosphorylated pRb, a decrease in cell number, and an increase in SA-¦Â-Gal positivity, was blocked in both p53?/?and p21?/?HCT116 human colon cancer cells.We confirmed that P APSS2 depletion induced augmented FGFR/AKT activation and p53/p21 accumulation in both A549 and HDF cells . The overexpression of a kinase-inActivate (KI) mutant of FGFR1 (FGFR1-KI) attenuated AKT phosphorylation and p53/p21 accumulation in PAPSS2-depleted cells.

Human

HL

apoptosis

26,250,908

Gene

0

1

0

1

0

1

0

1

0

PTEN

5728

protein coding

LN-18,U87,U251,U373,LN428

--

Glioma

Prevent

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

We first examined relative cell numbers and cellular morphologies, and observed that all cell types had decreased cell numbers in a dose-dependent manner following IR .Furthermore, microscopic analyses indicated that U87, U251, and U373 cells were positive for senescence-associated ¦Â-galactosidase (SA-¦Â-Gal), a hall-mark of senescence, and for senescent morphology (large flattened shape).Cyclin-dependent kinase inhibitor p21, one of senescence markers, was dramatically increased in PTEN-deficient U87, U251, and U373 cells, but not in PTEN-proficient LN18 and LN428 cells, and cleaved poly (ADP-ribose) polymerase (PARP), which is an indicator of the biochemical changes due to caspase activation during apoptosis was detected only in LN18 and LN428 cells .

--

Akt-ROS-p53-p21

--

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

U87 cells were characterized by gradual increases in phospho-AKT, wt p53, and p21. AKT depletion also shifted cells from premature senescence to apoptosis as a response to IR , as observed in wt PTEN-expressing cells. AKT depletion resulted in decreases in both SA-¦Â-Gal activity and intracellular ROS levels.In p53siRNA transfected cells before IR exposure, p21 expression was not induced , and cells showed decreased SA-¦Â-Gal activity. Cells transfected with p21 siRNA before IR exposure showed phenotypes similar to those transfected with p53 siRNA.

Human

L

apoptosis

21,072,054

Gene

0

1

0

1

0

1

0

UBTD1

80019

protein coding

HDF,MKN45

Tumor tissue

Gastric cancer

Accelerate

Flow cytometry//Knockdown//SA--gal activity assay//Trypan blue staining//Western blot//qRT-PCR

UBTD1 knockdown increased cell proliferation and down-regulated p53 protein. We further analyzed cell cycle and apoptosis by flow cytometry and dead cells by Trypan blue staining, and found that UBTD1 overexpression indeed induces G1-stage arrest, apoptosis and an increase in dead cells. Frozen tumor tissue slices were stained for SA-¦Â-gal marker and a higher proportion of senescent cells were present in the UBTD1 overexpression group (p<0.05).

--

Mdm2-p53

Downregulation

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

We treated MKN45 cells with nutlin-3, a known MDM2 antagonist that stabilizes p53, and found that nutlin-3 upregulated p53 and UBTD1 in a dose-dependent manner.We found that exogenous UBTD1 can pull down both endogenous Mdm2 and p53 , suggesting that these three proteins can form a complex and that UBTD1 might regulate p53 via Mdm2. We overexpressed Mdm2 in UBTD1-overexpressing cells, and found that Mdm2 overexpression could reverse UBTD1-mediated up-regulation of p53 expression .

Human

HL

apoptosis

25,382,750

Gene

0

1

0

1

0

1

0

1

0

1

0

1

0

RBX1

9978

protein coding

U87,H1299

--

Cancer

Prevent

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

Morphologic observation revealed that LT-ROC¨Cinfected U87 cells were in size with flattened shape. Indeed,¡«25% of LT-ROC¨Cinfected cells, but <2% of the control cells, were positively stained with SA-¦Â-gal.Our FACS analysis also revealed that among the remaining cell populations, not undergoing apoptosis, ¡«50% to 60% of LT-ROC1¨Cinfected U87 cells were arrested in the G2-M phase of the cell cycle, compared with ¡«15% to 20% of control cells at 120 hours postinfection.

--

Human

L

apoptosis

19,509,229

Gene

0

1

0

1

0

1

0

CYP24A1

1591

protein coding

Primary renal proximal tubule epithelial cell

Kidney

Diabetes

Accelerate

Flow cytometry//SA--gal activity assay

CYP24A1 overexpression induced senescence (increased G1 arrest) in normal conditions and induced apoptosis (increased sub-G1 fraction) in high glucose conditions. We did in fact see increased cellular senescence in cells overexpressing CYP24A1 that exceeded cellular senescence induced by high glucose alone, as shown by increased numbers of blue-stained cells .

--

Human

L

apoptosis

23,119,081

Gene

0

1

0

1

0

TERT

7015

protein coding

MSC

--

Aging

Prevent

CCK-8 assay//Flow cytometry//SA--gal activity assay//TUNEL assay

Compared with the control group, the downregulation of hTERT induced cellular senescence, decreased cell proliferation and the percentage of cells in the S phase and increased the percentage of apoptotic MSCs.

--

PI3K-Akt

Upregulation

Western blot

We found that the downregulation of hTERT significantly decreased p-PI3K and p-AKT expression in the MSCs, whereas the upregulation of hTERT significantly increased p-PI3K and p-AKT expression in the MSCs; no changes were observed in the levels of total PI3K and AKT in either group.The MSCs were treated with LY294002 (Sigma-Aldrich), a PI3K inhibitor, and the hTERT expression levels were then measured by western blot analysis. The results revealed that LY294002 significantly inhibited hTERT expression.

Human

HL

apoptosis

26,178,664

Gene

0

1

0

1

0

1

0

1

0

BMP7

655

protein coding

MCF-7

--

Breast cancer

Accelerate

Cell morphological analysis//Telomerase activity assay//Western blot

In the BMP7 treated cell cultures, we observed the cells characteristics of enlarged and flattened cell morphology, greater cytoplasm/nuclear ratio, and expressions of cell senescence markers such as ¦Â-galactosidase and p16 (Janzen et al., 2006;Molofsky et al., 2006).Treatment of MCF-7 cells with BMP7 (30 ng/mL, 15 h in every two-day for two weeks) resulted in a marked increase in the incidence of cell senescence. The increase in cell senescence in the BMP7-treated cultures was associated with reduced cell numbers and protein concentrations (not shown), decreased telomerase activity. The inhibition of telomerase activity in these cells was by 60%¨C70%.Consistent with cell senescence, BMP7-treated cell cultures showed increased p53, p21 and p16.

hTERT

Downregulation

Flow cytometry

The extent of BMP7-induced hTERT gene promoter inhibition was similar to that induced by BMP7 in MCF-7 cells.Overexpression of BMP RII receptor similarly blocked BMP7-induced hTERT gene repression in PMC42 breast cancer cells.

Smad3

--

Immunofluorescence//Luciferase reporter assay

Incubation of cultured MCF-7 cells with BMP7 for different periods of time from 10 min to 2 h resulted in Smad3 phospho rylation. Predominant cytoplasmic staining of phospho-Smad3 was observed in non treated MCF-7 cells, whereas predominant nuclear staining BMP7 treatment and in most cells 30 or 60 min after the BMP7 treatment.BMP7 or TGF-¦Âinduced marked increases in the luciferase reporter gene activity that is under the transcriptional control of the Smad3-specific promoter.

Human

L

apoptosis

27,696,331

Gene

0

1

0

1

0

1

0

BRD7

29117

protein coding

BJ

--

Aging

Accelerate

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

BRD7 depletion resulted in a striking increase in proliferation rate in both young and midpassage BJ fibroblasts, suggesting that BRD7 may function in proliferation control before the onset of senescence.BRD7 depletion significantly delays replicative senescence, as these cells continued to Activately proliferate while control firefly luciferase (FF) shRNA expressing cells ceased proliferation and stained positive for senescence-associated (SA) ¦Â-galactosidase .Overexpression of BRD7 slows proliferation and results in premature senescence in BJs fibroblasts .

p21//p53

Upregulation//--

qRT-PCR//Western blot

BRD7 depletion in HCT116 cells significantly decreased p21 induction in response to TGF-¦Â, which also occurred in p53 null-HCT116 cells,indicating p53 independence.BRD7 was also required for p21 induction by 1¦Á,25(OH)2D3(vitamin D) in human mammary epithelial cells.BRD7 depletion reduces both the basal and induced expression of p21 in response to p53 activation, although p53 was stabilized as expected,suggesting that BRD7 acts downstream of p53 stabilization. Expression of p21 was also elevated in BRD7-overexpressing cells, providing a mechanistic explanation for their slowed proliferation and premature senescence.

--

Human

HL

apoptosis

20,660,729

Gene

0

1

0

1

0

1

0

MAPK8

5599

protein coding

MCF-7

--

Lung cancer

Prevent

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

Cells treated with the JNK inhibitor,SP600125, showed senescence-specific large and flat cellular morphology and stained positively for SA-¦Â-Gal activity.Positive SA-¦Â-Gal activity gradually increased up to 7 days after SP600125 treatment.Analysis of cell cycle distribution revealed that only SP600125-treated cells were arrested in the G2/M phase and that the number of aneuploid cells (>4N) also increased.

--

Bcl-2-ROS-DDR

Activation

Colony formation assay//SA-¦Â-gal activity assay//Western blot//BrdU Assay//Trypan blue staining

When H460 lung carcinoma cells were treated with SP600125, cellular morphology, SA-¦Â-Gal activity and colony-formation ability were compatible with senescence, indicating that this biological process was induced . Loss of phospho-Bcl-2 and phospho-pRB was also detected . Furthermore, ROS generation and DDR were observed along with a decrease in S-phase entry .HEFs transfected with siRNA of JNK or Bcl-2 exhibited SA-¦Â-Gal activity and decreased cell numbers .

Human

L

apoptosis

19,855,432

Gene

0

1

0

1

0

1

0

GAS2

2620

protein coding

SK-HEP-1

--

Hepatocellular carcinoma

Accelerate

Flow cytometry//SA--gal activity assay

First,flow cytometry analysis revealed a significant elevation of sub-G1cell population upon overexpression of GAS2 .V ector-transfected SK-Hep1 cells were devoid of ¦Â-galactosidase (similar to mock-transfected control),whereas GAS2-overexpressed SK-Hep1 cells displayed induction of ¦Â-galactosidase.

p53//p21

Upregulation//Upregulation

Western blot

Quantitative analysis demonstrated that up-regulation of GAS2 increased the number of ¦Â-galactosidase-positive cells by almost four-fold,accompanied by elevation of the senescence regulators p53 and p21, as determined by western blot.

--

Human

HL

apoptosis

25,925,944

Gene

0

1

0

1

0

FASLG

356

protein coding

CRC29

--

Colorectal cancer

Accelerate

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

We found that chronic CD95L exposure suppressed the colony-forming potential of five out of nine of these cultures by more than 50% .FACS analysis of PI-stained cells did show a marked increase in the number of cells in G2.We found that CD95L stimulation induced expression of senescence-associated ¦Â-galactosi-dase (SA-¦Â-GAL).

Caspase

Activation

Western blot

A time course experiment showed that induction of ¦ÃH2AX following CD95L stimulation paralleled caspase-8 and caspase-3 activation, starting already 2?h following ligand stimulation.

p53

--

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

Knockdown of p53 did not prevent upstream events like caspase-8 and caspase-3 activation, iCAD processing or DNA damage induction. Rather, p53 knockdown increased CD95L-induced DNA damage, which is in line with its function as a ¡®guardian of the genome' by activating genes involved in cell cycle arrest and DNA repair.26 Despite the increased DNA damage in CD95L-stimulated p53 knockdown cells, these cells did not enter senescence and largely retained their clone-forming potential .

Human

HL

apoptosis

28,300,842

Gene

0

1

0

1

0

1

0

KRAS

3845

protein coding

L13

--

Colorectal cancer

Prevent

SA--gal activity assay

By contrast, chronic stimulation of KRAS-deficient C26 cells (L13 cells) caused a pronounced growth inhibition, which was associated with the induction of SA-¦ÂGAL.

--

Human

HL

apoptosis

28,300,842

Gene

0

1

0

SIRT7

51547

protein coding

NIH-3T3,U2OS

--

Aging

Prevent

EdU assay//SA--gal activity assay

The SIRT7GFP overexpressing cells showed a significant decrease in EdU incorporation indicative of fewer S-phase cells at 14 and 18 h .The control GFP overexpressing cells showed significantly more number of SA-¦Â-gal positive cells,while SIRT7GFP overexpressing cells were mostly SA-¦Â-gal negative.

p53

Downregulation

Western blot

We had noted that SIRT7GFP overexpressing cells when treated with doxorubicin at senescent inducing dose (0.25 mM for 2 h) showed lesser p53 accumulation compared to control.

--

Human

L

apoptosis

25,445,786

Gene

0

1

0

1

0

ERBB2

2064

protein coding

DLD1

--

Melanoma

Prevent

SA--gal activity assay//qRT-PCR

HER2 ablation exaggerated this damage-induced cellular senescence, which was alleviated by HER2 reconstitution, suggesting that HER2 is responsible for the regulation of damage-induced senescence.

TBK1

Downregulation

Western blot

HER2 expression markedly inhibited the damage-induced TBK1 activation, indicating a critical role for HER2 in the regulation of DNA sensing during the cellular-damage response .

cGAS-STING

Downregulation

Western blot

HER2 suppressed STING- or TBK1-initiated signalling in a dosedependent manner but failed to suppress the activated IRF3 (5SD).

Human

L

apoptosis

31,332,347

Gene

0

1

0

1

0

CDKN2A

1029

protein coding

WI-38

--

Aging

Accelerate

Immunofluorescence//SA--gal activity assay

We found that p16 overexpression or H2O2 treatment alone induced senescence in WI-38 cells and the combination of both resulted in an additive effect. Meanwhile, the senescence-associated heterochromatin foci (SAHF) assay confirmed the observations in senescence cell staining. Both 3MeK9H3 and HMGA1, two classic markers of SAHF, localized to the specific heterochromatic foci in cells transfected with p16 and treated with H2O2.

--

Human

L

apoptosis

28,120,917

Gene

0

1

0

1

0

RELA

5970

protein coding

HCT116,HCT116 p53-/-,MCF-7

--

Cancer

Accelerate

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

Senescence-associated (SA) ¦Â-galactosidase positive cells were up to 48.0 and 77.6% of the detected HCT116 p53 wt and HCT116 p53-/- cells, respectively. Using real-time qPCR, we examined the expression 12 senescencerelated genes, and found that in HCT116 cells p15, p21, and Rb were also upregulated in addition to p16.We further examined the senescence associated secretory phenotype (SASP) in MCF7, HCT116 and HCT116 p532/2 cells by real-time qRTPCR.IL-6, IL-8, IL-18, IL-1b, TNF-aCXCL1 and CXCR2 were all greatly induced, suggesting that the p65/S536D indeed has the function of wild type RelA/p65 phosphorylation at Ser536 in regulation of SASP phenotype.

--

p16

Upregulation

Western blot

In these cells p16 protein levels were elevated , suggesting that p16, not p53 mediates the senescence in HCT116 cells.

Human

HL

apoptosis

26,375,985

Gene

0

1

0

1

0

1

0

TFEB

7942

protein coding

Primary mice Chondrocyte,Primary human Chondrocyte

--

Osteoarthritis

Prevent

SA--gal activity assay//Western blot

The TBHP-treated chondrocytes exerted higher SA-¦Â-gal activity and p16INK4a protein level relative to the control group, whereas TFEB overexpression significantly prevents this increment.

--

Human

L

apoptosis

30,154,423

Gene

0

1

0

1

0

BUB1

699

protein coding

IMR-90

--

Aging

Prevent

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

We found that knockdown of either genes resulted in significant increase of SA-¦Âgal positive cells (p< 0.0001 for all cultures) when compared to EV (EV = 9.6% SD 1.9, shB2= 55.4 SD 7.1, shS2= 74.7% SD 2.86, SEN= 80.2% SD 3.42). Cells depleted of either gene also underwent drastic changes in morphology reminiscent of SEN, such as larger size and flatter morphology.When analyzed by FACS for a senescent-like phenotype (i.e. elevated size and/or AF)30, we observed significantly higher number of cells bearing senescent-like features in BUB1 and SMC1A-depleted cultures relative to EV (shB2: p=0.0013; shS2: p<0.0001), similar to what observed in SEN cultures (SEN: p<0.0001).

--

CDKN2A-Rb1//p53-CDKN1A

--//--

IF

Cultures depleted of BUB1 and SMC1A were significantly enriched for the frequency of CDKN2A and for CDKN1A positive cells.

Human

L

apoptosis

27,731,420

Gene

0

1

0

1

0

1

0

1

0

SMC1A

8243

protein coding

IMR-90

--

Aging

Prevent

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

We found that knockdown of either genes resulted in significant increase of SA-¦Âgal positive cells (p< 0.0001 for all cultures) when compared to EV (EV = 9.6% SD 1.9, shB2= 55.4 SD 7.1, shS2= 74.7% SD 2.86, SEN= 80.2% SD 3.42). Cells depleted of either gene also underwent drastic changes in morphology reminiscent of SEN, such as larger size and flatter morphology.When analyzed by FACS for a senescent-like phenotype (i.e. elevated size and/or AF), we observed significantly higher number of cells bearing senescent-like features in BUB1 and SMC1A-depleted cultures relative to EV (shB2: p=0.0013; shS2: p<0.0001), similar to what observed in SEN cultures.

--

CDKN2A-Rb1//TP53-CDKN1A

--//--

IF

Cultures depleted of BUB1 and SMC1A were significantly enriched for the frequency of CDKN2A and for CDKN1A positive cells.

Human

L

apoptosis

27,731,420

Gene

0

1

0

1

0

1

0

1

0

DNAJA4

55466

protein coding

HaCaT

--

Aging

Prevent

Flow cytometry//Knockdown//MTS assay

44¡æhyperthermia treatment significantly reduced the S phase fraction (P < 0.05) in wild-type HaCaT and NC group cells. By themselves, in the absence of hyperthermia, DNAJA4-deficient cells showed significant reductions in the S phase fraction as compared to both wild-type HaCaT and NC cells (P < 0.05).Results obtained from the MTS assay further substantiated those obtained from flow cytometry, that is,44¡æ hyperthermia treated DNAJA4-deficient cells show a significant decrease in cell viability as compared to the other cell groups. Moreover, PDTC pretreatment prior to hyperthermia significantly reduced the apoptosis ratio in wildtype and NC groups, as well as the senescence ratio in DNAJA4-knockout group as compared to hyperthermia treatment alone.

--

NF-¦ÊB

--

qRT-PCR//Western blot

The DNAJA4-deficiency group showed increased levels of P65 phosphorylation as compared to the wild-type and NC groups after hyperthermia stimulation.

Human

L

apoptosis

29,807,809

Gene

0

1

0

1

0

1

0

G6PD

2539

protein coding

HCC,G6PD,HCT116

--

Colorectal cancer

Prevent

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

Two non-overlapping shRNAs significantly reduced the expression of G6PD and decreased cell proliferation, which was rescued by overexpression of G6PD. Furthermore, G6PD knockdown significantly increased the number of cells positive for SA-¦Â-GAL (¦Â-galactosidase) staining.the expression of p21, one classic marker for senescence, was upregulated in G6PD knockdown cells and restored to normal level when mouse G6PD was overexpressed. Therefore, suppression of G6PD induced cellular senescence in HCC and HCT116 cells.

--

Human

L

apoptosis

24,352,616

Gene

0

1

0

1

0

1

0

1

0

MTDH

92140

protein coding

LNCaP,C4-2B

--

Prostate cancer

Prevent

Cell proliferation assay//SA--gal activity assay

Pretreatment with AEG-1-selective siRNA promoted senescence as indicated by increased ¦Â-galactosidase activity in LNCaP and C42B cells treated with toxic concentrations of DIM and ring-DIMs.Our results showed a significant inhibition of cell proliferation in LNCaP and C42B cells pretreated with AEG-1 siRNA and then treated with DIM compared to DMSO control cells.

--

Human

L

apoptosis

28,923,415

Gene

0

1

0

1

0

BMI1

648

protein coding

--

Kidney

Renal tubulointerstitial injury

Prevent

SA--gal activity assay

E-cadherin-positive renal tubules and the percentage of Ki67 positive renal cells were clearly decreased, whereas the percentages of senescence-associated-¦Â-gal (SA-¦Â-gal) positive area and TUNEL-positive cells were increased significantly in Bmi-1-/- mice, compared with wild-type mice.

¦Ã-H2AX//Pchk2//p16//p19//p53//p21

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

Western blot

Protein expression levels of c-H2A.X, pCHK2, p16, p19, p53, and p21 in kidneys were increased significantly in Bmi-1-/- mice, compared with wild-type mice.

--

Human

L

apoptosis

24,915,841

Gene

0

1

0

PMAIP1

5366

protein coding

H460

--

Lungcancer

Prevent

Cell morphological analysis

We further found that NOXAsilenced H460 cells displayed classical senescence morphology with an enlarged and flattened cellular shape when MLN4924-induced apoptosis was blocked; this indicated a conversion of the cell death phenotype from apoptosis to senescence in these cells.

--

Human

L

apoptosis

24,853,380

Gene

0

1

0

RBM38

55544

protein coding

SMMC-7721,HepG2

--

Hepatocellular carcinoma

Accelerate

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

The proportion of cells that were positive for ¦Â-galactosidase activity, an indicator of cell senescence, was significantly increased in the HepG2-RBM38-OE (p = 0.0077) and SMMC7721 RBM38-OE cell lines (p = 0.0421) compared to the corresponding controls.Up-regulation of RBM38 led to significantly decreased cell proliferation in HepG2-RBM38-OE cells (p = 0.032) and SMMC7721-RBM38-OE cells (p = 0.044) compared to their corresponding control cells. The colony formation assay showed that when RBM38 was over-expressed, the colony number and size were significantly reduced in HepG2-RBM38-OE (colony number, p = 0.01145; colony size, p = 0.0001) and SMMC7721-RBM38-OE cell lines (colony number, p = 0.0116; colony size, p = 0.0001) when compared to their corresponding control cells .

p53//MDM2

Upregulation//Downregulation

Western blot//Luciferase reporter assay

Up-regulation of RBM38 lead to a significant increase in wtp53 and inhibition of mdm2 protein levels compared to their corresponding controls.We found that the luciferase activity for a reporter carrying mdm2 3¡ä-UTR-B and -C was significantly repressed by RBM38 in SMMC7721-RBM38-OE cells. By contrast, the mdm2 3¡ä-UTR-A was not responsive to RBM38. In Hep-G2-RBM38-OE cells, the luciferase activities for reporters carrying mdm2 3¡ä-UTR-A, -B and -C were significantly repressed by RBM38.

--

Human

L

apoptosis

30,176,896

Gene

0

1

0

1

0

1

0

TFEB

7942

protein coding

BEAS-2B

Lung

Chronic obstructive pulmonary disease

Prevent

MTS assay

Our data shows that CSE-induced significant increase in ROS-activity is controlled by TFEB-induction, using GEM/FIS.Moreover, TFEBinduction by GEM/FIS also rescues the CSE-mediated decrease in cell viability as shown by the MTS assay.We and others have recently observed that tobacco-smoke/eCV exposure leads to cellular senescence ,via ROS-activation that accelerates lung aging and COPD-emphysema pathogenesis. We demonstrate here that CSE-mediated increase in number of senescent cells is significantly diminished by treatment with TFEB-inducing drugs, GEM/FIS.

--

Human

L

apoptosis

27,835,930

Gene

0

1

0

TFDP1

7027

protein coding

HeLa,A549,WI-38,Saos-2

--

Cervical cancer

Prevent

Knockdown//SA--gal activity assay

Based on SA-¦Â-Gal activity after DP1 or DP2 siRNA transfection, HeLa, A549 and WI-38 cells, but not p53-null Saos-2 cells, exhibited an increased percentage of SA-¦Â-Gal-positive cells only upon DP1 depletion. The acute loss o DP2 failed to induce senescence.

--

p53//p21

--//--

qRT-PCR//Western blot//Knockdown

In all cells examined, the acute loss of DP1, but not DP2, induced a p21Waf1/Cip1 mRNA level comparable to that in the control RNA-treated cells .The accumulation of p53 and p21Waf1/Cip1 protein was clearly detected in DP1-silenced cells, whereas the knock-down of DP2 alone had no effect on the p53 and p21Waf1/Cip1 protein levels in any of the cells that were examined, compared with the control RNA-transfected cells.

Human

HL

apoptosis

22,012,588

Gene

0

1

0

1

0

BRCA1

672

protein coding

HBLpS,MCF-7pS,HBLpS-BR,MCF-7pS-BR,HCC1937,HCC+BR

--

Breast cancer

Prevent

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

Accordingly to cytologic data, BRCA1-defective cells showed also a more pronounced induction of the cyclin-dependent kinase inhibitor p21 (CDKN1A) after challenging with MMC, supporting a cell cycle arrest.In fact, treatment with MMC or CDDP resulted in a statistically significant increase in the number of elements showing hallmarks of senescence(enlarged cytoplasm, polynucleation, senescence-associated ¦Â-galactosidase positivity, and negativity for BrdUrd incorporation) in BRCA1-defective cells compared with control cells.

--

Human

L

apoptosis

19,372,557

Gene

0

1

0

1

0

1

0

KDR

3791

protein coding

U373 MG

--

Glioblastoma

Prevent

Knockdown//SA--gal activity assay

VEGFR2 gene silencing induced a 4- to 5-fold increase in cells positive for SA-¦Â-Gal (P <0.001).

--

Human

L

apoptosis

26,420,897

Gene

0

1

0

1

0

SOCS1

8651

protein coding

IMR-90

--

Aging

Accelerate

SA--gal activity assay

Expression of SOCS1 inhibited cell growth and induced senescence, characterized by senescence-associated ¦Â-galactosidase staining and the accumulation of PML bodies.

p53

Activation

Western blot//Luciferase reporter assay

Intriguingly, during SOCS1-induced senescence, the increase in p53 levels was accompanied with serine 15 phosphorylation of p53.SOCS1 was not able to activate the p53 reporter alone, but it did increase the activity of added p53 to a similar extent as PML.

--

Human

HL

apoptosis

20,005,840

Gene

0

1

0

MECP2

4204

protein coding

MSC

--

Aging

Prevent

Knockdown//SA--gal activity assay

We observed signs of senescence in cells treated with Ad©\siRNA©\MECP2, as detected by acid ¦Â©\galactosidase, when compared with cells transduced with Ad©\siRNA©\CTRL.

--

Rb//p53

--//--

RT-PCR//Western blot

In cells transduced with Ad©\siRNA©\MECP2, we observed an increase in RB gene expression at both the mRNA and protein levels and an increase in RB2/P130 gene expression at only the mRNA level. On in vitro MECP2 down©\regulation, we obtained evidence for significant changes in P53 mRNA levels, but no modification of the protein levels occurred.

Human

HL

apoptosis

20,065,150

Gene

0

1

0

1

0

SIRT1

23411

protein coding

BJ

--

Aging

Prevent

SA--gal activity assay//Western blot

Accordingly transfection of specific siRNA oligos independently targeting SIRT1 and SIRT2 significantly decreased expression of SIRT1/2 and induced senescence as shown by increased SA-¦Âgal activity in BJ fibroblasts. Induction of senescence is mediated by DNA damage as evidenced by formation of-H2A.X foci and activation of p53-p21CIP1 pathway. A slight increase in levels of p16INK4A was also detected.

--

Human

L

apoptosis

25,924,011

Gene

0

1

0

1

0

SIRT2

22933

protein coding

BJ

--

Aging

Prevent

SA--gal activity assay//Western blot

Accordingly transfection of specific siRNA oligos independently targeting SIRT1 and SIRT2 significantly decreased expression of SIRT1/2 and induced senescence as shown by increased SA-¦Âgal activity in BJ fibroblasts. Induction of senescence is mediated by DNA damage as evidenced by formation of-H2A.X foci and activation of p53-p21CIP1 pathway. A slight increase in levels of p16INK4A was also detected.

--

Human

L

apoptosis

25,924,011

Gene

0

1

0

1

0

RUNX1

861

protein coding

AML

--

Acute leukemia

Prevent

Knockdown//SA--gal activity assay

After two or more electroporations with RUNX1-CBFA2T1 siRNA, but not with control siRNA, a significant fraction of the Kasumi-1 cells stain positive for ¦Â-galactosidase. Cell counting revealed up to 50% of senescent cells, whereas mock or control siRNA treatment caused only a minor increase in senescent cells compared to untreated cells.

--

Human

L

apoptosis

15,298,716

Gene

0

1

0

1

0

RUNX1T1

862

protein coding

AML

--

Acute leukemia

Prevent

Knockdown//SA--gal activity assay

After two or more electroporations with RUNX1-CBFA2T1 siRNA, but not with control siRNA, a significant fraction of the Kasumi-1 cells stain positive for ¦Â-galactosidase. Cell counting revealed up to 50% of senescent cells, whereas mock or control siRNA treatment caused only a minor increase in senescent cells compared to untreated cells.

--

Human

L

apoptosis

15,298,716

Gene

0

1

0

1

0

ZEB1

6935

protein coding

HUVEC

--

Aging

Prevent

SA--gal activity assay//Western blot

Finally, HUVEC depleted of ZEB1 showed an induction of senescence, displaying a higher percentage of SA-¦Â-gal-positive cells , and an increase of p21 protein.

--

Human

L

apoptosis

21,527,937

Gene

0

1

0

1

0

TP53

7157

protein coding

HN 30,HN 31

--

Squamous cell carcinoma

Prevent

SA--gal activity assay//Western blot

Expression of disruptive TP53 mutations (C176F and E336X) led to decreased SA-¦Â-gal activity compared with empty vector control.metformin decreased clonogenic survival following radiation and increased radiation-induced SA-¦Â-gal activity in HN 31 cells (C176F, disruptive TP53), but had little effect in HN 30 cells (wild type TP53) . Furthermore, after inhibition of wild type p53 expression, metformin was found to potentiate SA-¦Â-gal activity and decrease clonogenic survival.

--

Human

L

apoptosis

22,090,360

Gene

0

1

0

1

0

ILK

3611

protein coding

Rb116,Y79

--

Human retinoblastoma

Prevent

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

ILK knockdown significantly increased SA-¦Â-gal activity in Rb116 cells as compared to control. As has been shown previously for Rb-negative retinoblastoma cell lines, a 5-day exposure to QLT-0267 significantly increased the fraction of Y79 cells in G2+M-phase. The percentage of cells in G2+M-phase was 39.6 ¡À 5% as compared to 24.6 ¡À 2.1% in vehicle control.

Rb

--

SA-¦Â-gal activity assay

ILK siRNA treatment increased SA-¦Â-gal activity in Rb positive cells but not Rb deficient cells.

--

Human

L

apoptosis

26,176,204

Gene

0

1

0

1

0

1

0

IL6

3569

protein coding

HCT116

--

Tumor

Prevent

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

Senescence was induced by either gp130 knockdown, IL-6 neutralization,or IL-6 knockdown.inhibition of the IL-6-dependent activation of the JAK1¨C STAT3 pathway induced senescence following DNA damage.

--

JAK1-STAT3

Activation

Western blot

To confirm that IL-6 is responsible for the phosphorylation of STAT3 and JAK1, an IL-6 neutralizing antibody (Jo-2) was added to the culture medium 1 day after treatment with doxorubicin.Western blot analysis showed that the Jo-2 antibody blocked the phosphorylation of JAK1 and STAT3 but not of p53 and H2AX.These results suggest that autocrine IL-6 activates the JAK1¨CSTAT3 signaling pathway.

Human

L

apoptosis

22,521,547

Gene

0

1

0

1

0

1

0

NGF

4803

protein coding

Res 186,Res 259,U87,A172

--

Glioma

Accelerate

SA--gal activity assay

Microscopic analyses indicated that after 72 hr of exposure to NGF, the HGG cells showed an increase in SA©\¦Â©\Gal positive staining compared with control, suggesting the induction of senescence.

p27//p21//Cyclin D1//p53//p16 //Rb

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

Western blot

In our studies following NGF treatment in A172 cells (expressing p53) we observed p27, p21, cyclin D1, pp53, and p16 enhanced expression and the decrease of phospho©\Rb that directly or indirectly lead to accelerated cell senescence.

NF-¦ÊB

Activation

Western blot

In addition, we have found the involvement of NF©\kB in promoting senescence in PLGG cells, in fact the NGF is able to activated NF©\kB signaling after 24 hr of treatment.

Human

L

apoptosis

30,417,351

Gene

0

1

0

TRIM32

22954

protein coding

HCT116 p53+/+,HCT116 p53-/-

--

Tumor

Prevent

Knockdown//SA--gal activity assay

While ectopic TRIM32 reduced doxorubicin-induced senescence in HCT116 p53+/+ but not in p53?/? cells, TRIM32 knockdown induced senescence in HCT116 p53+/+ but not in p53?/? cells.

p53

Downregulation

Western blot//IP

Western blot assays confirmed that TRIM32 overexpression downregulated p53 in p53+/+ HCT116 and RKO tumors.TRIM32-Flag was co-precipitated by the p53 antibody, and vice versa, indicating that the ectopic TRIM32 interacts with p53 in cells.

--

Human

L

apoptosis

25,146,927

Gene

0

1

0

1

0

EPO

2056

protein coding

DA3/EPOR

--

Leukemia

Accelerate

SA--gal activity assay

SA¦Âgal staining was detected in nearly all cells treated with DNR and EPO but not in the untreated control cells.

--

Human

L

apoptosis

30,622,244

Gene

0

1

0

HRAS

3265

protein coding

HuDE

--

Aging

Accelerate

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

Results confirmed that many H-RasV12 expressing cells showed premature aging within one day from the transfection.Microscope analysis of cells transfected with H-RasV12 showed a senescent phenotype within one day from the end of the selection, with a flattened cellular morphology, followed by detachment from the adhesion surface. Besides, H-RasV12 transfected cells showed a cell growth arrest that was already evident one day after the end of the selection, compared to cells transfected with the empty vector alone as control. Interestingly, analysis by flow cytometry showed that significant fractions of H-RasV12 expressing cells are still in G0/G1 and S-phase of the cell cycle, despite their arrested state and altered morphology. Indeed, cell cycle analysis showed that transfected cells had a 14.3% reduction of G0/G1 phase cells and a 6.4% increase of G2/M phase cells, in comparison with control cells. Moreover a 5.2% increase of cells in sub-G0 phase was present in H-RasV12 expressing cells compared to the control, suggesting a possible apoptosis induction.

GAPDH

Downregulation

Biochemical assay

We observed a significant depletion of GAPDH activity in H-RasV12 transfected cells compared to the control cells.

--

Human

L

apoptosis

23,284,910

Gene

0

1

0

1

0

1

0

NUDT5

11164

protein coding

IMR-90

--

Aging

Prevent

SA--gal activity assay//Western blot

Suppressed NUDT5 expression induced significant rates of senescence within one population doubling, as determined by the senescence-associated ¦Â-galactosidase (SA-¦Â-gal) activity.Additionally, an immunoblotting analysis showed that the apoptosis-related proteins p53, p21, and caspase-3 were activated in shNUDT5 cells .

--

Human

L

apoptosis

23,581,889

Gene

0

1

0

1

0

PDZD2

23037

protein coding

DU 145

--

Prostate cancer

Accelerate

SA--gal activity assay

Furthermore, 108 and 107 M sPDZD2 induced senescence of DU145 cells after 6 days of incubation.

p21/WAF1

Upregulation

Western blot

Similarly, up-regulation of p21CIP1/WAF1 expression to 2.6-, 2.9- and 3.2-fold were observed after the cells were treated with 109, 108 and 107 M sPDZD2, respectively.

p53

Upregulation

Western blot

Treatment of MCF-7 cells with 108 and 107 M sPDZD2 for 48 h resulted in 2.4- and 2.2-fold increases in p53.

Human

L

apoptosis

18,639,375

Gene

0

1

0

BCL2L12

83596

protein coding

MEF

--

Glioblastoma

Prevent

Flow cytometry//SA--gal activity assay

Bcl2L12-expressing MEFs exhibited unabated growth for >35 passages, whereas control cultures showed significantly reduced growth rates or underwent growth arrest with coincidental increase in senescence-associated ¦Â-galactosidase (SA-¦Â-Gal) activity .

--

p53

Downregulation

Western blot

Prompted by the molecular p53 pathway analyses in DNA-damaged MEFs showing reduced p53 accumulation in the presence of Bcl2L12.

Human

L

apoptosis

20,837,658

Gene

0

1

0

1

0

HIF1A

3091

protein coding

IMR-90

--

Aging

Accelerate

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

The knockdown of HIF-1¦Á also strongly suppressed the conversion of the growth-arrested state in Ni-treated cells into senescence,HIF-1¦Á enhanced protein expression of the CDK inhibitor p21 but moderately diminished upregulation of p53 protein or its Ser15 phosphorylation by Ni. we found that its knockdown helped preserve the expression of the S/G2-specific cyclin A at the midrange of our Ni doses and inhibited upregulation of the CDK inhibitor p27.

--

Human

L

apoptosis

28,552,779

Gene

0

1

0

1

0

1

0

1

0

TWIST1

7291

protein coding

H460,A549,H358,H727,Calu-1,Calu-6,H1975

--

Lung cancer

Prevent

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

Representative photomicrographs (20¡Á) of increased SA-¦Â-gal staining in 3 representative NSCLC cell lines following shRNA mediated TWIST1 knockdown(above).We observed a dramatic activation of latent OIS as demonstrated by positive SA-¦Â-Gal staining and characteristic enlarged, flattened cytoplasm morphology in three KRAS mutant NSCLC cells lines (H460, A549, and H358). These findings were further supported by profound cell cycle arrest in these cell lines. as well as by induction of the senescence markers, p21 and/or p27 in all cell lines examined .

--

Human

HL

apoptosis

23,364,532

Gene

0

1

0

1

0

1

0

1

0

1

0

RACK1

10399

protein coding

CaSki

--

Cervical cancer

Prevent

SA--gal activity assay

The number of ¦Â-galactosidase-positive cells in control CaSki-vector cells was much higher than RACK1 overexpression CaSki cells.

--

Human

L

apoptosis

29,048,616

Gene

0

1

0

POT1

25913

protein coding

IMR-90

--

Aging

Prevent

SA--gal activity assay

A premature senescent phenotype was observed in IMR90 cells expressing POT1-RNAi, as assayed by SA-¦Â-Gal staining.

TRF2

--

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

The expression of POT1 decreased the loss of telomeric overhang induced by TRF2¦¤B¦¤M.(B) POT1 blocked the senescent phenotype induced by TRF2¦¤B¦¤M.

--

Human

L

apoptosis

15,657,433

Gene

0

1

0

TERF2

7014

protein coding

IMR-90

--

Aging

Accelerate

SA--gal activity assay

We found that Myc-TRF2¦¤B¦¤M induced premature senescence 16 days after infection in IMR90 cells.

--

Human

L

apoptosis

15,657,433

Gene

0

1

0

TMSB4X

7114

protein coding

Nucleus pulposus cell

Nucleus pulposus tissue

Aging

Prevent

SA--gal activity assay

Furthermore, cell aging and apoptosis were also investigated. In situ SA-¦Â-Gal cell staining was conducted for both control and transfected cells, and the P3 generations of both cell groups were compared. The TB-4 recombinant AAV-transfected cells showed less staining than cells from the control group, which indicated that the transfected cells underwent slower cellular aging.

--

Human

L

apoptosis

26,021,512

Gene

0

1

0

PIM1

5292

protein coding

hCPC

Heart left ventricle

Heart failure

Prevent

Cell morphological analysis//SA--gal activity assay

SA ¦Â-gal positive cells were least prevalent in Nuc-Pim1 hCPCs, with a ?30.25% decrease (p < 0.001) as compared with Nuc-GFP.Longer, spindle-shaped hCPCs were evident after Nuc-Pim1 modification as measured by the length to width ratio and cell roundness. Concurrently, Nuc-Pim1 modification increased the Hayflick limit of hCPCs as compared with PimWT, prolonging the post-mitotic phenotype as demonstrated by the extended expansion capability of modified cells .PimWT and Mito-Pim1 modification resulted in a reduction of SA ¦Â-gal+ cells.

p53//p16//NS

Downregulation//Downregulation//Upregulation

Western blot//qPCR

Gene and protein expression of the cell cycle arrest/senescence marker p53 decreased after Nuc-Pim1 modification with a 1.15-fold reduction of mRNA and a 1.45-fold reduction of protein, as measured by qPCR and immunoblot analysis, respectively.Concurrent down-regulation of the p16 gene and protein expression occurred in Nuc-Pim1 hCPCs as evident by 1.85- and 1.96-fold reductions, respectively. A highly significant increase in NS gene expression resulted from Nuc-Pim1 expression in hCPCs (2.59-fold, p < 0.001) compared with PimWT and Mito-Pim1, as measured by qPCR analysis. Concurrent up-regulation of Ns protein expression was also detected in Nuc-Pim1 hCPCs (2.17-fold, p < 0.01) compared with controls .

--

Human

HL

apoptosis

25,882,843

Gene

0

1

0

1

0

TERF2

7014

protein coding

SH-SY5Y

--

Neuroblastoma

Prevent

BrdU assay//SA--gal activity assay

DN-TRF2 expression in neuroblastoma cells resulted in a significant reduction in their proliferation as indicated by a decrease of BrdU incorporation, limited growth rate and induction of expression of ¦Â-galactosidase.

p21//p53

Upregulation//Upregulation

Western blot

Levels of p21 and p53 were increased by more than 10-fold in neuroblastoma cells expressing DN-TRF2.

--

Human

L

apoptosis

16,539,655

Gene

0

1

0

1

0

KDM4B

23030

protein coding

HCT116,SW480

--

Colorectal cancer

Prevent

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

Positive SA-¦Â-gal staining was observed at 72 h post-transfection in about 49% and 20% of JMJD2B-silenced HCT116 and SW480 cells, respectively, but only <2% of the control group were SA-¦Â-gal positive.Compared with the control group, there was a significant increase of HCT116 cells with maximum 4N content at 12?h, and an increase of SW480 cells with maximum 2N content at 24?h after JMJD2B silencing in hypoxia, indicative of G2 and G1 phase arrest, respectively. Morphologically, JMJD2B-silenced cells were larger and appeared flattened (data not shown), which is a feature of senescence (Itahana et al, 2007).

--

STAT3

--

Western blot

Although a slight decrease of total STAT3 was visually observed, p-STAT3 expression significantly decreased in JMJD2B-silenced cells in both normoxia and hypoxia, which was confirmed upon quantification.

Human

HL

apoptosis

24,473,398

Gene

0

1

0

1

0

1

0

VASH1

22846

protein coding

HT29

--

Colorectal cancer

Accelerate

SA--gal activity assay

We found significantly increased SA-¦Â-Gal positive cell populations in HT29 tumor cells after transfection with VASH1-A, indicating the induction of tumor cell senescence.

--

Human

L

apoptosis

25,797,264

Gene

0

1

0

CDK2AP1

8099

protein coding

HDF

--

Aging

Prevent

Knockdown//SA--gal activity assay

We found that 91.7% of HDFs transduced with CDK2AP1-shRNA1 and 70.8% of CDK2AP1-shRNA2 transduced cells displayed appearance of enlarged blue cells in contrast to the control EV transduced cells, which failed to exhibit a detectable blue appearance.

p53//p21//BAX//PUMA

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

Western blot//qPCR

Indeed we have found that HDFs in which CDK2AP1 was down regulated had a significant increase in p53 protein levels. we found that p21 mRNA levels increased by 2.1 fold in the CDK2AP1-shRNA1 cells and by 1.8 fold in the CDK2AP1-shRNA2 HDFs when compared with EV transduced cells. Western blot analysis further confirmed the increase in p21 protein levels.QPCR analysis indicated that BAX mRNA levels were increased by 1.5 fold in the CDK2AP1-shRNA1 transduced HDFs and by 2.4 fold in the CDK2AP1-shRNA2 when compared with EV transduced cells. Similarly, we observed that PUMA mRNA levels were increased by 2.0 fold in the CDK2AP1-shRNA1 cells and by 3.6 fold in cells transduced with CDK2AP1-shRNA2 when compared with EV cells.

--

Human

L

apoptosis

25,785,833

Gene

0

1

0

1

0

PLA2R1

22925

protein coding

HMEC

--

Breast cancer

Accelerate

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

HMECs ectopically expressing PLA2R1 preferentially underwent proliferation arrest as measured by a colony formation assay, a decrease in the phosphor Ser10 of histone 3 (H3-pSer10), and an increased staining in the senescence-associated-¦Â-galactosidase assay.

--

Human

L

apoptosis

23,994,771

Gene

0

1

0

1

0

1

0

GPC3

2719

protein coding

HuH-7,HepG2

--

Hepatocellular carcinoma

Prevent

Flow cytometry//SA--gal activity assay

Suppression of GPC3 caused a significant increase in G1 peak in HepG2 cells (P =0.04 compared with siRNA-N), together with a decrease in S-phase cells.Transfection with siRNA-GPC3 alone caused marked accumulations of SA-¦Â-Gal in both cell lines.

TGF-¦Â2

--

Western blot//Semi-RT-PCR

A member of theTGFBR pathway, TGF-¦Â2 (but not TGF-¦Â1 or TGF-¦Â3), was significantly upregulated by GPC3 suppression in both Huh7 and HepG2 cells.The up-regulation of TGF-¦Â2 by siRNA-GPC3 was independently validated using semiquantitative RT-PCR and Western blot analysis to confirm the negative correlation at both transcript and protein levels, respectively.

--

Human

HL

apoptosis

21,847,365

Gene

0

1

0

1

0

TGFB2

7042

protein coding

HuH-7

--

Hepatocellular carcinoma

Accelerate

Flow cytometry//SA--gal activity assay

We also observed that addition of rhTGF-¦Â2 induced cellular senescence in both Huh7 and HepG2 cells as shown by enhanced accumulation of SA-¦Â-Gal. Huh7 and HepG2 cells were grown in culture medium supplemented with rhTGF-¦Â2 (1 or 5 ng/ml) for a period of 48 hours before flow cytometry analyses. Similar to the effects of GPC3 suppression, significant increases in the G1 peak , together with a decrease in S- and G2-phase cells were observed in both cell lines compared with control cells not treated with rhTGF-¦Â2 .

Cyclin A//Cyclin D1//Rb//p15INK4b//p21//Bcl-xL//Mcl-1//Bcl-2

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

Western blot

Western blot analysis further showed that rhTGF-¦Â2 decreased the expression of cell cycle regulators cyclin A and cyclin D1, as observed with GPC3 suppression . In addition, phosphorylation of retinoblastoma (Rb) protein was decreased, whereas the expression of p15Ink4b and p21Cip1 were increased in both Huh7 and HepG2 cells, indicating a G1 cell cycle arrest. The addition of TGF-¦Â2 also increased the apoptotic response, demonstrated by the reduced expression of antiapoptotic proteins (Bcl-xL and Mcl-1) in both cell lines, and the reduced expression of Bcl-2 in HepG2 cells only (Huh7 cells do not express Bcl-2).

--

Human

HL

apoptosis

21,847,365

Gene

0

1

0

1

0

ERBB4

2066

protein coding

Saos-2,MG63.2

--

Osteosarcoma

Prevent

Knockdown//SA--gal activity assay

After 3, 5 and 7?days in monolayer culture, knockdown of endogenous HER4 significantly inhibited the proliferation of OS cells. Staining of tumor sections with SA-¦Â-gal showed much higher senescence levels in shHER4 derived xenografts. Cytochemical analysis on SA-¦Â-gal activity revealed that there was a dramatic increase of SA-¦Â-gal-positive cells in shHER4 colonies as compared with scramble shRNA colonies.

PERK//p21//p27

--//--//--

Western blot//Knockdown

We detected that the results showed that the senescence-associated protein p-ERK, p21 and p27 levels were increased in cells with HER4 knockdown.

--

Human

HL

apoptosis

29,524,631

Gene

0

1

0

1

0

SOD1

6647

protein coding

Mammary epithelial cell

Mammary Gland

Breast cancer

Prevent

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

We found an increase in ¦Â-gal staining in the SOD1+/?/ErbB2 cells .Using p53 and p16 as markers of senescence, we found a significant increase in both markers in the SOD1?/?ErbB2 mammary glands. To confirm that senescence was induced in the epithelial cells, we performed immunohistochemistry of p16 and found a significant increase in p16 in the SOD1?/?/ErbB2 mammary glands.

--

Human

L

apoptosis

31,222,103

Gene

0

1

0

1

0

1

0

1

0

SIRT1

23411

protein coding

A549

--

Idiopathic pulmonary fibrosis

Prevent

Western blot

We found that Sirt1 overexpression partially rescued miR-34a-induced cellular senescence in A549 cells, as evidenced by reduced expression of PAI-1 and p21 in these cells.

--

Human

L

apoptosis

27,979,858

Gene

0

1

0

ZBTB7A

51341

protein coding

U87 MG

--

Glioma

Prevent

SA--gal activity assay

We examined the role of Pokemon in RSVinduced cell senescence by detecting the senescenceassociated ¦Â-galactosidase (¦Â-gal) activity. We found that overexpression of Pokemon reduced RSV-induced cell senescence in U87MG cells.

--

Human

L

apoptosis

25,875,864

Gene

0

1

0

TP53

7157

protein coding

HSC-1

--

Skin cancer

Accelerate

SA--gal activity assay

Clones in which PA treatment induced p53 protein expression showed a Xat and extended morphology such as is commonly observed in senescent cells.we examined whether p53-expressing clones execute senescence-like growth arrest by the expression of a senescence marker, SA-¦Â-gal.A large number of the PAtreated Clone 4 cells stained positive for SA-¦Â-gal at 7 days, while none of the cloned cells not treated with PA were stained blue.

--

Human

L

apoptosis

19,009,304

Gene

0

1

0

AKAP4

8852

protein coding

COLO 205 ,HCT116

--

Colorectal cancer

Prevent

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

The percentage of senescent cells was significantly higher in COLO 205 (44.2 %) and HCT 116 cells (48.4 %) when transfected with AKAP4 shRNA3 as compared to NC shRNA staining . AKAP4 ablation also resulted in the upregulation of decoy receptor 2 (DCR2) protein expression which is a marker for cellular senescence.

--

Human

L

apoptosis

26,590,805

Gene

0

1

0

1

0

1

0

POT1

25913

protein coding

BGC-823

--

Gastric cancer

Prevent

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

After 24 h of culturing, the cells were incubated with ¦Â-galactosidase and observed microscopically. In the control cells, only one or two cells showed weak staining. However, after silencing hPOT1 with RNAi, the cells appeared large and flat, some of which were strongly stained with blue. The ratio of senescent cells in blue was 144 ¡À 12.4/105 (n = 3).The telomere length assay showed that the T/S value = [2Ct (telomere)/2Ct (36B4)] ¨C 1 of the hPOT1 RNAi cells (1.383 ¡À 0.091) was statistically less than that of the control cells (3.043 ¡À0.548) (P= 0.007, n= 3), indicating that the loss of hPOT1 in BGC823 cells leads to the shortening of the telomere.

--

Human

HL

apoptosis

20,517,159

Gene

0

1

0

1

0

1

0

PPP1R13L

10848

protein coding

OCCC,OVTOKO,KK

--

Ovarian cancer

Prevent

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

After 72 hr of lentivirus infection, both OVTOKO and KK cells showed more SA©\¦Â©\Gal staining in iASPP knockdown cells than the scramble control.The expression of p21WAF1/Cip1, an inducer for senescence, also increased in OCCC cells with iASPP silencing.

--

Human

L

apoptosis

29,663,364

Gene

0

1

0

1

0

1

0

BTG1

694

protein coding

HCT-15,HCT116

--

Colorectal cancer

Accelerate

PI staining//SA--gal activity assay

BTG1 overexpression caused G2 arrest in HCT-15 cells (p<0.05), while G1 arrest in HCT-116 cells (p<0.05) by PI staining.A lower mitochondrial membrane potential by JC-1 staining and a higher senescence by ¦Â-galactosidase staining were detectable in HCT-116 transfectants than the mock and control, while there was no significant alteration in HCT-15 transfectants.

--

Human

HL

apoptosis

27,447,746

Gene

0

1

0

1

0

DEK

7913

protein coding

Melanocyte,SK-Mel-28,SK-Mel-94,G-361,UACC-62,UACC-257,MM576

--

Melanoma

Prevent

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

In 6 of 12 lines analyzed (SK-Mel-28, SK-Mel-94, G-361, UACC-62, UACC-257, and MM576), DEK depletion led to a progressive cell cycle arrest, eventually resulting in a near complete inhibition of cell proliferation as determined by total cell counting and flow cytometry.The reduction of cells in S phase of the cell cycle was accompanied by cell enlargement, flattening, and vacuolization reminiscent of classic senescent phenotypes . In fact, staining for senescence-associated ¦Â-galactosidase activity was positive in DEK-depleted melanoma cells.

--

Human

HL

apoptosis

19,679,545

Gene

0

1

0

1

0

1

0

RHEB

6009

protein coding

MEF

--

Lymphoma

Accelerate

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

Rheb expression causes morpho logical changes (data not shown), induction of senescence-associated ¦Â-galactosidase (SA ¦Â-Gal) ,early growth arrest (Vector vs. Rheb, P < 0.0001),and p16 protein induction .

p53//mTORC1

--//--

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

As expected, these effects depended on p53 and did not occur in the context of c-Myc expression.Notably, pharmacologic inhibition of mTORC1 with rapamycin could prevent many phenotypic changes associated with senescence induced by Akt and Rheb and had a partial effect on Ras-induced senescence.

--

Human

L

apoptosis

18,708,578

Gene

0

1

0

1

0

1

0

1

0

BRAF

673

protein coding

Melanocyte

--

Melanoma

Accelerate

Cell morphological analysis//SA--gal activity assay

Expression of BRAF(V600E) induced senescence in melanocytes and led to an increase in the percentage of senescent cells with the characteristic enlarged cell body and positive staining of senescence-associated ¦Â-galactosidase (SA-¦Â-Gal) activity.

--

Human

HL

apoptosis

19,805,117

Gene

0

1

0

1

0

TYRO3

7301

protein coding

Melanocyte

--

Melanoma

Prevent

Colony formation assay//SA--gal activity assay

Expression of TYRO3 alone did not have clear effect compared to control virus infected melanocytes. Expression of BRAF(V600E) induced senescence in melanocytes and led to an increase in the percentage of senescent cells with the characteristic enlarged cell body and positive staining of senescence-associated ¦Â-galactosidase (SA-¦Â-Gal) activity . However, when TYRO3 was co-expressed with the BRAF(V600E) mutant, primary melanocytes formed colonies in which non-senescent cells dominate the population, as indicated by a strong reduction in the percentage of SA-¦Â-Gal positive cells.

--

Human

HL

apoptosis

19,805,117

Gene

0

1

0

1

0

TIGAR

57103

protein coding

OVCA420

--

Ovarian cancer

Prevent

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

Within 3?h of the pulse-chase, most of the cells in TIGAR-proficient, scrambled siRNA-transfected cells were progressed into G2/M phase whereas the majority of TIGAR KD cells remained in S-phase. In addition to delay in S-phase, G2/M transition is also affected in TIGAR KD cells because remarkably lower number of phospho-H3-positive M-phase cells were observed in TIGAR KD cells compared to TIGAR-proficient counterparts. we observed an increase in SA-¦Â-gal staining in the TIGAR KD cells and the extent of cells undergoing senescence is inversely correlated with TIGAR expression level. Similar results were observed in A2780 cells.

--

Human

HL

apoptosis

31,508,509

Gene

0

1

0

1

0

1

0

1

0

GRB2

2885

protein coding

H1299,SPCA1

--

Lung cancer

Prevent

Flow cytometry//SA--gal activity assay

Up©\regulation of GRB2 effectively counteracted the inhibitory effect of miR©\1258 up©\regulation on H1299 cell proliferation. Similarly, GRB2 down©\regulation in SPCA1 cells reversed the proliferation©\promoting effect of low expression levels of miR©\1258. Moreover, it was found that up©\regulation of GRB2 suppressed cellular senescence and apoptosis, whereas the down©\regulation of GRB2 had an opposite effect.

--

Human

L

apoptosis

30,069,987

Gene

0

1

0

1

0

ZBTB7A

51341

protein coding

FS090,SW 13 53

--

Chondrosarcoma

Prevent

Flow cytometry//SA--gal activity assay

When compared with wild-type and EGFP-siRNA control cells, LRF-siRNA-expressing cells exhibited a significant accumulation of cells in the G 1 phase, with a corresponding reduction of cells in the S and G 2 /M phases .the percentages of ¦Â-galactosidase-positive cells were markedly increased in LRF-depleted FS090 (~50%) and SW1353 (~80%) cells .

p53//p21

--//--

Western blot

The western blot results demonstrated that p53 was markedly elevated in LRF-depleted FS090 and SW1353 cells.The expression of p21 was also markedly increased.

--

Human

HL

apoptosis

22,847,180

Gene

0

1

0

1

0

TBPL1

9519

protein coding

HCT116

--

Colorectal cancer

Accelerate

Knockdown//SA--gal activity assay

TLP-depleted cells exhibited resistance to senescence after UV irradiation.

p53

--

Western blot//qRT-PCR

The protein levels of both total p53 and Lys-382-acetylated p53, which is an activation marker for p53 transcriptional activity, were elevated within 4 h and decreased around 32¨C36 h after UV irradiation. Notably, TLP was significantly elevated around 32¨C36 h, and depletion of TLP accelerated the decrease of both total p53 and Lys-382-acetylated p53 during these time points . TLP-depleted cells clearly showed the reduction of p53 transcriptional activity in the late phase (at 32¨C48 h) of the UV response.

--

Human

L

apoptosis

28,082,682

Gene

0

1

0

1

0

YAP1

10413

protein coding

A549

--

Lung cancer

Prevent

SA--gal activity assay

YAP plus TEAD completely blocked NCTD-induced senescence enhancement.

--

Human

L

apoptosis

27,903,989

Gene

0

1

0

TEAD2

8463

protein coding

A549

--

Lung cancer

Prevent

SA--gal activity assay

YAP plus TEAD completely blocked NCTD-induced senescence enhancement.

--

Human

L

apoptosis

27,903,989

Gene

0

1

0

DDX24

57062

protein coding

U2OS

--

Cancer

Prevent

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

43% of the total control cells arrested at G1 phase, and DDX24 knockdown led to the significant increase of G1 population to 62%. In accordance, the reduction of S phase is drastic when DDX24 is depleted from cells.DDX24 siRNA treated cells, under same treatment, did not undergo apoptosis but showed even more predominant G1/S arrest.prolonged culturing of DDX24 siRNA treated U2OS cells ultimately resulted in enlarged and flattened cell shape, the phenotypes typical of senescence45. DDX24 knockdown cells appeared nearly 100% positive for ¦Â-galactosidase staining, which proves the occurrence of senescence in cells lacking DDX24 protein.

p53

--

Knockdown//Western blot

RNAi mediated knockdown of DDX24 in U2OS cells significantly augmented expression level of p21 and PUMA, the classic p53 targets in regulating cell cycle arrest and apoptosis. Therefore, DDX24 acts as a negative modulator of p53 transcriptional activity.

--

Human

L

apoptosis

25,867,071

Gene

0

1

0

1

0

1

0

1

0

1

0

MAPK1

5594

protein coding

EC109,EC109/CDDP

--

Esophageal cancer

Accelerate

Annexin V binding assay//PI staining//SA--gal activity assay

Inhibition of ERK1/2 significantly suppressed cisplatin-induced apoptosis and senescence in EC109 and EC109/CDDP cells.

--

Human

L

apoptosis

25,236,911

Gene

0

1

0

1

0

1

0

POT1

25913

protein coding

HT-1080,IMR-90

--

Aging

Prevent

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

Microscopic examination revealed a fraction of the hPot1 knockdown HT1080 cells with senescent phenotypes (not shown)microscopic examination revealed a fraction of the hPot1 knockdown HT1080 cells with senescent phenotypes (not shown).The Pot1 shRNA elicited a more severe senescent phenotype in these cells; many cells exhibited an enlarged and flattened morphology and stained strongly positive for ¦Â-galactosidase (¦Â-gal), a marker of senescence.

--

Human

L

apoptosis

15,620,654

Gene

0

1

0

1

0

1

0

RING1

6015

protein coding

HepG2,HCT116

--

Hepatocellular carcinoma

Prevent

Flow cytometry//SA--gal activity assay

Significant induction of senescence was observed, as 3- to 7-fold more cells were positively stained in the RING1-deficient group showed than in the controls. FACS analysis also indicated that, upon RING1 depletion, increased cell subpopulations were found to retain in G1 phase (increased from 60.0% to 68.4% in HepG2 cells, and from 53.1% to 58.9% in HCT116 cells), suggesting that RING1 deficiency might at least cause partial G1 arrest.

p53

--

Knockdown//Western blot

RING1 knockdown decreased the ubiquitination of p53.In a reconstituted in vitro ubiquitination assay that included E1, UbcH6 (E2), ATP and Ub, RING1 was also found to ubiquitinate p53.

--

Human

HL

apoptosis

29,187,402

Gene

0

1

0

1

0

RANBP9

10048

protein coding

A549,H460

--

Lung cancer

Prevent

Knockdown//SA--gal activity assay

Silencing of RanBP9 resulted in a significant increase of SA-¦Â-gal positive cells following IR exposure in both RanBP9 stably-silenced A549 and H460 cells, compared to their negative controls, respectively.

--

Human

L

apoptosis

26,943,034

Gene

0

1

0

1

0

TP53

7157

protein coding

Saos-2

--

Cancer

Accelerate

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

Bright©\field microscopic images of COE alone, COE + vector and COE + p53 (V138) SKOV©\3 cells at 32 ¡ãC and 37 ¡ãC showed growth arrest in COE + p53 (V138) at 32 ¡ãC only .We examined HP1¦Ã nuclear heterochromatin foci in p53 (V138)©\transfected COE Saos©\2 and SKOV©\3 cells (data not shown) at 32 ¡ãC and 37 ¡ãC. HP1¦Ã foci were observed only at 32 ¡ãC in both the cell lines. We observed ¦Â©\galactosidase positive senescence in p53 (V138)©\transfected COE cells at 32 ¡ãC. Whereas a significant increase in ¦Â©\galactosidase positive cells was seen in p53 (V138)©\transfected cells cultured at 32 ¡ãC, the number of Ki©\67 positive cells was low.

--

Human

HL

apoptosis

26,278,998

Gene

0

1

0

1

0

1

0

IFITM3

10410

protein coding

ORL-150,ORL-204

Human OSCC primary tumour

Oral cancer

Prevent

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

When examining cells that did not undergo cell death following IFITM3 knockdown, we noted that the cell morphology appeared to be flattened with an enlarged cytoplasm as well as an accumulation of vacuoles. This significant increase in senescence-associated ¦Âgalactosidase staining indicates that IFITM3 knockdown induces senescence in ORL-150 (48%) and ORL-204 (14.2%) cells compared to the respective NT control cells.By doing so, we indeed observed a significant increase of cells in the G1 phase following IFITM3 knockdown in ORL-150 (86.7%) and ORL-204 (62.5%) cells compared to NT control cells, thus confirming that IFITM3 knockdown induces senescence and accumulation of cells in the G1 cell cycle phase.

--

CCND1-CDK4-pRb

Upregulation

Western blot//Knockdown

We found that the expression of CCND1 was markedly reduced following IFITM3 knockdown in both cell lines. Similarly, we found that the CDK4 expression levels were reduced, but recovered at day 6 post-transfection when IFITM3 levels increased again.The reductions in both CCND1 and CDK4 following IFITM3 knockdown eventually resulted in a reduction in RB phosphorylation (pRB) and to a lesser extent in total RB expression.

Human

L

apoptosis

30,949,979

Gene

0

1

0

1

0

1

0

1

0

SIRT3

23410

protein coding

Nucleus pulposus cell

Nucleus pulposus tissue

Disc degenerative disease

Prevent

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

The results of the Western blot analysis showed that SIRT3 knockdown augmented the senescence marker protein expression of p16INKa, while SIRT3 overexpression suppressed the p16INKa level under TBHP-induced oxidative stress conditions.Compared to the NPCs transfected with scramble lentivirus, the percentage of SA-¦Â-gal-positive staining cells in the SIRT3 knockdown group was increased, whereas SIRT3 overexpression suppressed the percentage of senescent cells under conditions of oxidative stress.

--

Human

L

apoptosis

30,420,619

Gene

0

1

0

1

0

1

0

BECN1

8678

protein coding

U87

--

Glioma

Prevent

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

Beclin-1 knockdown was found to inhibit cell proliferation,apparently, due to induction of cell cycle arrest (in 61% to 84% of total cells) in G1 phase.Beclin-1 knockdown in U87 cells results in blue ¦Âgalactosidase staining, which is known to be associated with expression and pH-dependent activation of a lysosomal enzyme senescence-associated ¦Â-galactosidase (SA-¦Â-gal), a commonly used biomarker for cellular senescence.

--

Human

HL

apoptosis

29,991,800

Gene

0

1

0

1

0

1

0

1

0

1

0

KDM2B

84678

protein coding

MDA-MB-231,T47D,MCF-7

--

Breast cancer

Prevent

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

Flow cytometry of EtBr-stained semiconfluent cell cultures growing under normal tissue culture conditions revealed that the knockdown of NDY1 induces a partial G1 arrest in all the cell lines,and suggested that NDY1 contributes to progression from G1 to S. stained for ¦Â-galactosidase (¦Â-gal), revealed that the knockdown elicits a strong senescence phenotype, which, however, is limited to T47D cells.

--

Human

L

apoptosis

24,853,546

Gene

0

1

0

1

0

1

0

TP63

8626

protein coding

keratinocyte,JHU-011,JHU-029

Foreskin

Skin cancer

Prevent

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

Induction of the cell cycle inhibitor p21CIP1 was observed following knockdown of p63 expression by RNAi in primary human keratinocytes and was associated with an increase in G1-phase cells and a marked decrease in S phase.Little or no evidence of cell death was observed in primary human keratinocytes following lentiviral p63 knockdown. Instead, morphologic features of cellular senescence were observed in a subset of cells, and these changes correlated with staining for SA-¦Â-gal.

--

Human

L

apoptosis

17,018,588

Gene

0

1

0

1

0

1

0

1

0

BECN1

8678

protein coding

HT22

--

Aging

Prevent

SA--gal activity assay//Western blot

SA-¦Â-gal staining gradually increased in a time-dependent manner when HT22 cells were treated with glutamate or H2O2. Also, levels of some senescence markers including the cyclin-dependent kinase inhibitors p16INK4a, p21, p53 and p27Kip1 increased over time in HT22 cells treated with glutamate or H2O2.Also, treatment of ALLN calpain inhibitor caused a decrease of these senescence markers in 0.5 mM H2O2 -treated HT22 cells.

--

Human

L

apoptosis

30,807,795

Gene

0

1

0

1

0

EP400

57634

protein coding

U2OS,IMR-90

--

Aging

Prevent

DAPI staining//Flow cytometry//Knockdown//SA--gal activity assay//Western blot//qPCR

We found that p400 knockdown induces the activation of the gene encoding the p21 cell cycle-dependent kinase inhibitor, as well as the concomitant accumulation of cells in the G1 phase of the cell cycle. As already demonstrated [11], transfection of p400 siRNA leads to senescence of IMR90 cells, as observed by the appearance of the so-called SAHF (Senescence-Associated Heterochromatin Foci), the induction of Senescence¨CAssociated ¦Â-Galactosidase activity and induction of p16 mRNA expression.

ATM

--

Immunofluorescence//Knockdown//Flow cytometry

We observed by immunofluorescence staining that p400 knockdown induces ATM phosphorylation both in IMR90 cells and HCT116 cells.We found that the knockdown of p400 leads to an increase of ROS levels (measured by calculating the mean fluorescence from 25,000 cells) detectable 48 hours and 72 hours following siRNA transfection.

--

Human

HL

apoptosis

20,548,951

Gene

0

1

0

1

0

1

0

1

0

1

0

1

0

SRC

6714

protein coding

HT-1080

--

Aging

Prevent

Flow cytometry//SA--gal activity assay

Surprisingly, the fraction of SA-¦Â-gal¨Cpositive cells was greatly reduced in the Src-expressing population, As expected, most of the control cells were arrested in the fraction with 4N DNA content.By contrast, BrdUrd incorporation was seen in the Src population with DNA content between 4N and 8N, suggesting that these cells exit mitosis and then re-replicate their DNA.

p21

--

RT-PCR//Western blot

As expected, RT-PCR analysis indicated that p21waf1 accumulated in control cells 36 to 72 hours after drug treatment. Surprisingly, p21waf1 was not significantly expressed in Src-expressing cells.

--

Human

L

apoptosis

16,204,065

Gene

0

1

0

1

0

IL22

50616

protein coding

HSC

Liver

Liver disease

Accelerate

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

Immunohistochemistry staining revealed an increase in the number of SA©\¦Â©\Gal+ cells in livers of Ad©\IL©\22©\treated mice than in the livers of Ad©\GFP©\treated mice, and these cells tended to reside within fibrotic scar tissue.Approximately 60% of HSCs from Ad©\IL©\22©\treated mice were positive for SA©\¦Â©\Gal staining, whereas only 20% of HSCs from Ad©\GFP©\treated mice were positive.IL©\22 treatment increased the number of SA©\¦Â©\Gal+ HSCs, but decreased telomerase activity in HSCs. IL©\22 exposure up©\regulated the expression of several cellular senescence©\associated proteins, including phosphorylated p53 at serine 15 (p©\p53ser15), p53, p21, and SOCS3.

STAT3//p53//SOCS3

Activation//Upregulation//Upregulation

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

STAT3 protein deletion was confirmed by western blotting. IL©\22 treatment up©\regulated SA©\¦Â©\Gal activity and the expression of p©\p53ser15, p53, and p21 in WT HSCs, but not in STAT3?/? HSCs.The expression of Flag©\caSTAT3 was confirmed by western blotting. Infection with Ad©\Flag/caSTAT3 markedly decreased ¦Á©\SMA protein expression , but increased SA©\¦Â©\Gal staining in HSCs . Moreover, the introduction of Flag©\caSTAT3 increased the expression of p©\p53ser15, p53, and p21 in HSCs .IL©\22 exposure up©\regulated the expression of SOCS3 mRNA and protein in HSCs.IL©\22 treatment or caSTAT3 overexpression up©\regulated both p53 and SOCS3 proteins.The effects of IL-22 on the signaling pathways in HSCs. IL-22 exposure significantly activated STAT3 in all samples with peak effects observed at 30-60 min. Furthermore, IL-22-dependent STAT3 activation in HSCs was further confirmed by immunostaining for pSTAT3 in the nuclei of HSCs.

--

Human

L

apoptosis

22,473,749

Gene

0

1

0

1

0

1

0

1

0

PRMT5

10419

protein coding

GBMNS

Brain

Glioblastoma

Prevent

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

PRMT5 depletion increased the G1 population of GBMNS by atleast 30% implicating G1/S cell cycle arrest. Although control cells had undetectable ¦Â-gal-positive cells, there were more than 100 ¦Â-gal-positive cells per view field in the PRMT5-depleted GBMNS.

p53//p21//Rb

--//--// --

RT-PCR//Western blot//Knockdown

PRMT5 knockdown increased the relative mRNA expression of p21 and p53. Further we also probed for the expression of Rb, p53 and p21 proteins with or without PRMT5 knockdown .Consistent with senescence phenotype, we found decreased pRb and increased expression of p53 and its target gene p21.

--

Human

HL

apoptosis

27,292,259

Gene

0

1

0

1

0

1

0

PTEN

5728

protein coding

GBMNS

Brain

Glioblastoma

Accelerate

Knockdown//SA--gal activity assay

We tested the impact of PTEN overexpression in GBMNS on senescence.Similar to the results obtained with PRMT5 knockdown, PTEN overexpressing cells showed a significant increase in ¦Â-gal-positive GBMNS.

--

Human

HL

apoptosis

27,292,259

Gene

0

1

0

1

0

RB1

5925

protein coding

MSC

--

Retinoblastoma

Prevent

Flow cytometry//SA--gal activity assay

In samples with silenced RB1, the percentage of cells in S-phase is lower than the unirradiated wild type MSCs. In cells with down-regulated RB1, we observed a significant reduction of clones in basal condition vs. controls. This agrees with the high levels of DNA damage and senescence.

--

Human

L

apoptosis

27,124,644

Gene

0

1

0

1

0

RBL1

5933

protein coding

MSC

--

Retinoblastoma

Prevent

Flow cytometry//SA--gal activity assay

Cells lacking P107 gene expression showed a reduction in S-phase cells only at 48 hours after irradiation. irradiation of wild-type MSCs increased the percentage of senescent cells. A similar pattern was observed in cells lacking RB2 or P107.

--

Human

L

apoptosis

27,124,644

Gene

0

1

0

1

0