Venkatachalam/aging_reg_dataset · Datasets at Fast360

SETD1A

9739

protein coding

MDA-MB-231,MCF-7,MDA-MB-468,BT-549,HT1299,A549

Mammary Gland,Lung

Aging

Prevent

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

Remarkably, knockdown of SETD1A(SETD1A-KD) suppresses proliferation and triggers prompt (72h) and massive cellular senescence, with very large cells expressing characteristic ?--galactosidase (?-gal) activity. The cell cycle arrest resulting from SETD1A-KD is consistent with cellular senescence, and it is not associated with apoptosis as seen by the absence of the caspase-3 and PARP cleavage markers of apoptosis, following 3 and 7-days of SETD1A-KD23,24.

SKP2

--

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

Inducible expression of SKP2 in SETD1A-KD cells(using two different shSET1A constructs) effectively reduced the expression of p27 and p21 proteins.Staining for ?-Galactosidase shows that induction of SKP2 partially suppressed the emergence of ?-Gal-positive senescent cells by 50% following SETD1A-KD.

--

Human

HL

genomic instability

31,253,781

Gene

0

1

0

1

0

1

0

TRPC7

57113

protein coding

Keratinocyte

Lung

Aging

Accelerate

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

We found that after UVB irradiation, the knockdown of TRPC7 in keratinocytes significantly decreased the percentage of senescent cells, as determined by SA-¦Â-gal staining .Furthermore, the knockdown of TRPC7 inhibited UVB-induced p53 expression in keratinocytes.

--

Human

HL

genomic instability

31,755,176

Gene

0

1

0

1

0

1

0

AGT

183

protein coding

VSMC

Aorta

Atherosclerosis

Accelerate

SA--gal activity assay

SA-¦Â-gal activity was significantly increased in Ang II¨Ctreated VSMCs compared with control cells.Ang II also significantly increased the transcriptional activity of p53 compared with that in vehicle-treated cells, and its effect was dose dependent.Consistent with our in vitro data, treatment with Ang II enhanced SA-¦Â-gal activity in the aortas of apoE-deficient mice.

--

p53-p21

Upregulation

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

Expression of p21 and p53 was elevated in Ang II¨Ctreated VSMCs.Western blot analysis revealed that introduction of E6 effectively reduced the level of p53 protein and also markedly reduced the expression of p21, its target protein.Overexpression of p21 significantly increased the number of SA-¦Â-gal positive cells, whereas p21 knockdown with a small interfering RNA (siRNA) system effectively blunted the effect of Ang II on senescence.

Human

L

Others

16,908,765

Gene

0

1

0

AR

367

protein coding

DPC

Skin

Androgenetic alopecia

Accelerate

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

Overexpression of the AR increased both percentage of SA-¦Â-Gal¨Cpositive cells and cell size and DHT enhanced the AR effects.A quantitative analysis showed that DHT induced SAHF formation in DPCs, and overexpression of the AR reinforced the effects of DHT compared with vector control-transfected cells.Western blot analyses showed that H2AX had been converted to its phosphorylated form (¦Ã-H2AX) in DPCs in response to DHT, and the c-H2AX/total H2AX ratio was further increased in cells overexpressing the AR.

p16

Upregulation

Western blot

To gain insight into the mechanism of premature senescence induction in DPCs, we focused on the relationship between the AR and p16INK4aprotein, which is known to be involved in premature senescence and is upregulated in balding DPCs.

--

Human

L

Others

24,244,503

Gene

0

1

0

1

0

1

0

1

0

1

0

ARPC1B

10095

protein coding

Saos-2,HDF

--

Aging

Accelerate

Cell morphological analysis//SA--gal activity assay

Those cells became flattened and enlarged just like senescent cells as previously reported.£¾50% of cells became to express SA-¦Â-gal activity at 9 days after infection with the tTA-encoding virus(After infection with tTA virus, mRNA levels of p41-Arc was dramatically increased ,whereas control cells infected with the ¦¤E1 control virus showed no activity.

--

Human

L

Others

21,628,992

Gene

0

1

0

1

0

ASF1A

25842

protein coding

WI-38

--

Aging

Accelerate

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

HIRA and ASF1a each caused cells to assume markers of senescence, including a large flat morphology, expression of SA-¦Â-gal activity, and macroH2A-containing SAHF.The effect was more pronounced when both proteins were coexpressed.

HIRA

Binding

Co-IP

Endogenous HIRA and ASF1a coimmunoprecipitated from asynchronously growing WI38 cells, confirming that they directly or indirectly associate in cells.

--

Human

L

Others

15,621,527

Gene

0

1

0

1

0

1

0

BCL6

604

protein coding

MEF

--

Aging

Prevent

Survival curve

Importantly, when expressed in primary MEFs of FVB genetic background (at 37¡ãC), BCL6 was very efficient in inhibiting both spontaneous senescence and premature senescence induced by a RASV12oncogene .

Cyclin D1

Upregulation

Western blot

The cyclin D1 protein level is up-regulated by BCL6 in three different cell types.This up-regulation of cyclin D1 is at the level of transcription, as cyclin D1 mRNA was also increased by BCL6 in MEFs.

--

Human

L

Others

11,914,273

Gene

0

1

0

BMI1

648

protein coding

WI-38

--

Aging

Prevent

SA--gal activity assay

We infected near-senescent cells with pBabe-Bmi-1.Replicative life span increased by ~4 population doublings (PD),relative to control cells.Consistent with these results,Bmi-1 transiently lowered the fraction of cells expressing SA-¦Â-Gal,a marker of the senescent phenotype.

p16

Downregulation

Western blot

Most cells in Bmi-1-overexpressing cultures did not express p16.Western analysis confirmed that Bmi-1 overexpression rapidly decreased p16 levels in presenes -cent WI-38 cultures;moreover, p16 levels increased modestly when these cultures senesced.

pRb

--

Cell counting

By contrast, Bmi-1 had no significant effect on the replicative life span of cells expressing E7(pRb eliminated),E6 and E7, or SV40 large T antigen.

Human

L

Others

12,482,990

Gene

0

1

0

BRAF

673

protein coding

Melanocyte

Skin,Foreskin

Aging

Accelerate

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

Short-term expression of BRAFE600(3¨C7 days) led to enhanced melanocyte proliferation that was measured by a moderate but reproducible increase in 5-bromodeoxyuridine (BrdU) incorporation. However, this effect was transient¡ªsustained expression of BRAFE600resulted in marked cell cycle arrest.In most (roughly 80%) of the transduced melanocytes, this was associated with an intense activity of SA-¦Â-Gal, a marker for senescent or stressed cultured cells as well as for aged tissues in vivo6,15.We observed that low levels of BRAFE600also induced SAHF.

p16

Upregulation

Immunostaining

We found that BRAFE600-expressing melanocytes had elevated levels of p16INK4a.

--

Human

L

Others

16,079,850

Gene

0

1

0

1

0

1

0

CAV1

857

protein coding

Fibroblast

Foreskin

Aging

Prevent

Cell morphological analysis//Western blot

We observed that a simple reduction in the caveolin-1 status, as determined by the siRNA method, induced in senescent cells morphological changes corresponding to the young cell-like shape, i.e. a small and spindle shape.

FAK//Rac1//Cdc42

--//Activation//Activation

Western blot//Cell morphological analysis//IP

The phosphorylation of FAK was dramatically reduced by the down-regulation of caveolin-1 in caveolin siRNA-transfected senescent cells versus the control firefly luciferase siRNA-transfected senescent cells.Reductions of focal adhesion and actin stress fiber formation via the inactivation of FAK by caveolin-1 status reduction resulted in morphological alterations toward the young cell phenotype.Rho GTPases were recruited into caveolin fractions and were directly linked with caveolin-1 in senescent HDF cells by immunoprecipitation analysis.

--

Human

L

Others

15,263,006

Gene

0

1

0

1

0

CBX8

57332

protein coding

MEF,Fibroblast

--

Aging

Prevent

BrdU assay//Colony formation assay//Flow cytometry

We found that inhibition of CBX8 expression dramatically reduced the S-phase fraction of cells and led to a complete growth arrest.Overexpression of CBX8 bypassed senescence, and the growth rate of the CBX8 expressing cells did not change within the time frame of the experiment.We found that Cbx8 downregulation led to a significant reduction in the number of colonies in all the genetic backgrounds tested.

BMI1//INK4a//ARF

Binding//Downregulation//Downregulation

Western blot//IP//qRT-PCR

Western blot (WB) analysis of protein extracts before depletion (BD) and after depletion (AD) showed a reduction of about 60% of total CBX8 after BMI1 depletion and a reduction of about 20% of total BMI1 after CBX8 depletion.Consistent with binding to the Ink4a-Arf locus, CBX8 expression led to a decrease in Ink4a and Arf mRNA levels.

--

Human

HL

Others

17,332,741

Gene

0

1

0

1

0

1

0

IL1A

3552

protein coding

endothelial cell

--

Aging

Prevent

Cell counting//Cell morphological analysis

The daily addition of the IL-lot antisense oligomer to populationsofhuman endothelial cells at 20 population doublings resulted in asignificant extension of cell proliferation .Removal of the IL-la antisense oligomer from a cell population after an extended number of population doublings resulted in a reduction in the proliferative capacity of the monolayer and the generation of the senescent cell phenotype.

--

Human

L

Others

2,218,499

Gene

0

1

0

1

0

TGFB1I1

7041

protein coding

KMST-6,SUSM1

--

Aging

Accelerate

Cell morphological analysis//Colony formation assay

The number of colonies decreased as the ratio of CMV/S5 to CMV in the transfected plasmid mixture increased, suggesting that the forced expression of the hic-5 cDNA inhibited the growth of these immortalized human cells. Enlarged, flattened cells in a senescence-like state appeared and constituted about 20% of the population over 30 PD.

--

Human

L

Others

9,032,249

Gene

0

1

0

1

0

HRAS

3265

protein coding

IMR-90

--

Aging

Accelerate

SA--gal activity assay//Western blot

As expected,vector-transduced cells transiently induced c-fos 30 min after restimulation.No c-fos message was detected, however, in H-rasV12-transduced cells.H-rasV12-transduced IMR90 cells displayed a significant accumulation of PAI-1 mRNA,compared with vector-transduced cells (2.5-fold large tran script; 7.5-fold small transcript).The percentage of SA-¦Â-gal positive cells in H-rasV12-expressing populations was initially low, but increased at day 3 and reached 60% by day 6.

--

p53//p16-Rb

--//--

BrdU assay

In contrast to wild-type MEFs, p53-/-or p16-/-MEFs continued to incorporate BrdU and proliferate following introduction of H-rasV12.

Human

L

Others

9,054,499

Gene

0

1

0

1

0

VEGFA

7422

protein coding

HDMEC

Foreskin

Aging

Prevent

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

When cultures were supplemented with a single pulse of VPF/VEGF at a final concentration of 5 ng/ml at the time of each passage after PD26, many HDMECs continued to maintain a relatively small size even beyond PD56 and continued to proliferate.When VPF/VEGF was withdrawn HDMEC developed a senescent morphology within 7 days. Addition of VPF/VEGF to senescent PD36 HDMEC induced renewed cell proliferation and restored a more normal morphology.senescent cells subsequently cultured with VPF/VEGF reduced their expression of neutral ¦Â-galactosidase.

p21//p16//p27

Downregulation//Downregulation//Downregulation

Western blot

p16 and p21 were only weakly expressed in early passage HDMEC(lane 3)but both were strongly expressed in senescent cells (lane 2). The expression of both was markedly reduced when senescence was prevented by culture with VPF/VEGF (lane 4).p27/Kip1 was moderately expressed in senescent HDMEC but at very low or undetec -table levels in both early passage cells cultured without VPF/VEGF and in late passage cells cultured with VPF/VEGF.

--

Human

L

Others

9,160,882

Gene

0

1

0

1

0

1

0

CDKN1A

1026

protein coding

LF1

--

Aging

Accelerate

Southern hybridization analysis

Southern (DNA) hybridization revealed that the apparent extension of life-span was accompanied by loss of heterozygosity (LOH) at the p21 locus.

--

Human

L

Others

9,242,615

Gene

0

1

0

TP53

7157

protein coding

EJ

--

Tumor

Accelerate

BrdU assay//Cell morphological analysis//Flow cytometry//PI staining//SA--gal activity assay

Proliferation of EJ¨Cp53 cells overexpressing wt p53 (2tet) was almost completely inhibited.Tet removal led to obvious p53 nuclear staining by 24 h with the cells exhibiting increased size and a flattened morphology with elongated cellular processes, as well as enlarged nuclei.EJ¨Cp53 cells exhibited greatly reduced BrdUrd incorporation within 24 h after the removal of tet with the fraction in S phase declining to ~5% by 2¨C3 days .Greater than 95% of such cells showed positive staining for SA-¦Â-gal, which colocalized to cells possessing the senescent morphology .

--

Human

L

Others

9,275,177

Gene

0

1

0

1

0

1

0

1

0

1

0

CDKN2A

1029

protein coding

TIG-3

--

Aging

Accelerate

SA--gal activity assay

The both number of cells and [3H]thymidine incorporation are signi cantly decreased in H-ant-p16wt treated cells. Similar results were obtained using synchronized TIG-3 cells.The cells were then xed and stained for SA-¦Â-gal, described to be expressed by senescent cells [27]. The enlarged growth-arrested cells gave a positive staining reaction.

pRb

--

Western blot

pRB is phosphorylated following serum stimulation in cells treated.However, pRB phosphorylation was not observed in cells treated with the H-ant-p16wt protein.

--

Human

L

Others

9,607,312

Gene

0

1

0

RAF1

5894

protein coding

IMR-90

--

Aging

Accelerate

Cell counting//SA--gal activity assay

Activation of GFPDRaf-1:ER arrested the proliferation of IMR-90 cells.Activation of GFPDRaf-1[YY]:ER in IMR-90 cells elicited the expression of acidic ¦Â-galactosidase, which was apparent within 2 3 days and maximal by 5 6 days after addition of 4-HT.

p16

Upregulation

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

Following activation of GFPDRaf-1:ER, we observed induced expression of the CDK inhibitors p16Ink4a.However, the level of expression of p16Ink4a did not decrease after GFPDRaf-1:ER inactivation, which may reflect the long half-life of both p16Ink4a mRNA and protein.IMR-90 cells expressing p16Ink4a became positive for SA¨C¦Â-galactosidase activity.FACs can analysis revealed a threefold reduction in S-phase cells compared with the control infected populations.

MEK-MAPK

Activation

Western blot

As described above, activation of GFPDRaf-1[YY]:ER led to the phosphorylation and activation of the p42/p44 MAP kinases. In the presence of PD, however, phosphorylation of the MAP kinases was reduced to levels comparable to those found in uninduced IMR-90 cells.

Human

L

Others

9,765,202

Gene

0

1

0

1

0

MORF4

10934

protein coding

A9+F4

--

Aging

Accelerate

SA--gal activity assay

The MORF 4-transfected clones achieved between 19 and 35 PD before ceasing proliferation. The cells then morphologically resembled senescent cells and were positive for the senescence-associated ¦Â-galactosidase activity.

--

Human

HL

Others

9,891,081

Gene

0

1

0

E2F1

1869

protein coding

WI-38,NIH-3T3

--

Aging

Accelerate

SA--gal activity assay

Wild-type E2F1, when constitutively overexpressed, unexpectedly arrested cell proliferation within two population doublings (PD) after selection.wild-type and D423G proteins, but not the other E2F1 proteins, also induced .50% of the cells to assume a flat morphology, typical of senescent cells.

p14//p53

Upregulation//Upregulation

Semi-RT-PCR//Western blot

E2F1 proteins that had transactivation activity (wild type and D423G), but not the other E2F1 proteins, strongly induced p14ARF mRNA.p14ARF was also highly expressed by replicatively senescent cells.p14ARF and E2F1 did not arrest growth or induce SA-¦Â-Gal staining in cells that overexpress Mdm2(Mdm2 functionally inactivates p53).Indeed, wild-type E2F1 and D423G proteins, as well as the p14ARF protein, strongly induced p53.

--

Human

L

Others

10,594,030

Gene

0

1

0

CDKN2B

1030

protein coding

U-251 MG,Tp483 MG,U373 MG,SW1783

--

Aging

Accelerate

Cell counting//Cell morphological analysis//SA--gal activity assay//Telomerase Assay

Both inhibitors(p15 ,p16) were equally effective in causing complete growth arrest during a 7-day period.When U251 MG cells infected with Ad5CMVp15 or Ad5CMVp16 were stained for the SA-¦Â-gal marker 8 days p.i., the morphologically changed, enlarged, and flattened cells were strongly positive in contrast to mock-infected or to Ad5CMVlacZ infected cells .In contrast, telomerase activity was efficiently repressed in both Ad5CMVp15- and Ad5CMVp16- infected cells, reaching an undetectable level at day 6.

--

Rb

--

Cell morphological analysis//SA-¦Â-gal activity assay

U373 MG cells infected with Ad5CMVp15 or Ad5CMVp16 showed no morphological changes even after 7 days.The U373 MG cell line(absent pRB expression) did not become SA-¦Â-gal-positive after infection by adenoviruses expressing p15 or p16.

Human

L

Others

10,939,591

Gene

0

1

0

1

0

1

0

1

0

ETS1

2113

protein coding

HDF

--

Aging

Accelerate

Western blot

In normal circumstances, p16INK4a(senescence marker) levels increase substantially in senescent HDFs,we found that the Ets2 levelsdeclined .It was clear that the loss of Ets2 from senescent cells was in part compensated for by a reciprocal increase in Ets1 expression.

--

Human

L

Others

11,234,019

Gene

0

1

0

ID1

3397

protein coding

HDMEC

--

Aging

Prevent

Cell counting//Cell morphological analysis

HDMECs of transduced with LZRS-empty vector (HDMEC+vector) became senescent at approximately PD17 and demonstrated the enlarged, flattened morphology seen in the untreated cells.The HDMEC+Id-1 cells demonstrated a significant increase in replicative capacity and continued to divide until PD38.

--

Rb

Downregulation

Western blot

In contrast, HDMECs+Id-1 showed slightly lower, but sustained levels of both Rb forms until senescence, when levels of pRb and ppRb were barely detectable (not shown).

Human

L

Others

12,177,246

Gene

0

1

0

1

0

MAP2K6

5608

protein coding

U2OS

--

Aging

Accelerate

BrdU assay//Colony formation assay//Flow cytometry//Luciferase reporter assay//SA--gal activity assay

Relative to cells transfected with empty vector alone or with MKK6KA, £¼6% of the MKK6EE-transfected cells formed colonies.MKK6EE expression leads to a near complete loss of BrdUrd-positive cells, consistent with a G1/G0cell cycle arrest.We observed that 40% of MKK6EE-expressing cells are strongly positive for SA-¦Âgal, consistent with a senescent phenotype.At 6 days after transfection ,lipofuscin was markedly increased in fluorescence of MKK6EE-expressing cells compared with controls.

p21

Upregulation

Western blot

Trikingly,p21Cip1, a cdk inhibitora markediy increase.

p38

Activation

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

As expected, expression of MKK6EE in U2OS cells increased the activity, whereas kinase-dead MKK6 slightly reduced basal activity .Cellular Senescence was not observed with MKK6KA cells or MKK6EE-expressing cells, which were treated with SB202190.

Human

HL

Others

12,208,764

Gene

0

1

0

1

0

1

0

1

0

1

0

PNPT1

87178

protein coding

HO-1,melanoma cell,WM278,MEWO

--

Aging

Accelerate

MTT assay//Telomerase assay

Infection with Ad.hPNPaseold-35resulted in significant growth inhibition in all of the cells.Infection with Ad.hPNPaseold-35at an m.o.i.of 100 pfu/cell,but not with Ad.vec,inhibited telomerase activity by almost 60% at day 4 after infection.

c-Myc

Downregulation

Northern blot

The expression of c-myc mRNA began decreasing 2 days after Ad.hPNPaseold-35infection but not in uninfected or Ad.vecinfected cells even at 4 days after infection.

--

Human

L

Others

12,721,301

Gene

0

1

0

1

0

MXD4

10608

protein coding

293,HeLa

--

Aging

Accelerate

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

Growth was repressed by about 4- and 2.5-fold in 293- and HeLa-hMad4 infected cells, respectively. This repression of cell proliferation was accompanied by an increase in contact inhibition in a soft agar colony assay.In the case of hMad4-infected cells, following a small and very slow proliferative phase from day 1 to day 26 following replating, cells completely stopped dividing at which point they acquire the morphological features of replicative senescence such as flat and enlarged morphology.hMad4-infected cells did express s-¦Â-galactosidase expression while empty-vector- and hMad4L-P16-infected cells had a very low expression level.Only hMad4- infected cells had a high expression level of Pai-1 protein(senescence marker).

Max//c-Myc

Binding//Downregulation

Co-IP//CAT assay

Anti-Max and antihMad4 are able to co-immunoprecipitate hMad4 and Max, respectively.When c-Myc and Max were co-transfected in 293 cells, there was a 10-fold increase in CAT activity over cells transfected with the reporter construct alone. When hMad4 was co-transfected with c-Myc and Max at increasing amount, there was a 4-fold decrease in CAT activity, similar to the activity seen with transfection of c-Myc alone.

--

Human

HL

Others

12,761,891

Gene

0

1

0

1

0

1

0

1

0

SOD1

6647

protein coding

WI-38,WI38-SV40

--

Aging

Prevent

Cell morphological analysis//SA--gal activity assay

In contrast,the SOD1 RNAi-transfected cells no longer divided.In addition the SOD1 RNAi morphology was aberrant and cells had an increased cell area and flattened appearance .WI38 SOD1 RNAi cells exhibited significantly stronger activity of senescence-associated ¦Â-galactosidase than the control cells.

p53

--

Immunostaining

In contrast to control cells, SOD1 RNAi-transfected cells displayed the p53 protein in the nucleus and at elevated expression levels.

--

Human

L

Others

12,871,978

Gene

0

1

0

1

0

MAP2K1

5604

protein coding

HIEC,IEC-6

Small intestine

Aging

Accelerate

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

By contrast, caMEK-expressing HIEC cells did not proliferate and stopped accumulating well before reaching confluence.Ectopic expression of caMEK significantly inhibited thymidine kinase gene expression by 50%.Morphological examination of HIEC cells arrested by caMEK revealed that cells remained attached to the plates for at least 1 mo (data not shown) and acquired a large and flat morphology.Moreover, these HIEC cells accumulated SA-¦Â-galactosidase.

--

MEK-ERK

--

Western blot

Serum stimulation of HIEC and IEC-6 cells exhibited rapid and maximal activation of ERK1/2 within 10 min and persisted for up to 2 h. Nonstimulated caMEK expressing HIEC and IEC-6 cells showed a moderately increased basal level of ERK1/2 activities compared with pLXIN- and wtMEK-expressing cells.

Human

L

Others

14,701,721

Gene

0

1

0

1

0

1

0

1

0

NINJ1

4814

protein coding

HuH-7

HCC tissue,Liver

Hepatocellular carcinoma

Accelerate

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

Injurin1 overexpression increased the percentage of the total cell population in the G0¨CG1 phase, decreased the percentage of cells in the S phase. The percentage of SA-¦Â-galactosidase-positive cells in ninjurin1-overexpressing populations was 26% in Nin1 and 21% in Nin2. In contrast, less than 1% of the parental and mock clones were SA-¦Â-galactosidasepositive.Nin1 and Nin2 cells displayed intense cytoplasmic, granular fluorescence.Flow cytometric analysis revealed that both Nin1 and Nin2 cells showed a discernable increase in fluorescence in comparison to the mock clones.

p21//Cyclin A//CDK2//CDK4//CDK6

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

Western blot

There was no change in the level of p27, but the level of expression of p21 was greatly upregulated.The level of expression of cyclinA declined.The levels of expression of all CDKs(CDK2,CDK4,CDK6) were consistently suppressed by ninjurin1 overexpression.

--

Human

L

Others

15,464,245

Gene

0

1

0

1

0

1

0

SMURF2

64750

protein coding

BJ,WS1,WI-38

Foreskin,Skin,Lung,Mammary Gland

Aging

Accelerate

Cell counting//SA--gal activity assay

Adventitious expression of Smurf2 to the level observed physiologically during replicative senescence resulted in arrest of proliferation of all three lines of early passage fibroblasts in a subconfluent state.During this acute transition, the early passage Smurf2-expressing cells acquired the property of positive staining for ¦Â-galactosidase activity at pH 6.

--

p53//Rb

--//--

Cell counting//Cell morphological analysis//SA-¦Â-gal activity assay

The concurrent expression of both E6 and E7 prevent Smurf2 s effects as indicated by cell growth arrest in a subconfluent state, enlarged and flat cell morphology, and positive staining for -galactosidase activity at pH 6.

Human

HL

Others

15,574,587

Gene

0

1

0

1

0

DHCR24

1718

protein coding

REF,WI-38

--

Aging

Accelerate

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

In contrast, upon Ras introduction, REF cells with Seladin-1 knockdown continued to proliferate, exhibited a condensed cell morphology, and were not stained in SA-¦Â-gal assay .We observed that cells with Seladin-1 knockdown continued to show BrdU incorporation, cell proliferation and approximately fourfold less SA-¦Â-gal staining after H2O2 treatment.

p53//MDM2

Binding//--

Western blot

Silencing of Seladin-1 prevented Ras-induced p53 accumulation. We found that, first, Seladin-1 binds p53 and displaces Mdm2 from p53 in vitro.Second, recombinant Seladin-1 inhibits in vitro ubiquitination of Flag-p53 by Mdm2.

Ras

Upregulation

Western blot

Seladin-1 levels increased in response to Ras expression.

Human

HL

Others

15,577,914

Gene

0

1

0

1

0

1

0

LEO1

12319

protein coding

2BS

--

Aging

Accelerate

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

2BS/Leo cells showed complete growth inhibition similar to senescent cells (PD56).In contrast to the significantly increased G1 DNA content of 2BS/Leo, which was somewhat like senescent cells, RDL-Leo¨Creduced the G1 content of transfected cells.2BS/Leo cells showed increasing gross enlargement,flattening, and accumulation of granular cytoplasmic inclusions, like senescent cells (PD56).No SA-¦Â-Gal activity was observed in 2BS/RDL-Leo¨C(PD48) and young (PD 27) cells, whereas almost all 2BS/Leo(PD 48) cells were strongly stained, as were senescent 2BS control cells (PD 56). In contrast, 2BS/Leo ceased cell division 7¨C12 PD (CPD 48-50) earlier than did normal cells.

--

p16//p21//PTEN

Upregulation//Upregulation//Upregulation

Western blot

The results showed that introduction of Leo into 2BS cells affected all three pathways by increasing the expression of p16INK4a, p21WAF1, and PTEN.

Human

HL

Others

15,791,002

Gene

0

1

0

1

0

1

0

1

0

1

0

ING2

3622

protein coding

MRC-5

--

Aging

Accelerate

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

ING2 protein level was four- to sixfold higher in senescent fibroblasts.ING2 overexpression induced premature senescence in PDL28 fibroblasts. The downregulation of ING2 resulted in a statistically significant increase only in the number of small colonies (containing 10 to 50 cells). Thus, suppression of ING2 expression results in an extension of the life span of human fibroblasts for a limited number of divisions.

p53//p300

--//--

Immunostaining//Co-IP//Western blot

The colocalization of ING2 with p53 (Ser15) was dramatically enhanced in senescent fibroblasts.The complex formation between p53, ING2, and p300 was further corroborated in transient transfections in wild-type p53-containing U2OS cells, where immunoprecipitation of endogenous p300 also contained p53 and transfected Flag-ING2.With a constant amount of p300, increasing the amounts of purified ING2 protein led to a further two- to fourfold increase in the acetylation of lysine 382 on p53, as determined by the two detection methods. No p53 acetylation was detected with ING2 protein alone.

--

Human

L

Others

16,024,799

Gene

0

1

0

1

0

1

0

1

0

PRPF19

27339

protein coding

HUVEC,I38-neo-1 ,I38-SNEV-1

--

Aging

Prevent

Comet assay//SA--gal activity assay

I38-neo-1 cells entered growth arrest after around 8 PDs post-infection, where the cells showed the typical morphology of senescent cells and stained positive for SA-¦Â-Gal . In contrast, I38-SNEV-1 cells at the same PD were characterized by a young morphology with no positive SA-¦Â-Gal staining and entered replicative senescence around PD18pI.These data indicate that a high SNEV level correlates with a decrease of accumulated DNA damage during in vitro cultivation.

--

Human

L

Others

16,388,800

Gene

0

1

0

1

0

CSNK2A1

1457

protein coding

IMR-90

Liver and Testis

Aging

Prevent

SA--gal activity assay

These reindicate that CKII activity decreases in IMR-90 cells during both replicative and H2O2-induced senescence.SA-¦Â-gal staining in cells tended to be an increase in a dose-dependent manner by treatment with DRB.33 PDL of IMR-90 cells transfected with anti-CKIIa siRNA exhibited a higher rate of SA-¦Â-gal staining comparedwith control cells. The increase rate in SA-¦Â-gal staining from control was approximately 15-fold for CKIIa siRNA.

p53//p21

--//--

Western blot

When the protein levels of p53 and p21Waf-1 were determined by immunoblot, expression of both proteins was upregulated in CKII inhibitor-treated cells, like in H2O2- induced senescent cells.

--

Human

L

Others

16,442,104

Gene

0

1

0

IRF3

3661

protein coding

BJ,IMR-90

--

Aging

Accelerate

Cell counting//Knockdown//SA--gal activity assay

overexpression of IRF3 in BJ and IMR90 cells was shown to decrease the rate of cell proliferation and increase the senescence cell population,as determined by senescence-associated ¦Â-galactosidase staining assay.When the cellular life span was determined by 3T6 cell culture protocol, IRF3-knockdown BJ cells were found to increase the cell proliferation rate and life span.

p53

Activation

Western blot

We found that p53 mRNA was not increased by IRF3, but p53 protein and its downstream target p21WAF1 were upregulated in IRF3-overexpressing BJ cells.

--

Human

L

Others

16,513,254

Gene

0

1

0

1

0

1

0

MYC

4609

protein coding

BJ,IMR-90

--

Aging

Prevent

Colony formation assay

We introduced into the c-myc+/- cells a retrovirus vector expressing human telomerase reverse transcriptase (hTERT) to immortalize them. Although hTERT clearly extended their lifespan, several attempts with different vectors failed to elicit long term immortalization, whereas the same vectors readily immortalized c-myc+/+ cells in parallel experiments.

Bmi-1//p16

Binding//--

qRT-PCR//CHIP

As expected, retrovirus- mediated overexpression of c-Myc in normal HDFs resulted in Bmi-1 mRNA induction.we demonstrated direct binding of c-Myc protein to the E-box in the bmi-1 promoter by chromatin immunoprecipitation (ChIP) analysis.In the absence of ectopic Bmi-1, lentivirus vector-expressed c-Myc shRNA elicited a 2-fold up-regulation of p16 mRNA within 3 days of infection. Ectopic Bmi-1 expression alone resulted in repression of p16 mRNA levels, which remained low after c-Myc knockdown.

--

Human

L

Others

16,537,449

Gene

0

1

0

PIAS4

51588

protein coding

WI-38

--

Aging

Accelerate

Cell morphological analysis//Flow cytometry//Growth curve assay//Immunostaining//SA--gal activity assay//SAHF

We found that cells constitutively expressing PIASy irreversibly arrested growth 7 10 days postselection, whereas other PIAS proteins had no effect available with this article online).PIASy-overexpressing cells also exhibited a flat cell morphology, and more than 80% of the cell population stained SA-¦Â-gal positive.These assays clearly demonstrated that PIASy-expressing cells were completely devoid of proliferative potential and predominantly arrested in G1 .We also observed formation of SAHFs in PIASy-expressing cells.

E6

--

Western blot

Addition of PIASy, but not of other PIASs, to the reaction strongly enhanced the formation of HDAC1-SUMO conjugates, an effect that was abrogated by the presence of E6 or its 45/47/49 mutant.11E6 was also unable to inhibit PIASy-induced senescence, whereas E6 mutant 45/47/ 49 still did.

p53-p21//Rb-p16

Activation//Activation

Western blot

These data corroborate the increased p21 protein levels we observed in PIASy-transduced fibroblasts and suggest that sumoylation might contribute to PIASy-mediated activation of p53 in the context of PIASy-dependent senescence.PIASy corepressed the cyclin E promoter in an Rb- and dosedependent manner.Loss of PIASy using siRNA in SAOS-2 cells caused a significant derepression of the cyclin E promoter in the presence of Rb.

Human

L

Others

16,793,547

Gene

0

1

0

1

0

1

0

1

0

1

0

1

0

KL

9365

protein coding

MRC-5

--

Aging

Prevent

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

Downregulation of Klotho expression by RNAi significantly shortened replicative lifespan and reduced the growth rate of MRC-5 cells compared with mock-infected cells.Similar to the phenotype of senescent cells, Klotho RNAi cells were flattened and enlarged .we found a 15-fold increase in ¦Â-Gal activity in Klotho RNAi (1)-transduced cells compared to mock control and a sixfold increase for Klotho RNAi (2).

--

Insulin-IGF-1//p53-p21

--//Downregulation

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

The level of phosphorylated IRS1 is higher in MRC-5 cells treated with Klotho RNAi, suggesting an upregulation of signaling at this early step in the pathway. the levels of phosphorylated AKT and phosphorylated FOXO3 (the inActivate cytoplasmic form) are higher.Relative to control cells Klotho RNAi infected cells have similar levels of p16 protein but elevated expression of p21.Confirming the ability of p53 attenuation to restore replicative potential of Klotho RNAi cells, staining for ¦Â-Gal shows approximately the same number of positive cells in the double knockdown compared to the vector control or p53 RNAi cells .

Human

L

Others

17,014,852

Gene

0

1

0

1

0

1

0

CXCL1

2919

protein coding

NOF150,NOF151

--

Ovarian cancer

Accelerate

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

We found that the cells became enlarged andflattened, showing growth arrest and increased SA-¦Â-gal activity after 10 days of treatmentas compared with cells grown in normal medium without Gro-1,cells cultured in serum-free medium, or cells treated with the same concentration of mouse IgG.Gro-1 treatment of NOF151 cells led to expression of HP1¦Â in granular foci and marked increases in cytoplasmic p16INK4A levels.

--

p53

--

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

Treatment of parental NOF150 cells with Gro-1 led to strong blue staining on day 10, demonstrating activation of cellular senescence by Gro-1, but disruption of p53 suppressed or eliminated this effect.Fibroblasts immortalized by the introduction of p53siRNA and hTERT in particular completely lost the senescent response to Gro-1 .Disruption of p53 also markedly attenuated p16INK4Aexpression, particularly in the presence of hTERT.

Human

L

Others

17,060,621

Gene

0

1

0

1

0

1

0

1

0

RNASEL

6041

protein coding

MEF,WI-38,HDF,SV-WI38,Swiss 3T3,NIH-3T3

--

Aging

Accelerate

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

We found that RNase-L-expressing cells did not display nuclear labeling, suggesting that ectopic expression of RNase-L inhibited proliferation. In contrast,the nuclei of cells with low,background staining for RNase-L expression were strongly labeled.RNase-L induction resulted that these cells exhibited an increased cytoplasmic :nuclear volume and vacuolization, and a flattened, multinucleate morphology.Counting of the stained cells revealed that 62% of RNase-L-transduced WI38 HDFs were ¦Â-gal positive as compared to only 18% in the vector control cells. Replicative senescence was accelerated by nearly three weeks in the RNase-L WI38 as compared to vector control cells.

--

Human

L

Others

17,130,839

Gene

0

1

0

1

0

1

0

PCGF2

7703

protein coding

MRC-5

--

Aging

Accelerate

SA--gal activity assay

Mel-18 overexpression led to inhibition of cellular proliferation and induction of premature senescence in MRC-5 fibroblasts as determined by SA-¦Â-gal staining.

Bmi-1//c-Myc

Downregulation//Downregulation

Western blot//Knockdown

Our results indicated that similar to MRC-5 fibroblasts, stable overexpression of Mel-18 results in Bmi-1 down-regulation in MCF10A and MCF7 cells.Mel-18 overexpression led to down-regulation of c-Myc in multiple cell types.Western blot analysis of cells expressing Mel-18 shRNAs showed significant down-regulation of Mel-18 and up-regulation of Bmi-1 in these multiple cell types.As expected, knockdown of Mel-18 also up-regulated c-Myc expression.

--

Human

L

Others

17,151,361

Gene

0

1

0

MAPKAPK5

8550

protein coding

MEF

Skin

Aging

Accelerate

SA--gal activity assay//Western blot

PRAK+/+ primary mouse skin fibroblasts (MSFs) became growth arrested and accumulated SA-¦Â-gal upon transduction of an activated ras allele,Ha-RasV12, indicating that ras triggered premature senescence.Similarly, PRAK deletion abolished ras-induced senescence in primary mouse embryonic fibroblasts (MEFs£©.

--

Ras//p38//p53-p21

--//--//Activation

Western blot//Luciferase reporter assay

Ras-induced PRAK activation was also detected in primary .Ras-and MKK3/6E-induced PRAK kinase activity was greatly reduced in both BJ and MEF cells by a specific p38 inhibitor, SB203580.This indicates that PRAK is activated by p38 during ras-induced senescence.In both BJ and wild-type MEF cells containing this p53 reporter, luciferase activity was stimulated significantly by Ha-RasV12 or MKK3E,confirming the induction of p53 transcriptional activity in senescence. The regulation of p53 activity by PRAK during senescence was confirmed by the observation that ras-induced expression of p21WAF1, an endogenous p53 target previously implicated in rasinduced senescence, was abolished in PRAK-deficient BJ and MEF cells.

Human

L

Others

17,254,968

Gene

0

1

0

1

0

PEX19

5824

protein coding

U2OS,Human immortalized postcrisis cell,Fibroblast

Human tumor tissue and normal tissue

Tumor

Accelerate

Cell counting

Examination of 5AZA-dC response in Pex19p-overexpressing cells in comparison to the vector-transfected control cells revealed that the overexpression of Pex19p made the cells more sensitive to 5AZA-dC-induced senescence.Consistent with their stronger growth arrest.

--

p16

Upregulation

Western blot

Pex19p-overexpressing cells showed an enhanced induction of p16INK4A expression in response to 5AZA-dC treatment.

Human

L

Others

17,389,721

Gene

0

1

0

HSPA9

3313

protein coding

U2OS,Human immortalized postcrisis cell,Fibroblast

Human tumor tissue and normal tissue

Tumor

Accelerate

Cell counting//Western blot

Noticeably, mortalin-overexpressing cells were significantly more sensitive to 5AZA-dC, and showed a stronger induction level of p16INK4A.

--

p53-p21

Activation

Western blot

5AZA-dC-treated cells indeed showed upregulation of p53 and its downstream effector, p21WAF1, in addition to p16INK4a, and exhibited nuclear translocation of the p53 protein. Whereas only 10% 20% of control cells showed nuclear p53, 90% of the 5AZA-dC-treated cells showed strong p53 staining in the nucleus .

Human

L

Others

17,389,721

Gene

0

1

0

1

0

MMP9

4318

protein coding

Daoy medulloblastoma cell line

Tumor tissue,Brain

Medulloblastoma

Accelerate

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

Ad-MMP-9 infection led to a dosedependent decrease in cell proliferation.FACS analysis for nuclear DNA content by propidium iodide staining showed that cell growth was arrested in the G0-G1 cell cycle phase.Senescence, as indicated by ¦Â-gal staining (blue color), increased in a dose-dependent manner with Ad-MMP-9 infection.

p21CIP//p16

--//--

Western blot

We observed a significant induction of p21 with Ad-MMP-9 infection.The expression of p16 increased in a dose-dependent manner as cells reached senescence.

ERK-MAPK

--

Western blot

We found that both ERK and pERK levels were increased in Daoy cells infected with Ad-MMP-9 compared with the controls.

Human

L

Others

17,510,426

Gene

0

1

0

1

0

1

0

1

0

EHF

26298

protein coding

WI-38

--

Aging

Accelerate

SA--gal activity assay

When cells were maintained by regular feeding of fresh media,ESE-3b-expressing cells stopped growing significantly earlier than the mock-treated cells did.ESE- 3b-expressing cells also showed a higher level of SA-¦Â-gal-positive cells than the mock-infected cells.

p16

Upregulation

Western blot

The amount of p16 but not that of p21 was increased in ESE-3b-expressing cells.

--

Human

HL

Others

17,627,613

Gene

0

1

0

WNT2

7472

protein coding

RPE,WI-38

--

Aging

Prevent

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

There was a good correlation between knockdown of Wnt2 and recruitment of HIRA to PML bodies, formation of SAHF judged by DAPI staining, and expression of SA ¦Â-gal.Strikingly, Wnt3aconditioned medium or purified recombinant Wnt3a delayed HIRA s localization to PML bodies and formation of SAHF in cells passaged toward senescence .Likewise, exogenous Wnt3a delayed onset of cell senescence caused by extended growth of WI38 cells in culture.

--

Human

L

Others

17,643,369

Gene

0

1

0

1

0

1

0

1

0

ITGB4

3691

protein coding

HUVEC

--

Aging

Accelerate

SA--gal activity assay

The percentage of positive cells increased significantly during VEC aging.The percentage of SA-¦Âgal positive cells in integrin ¦Â4 siRNA treatment group (43.84% 2.17%) was much lower than that in the control group (59.65% 2.74%).

PC-PLC//p53

Downregulation//Upregulation

PC-PLC activity assay//Western blot

As a result, Ca2+-dependent PC-PLC was nearly not changed, but the activity of Ca2+-independent PC-PLC decreased obviously during senescence.Down-regulation of integrin ¦Â4 depressed P53 and ROS levels and inhibited the activity of Ca2+-independent PC-PLC significantly.

--

Human

L

Others

17,964,297

Gene

0

1

0

HSPB2

3316

protein coding

MCF 10A

--

Aging

Prevent

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

Hsp27 overexpression led to a significant increase in survival after doxorubicin treatment. significantly fewer Hsp27-MCF10A cells showed expression of acidic ¦Â-galactosidase and senescence morphology after treatment with doxorubicin, compared with control MCF10A cells.

--

p53-p21

Downregulation

Western blot

We observed a suppression of known downstream targets of p53, survivin, and cdc2 in shHsp27 cells.In fact, direct measurement of the degradation rates showed that the half-life of p53 in shHsp27 cells increased from 26to 42 min .Doxorubicin led to a pronounced accumulation of p21 in control cells but this increase was markedly suppressed in Hsp27-MCF10A cells.Nutlin-3 treatment for 48 h led to accumulation of p21 in control cells, but this accumulation was suppressed in Hsp27-overexpressing cells.

Human

L

Others

18,089,808

Gene

0

1

0

1

0

1

0

SPIN1

10927

protein coding

HeLa

--

Aging

Accelerate

CCK-8 assay//DAPI staining//Flow cytometry//PI staining//SA--gal activity assay

SPINDLIN1 over-expressing cells showed a heterogeneous DNA content, with broader G1 and G2 peaks and a substantial fraction of cells with 4N DNA content, at 29 days after transduction.Numerous large cells with multiple nuclei were easily visible under phase contrast microscope, and multinucleation was also clearly illustrated by DAPI staining .HeLa cell growth was delayed significantly by overexpression of SPINDLIN1.We indeed found that almost all adherent multinucleated cells were SA-¦Âgal positive (3.2%( 1.4%) in SPINDLIN1 overexpressing cells verse 0.8% ( 0.3%) in mock transduction cells).

--

Human

HL

Others

18,201,843

Gene

0

1

0

1

0

1

0

1

0

1

0

ID1

3397

protein coding

MEF

Embryo

Aging

Prevent

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

3T9 protocol revealed premature growth inhibition of Id1-/- MEFs beginning at P-5.Morphologic evaluation of these cells demonstrated cytoplasmic enlargement and flattening of Id1-/- MEFs and 80¨C90% positive staining of these cells with senescence-associated ¦Â-gal(16) at P-8, indicating premature cellular senescence in contrast to normal (+/+) MEFs that were 10¨C20% positive.

CDK2//CDK4//p16

--//--//Downregulation

Western blot

Evaluation of cdk2 and cdk4 functions revealed 75% and 50% inhibition of kinase activity, respectively, in early passage MEFs lacking Id1.We analyzed human foreskin keratinocytes that constitutively expressed Id1 for p16 expression levels and found that p16 expression inversely correlated with Id1 expression in these cells. Furthermore, we detected no p16 expression by Western analysis in Id1-immortalized human foreskin keratinocytes.

--

Human

L

Others

11,427,735

Gene

0

1

0

1

0

1

0

NANOG

79923

protein coding

HF-MSC

Hair follicle

Aging

Prevent

Cell counting//SA--gal activity assay

Cell growth assays showed that the ectopic expression of NANOG significantly increased the quantity of HF-MSCs by 1.4- and 1.2-fold,in comparison to cells in the vector group (control) on day 8.Ectopic NANOG expression significantly decreased the percentage of SA-¦Â-gal-positive cells.

p16//p21//p53//PBX1

Downregulation//Downregulation//Downregulation//Upregulation

Western blot//Luciferase reporter assay

Western blotting showed that ectopic NANOG expression significantly upregulated PBX1 expression by 4.11¡À0.54-fold in comparison to that of the control group. Dual-luciferase reporter gene assays showed that ectopic NANOG expression significantly increased PBX1 promoter activity by 1.99¡À0.43-fold incomparison to that of the control group (P < 0.05). HNANOG decreased p16 expression by 0.38¡À0.18-fold (P < 0.01),p21 expression by 0.57¡À0.24-fold (P < 0.05) and p53 expression by 0.60¡À0.16-fold (P < 0.05).

Akt

Activation

Western blot

Western blotting showed that NANOG significantly increased PARP1 expression by 1.64 ¡À 0.09-fold (P < 0:01) and the expression of p-AKT/AKT by 3.14 ¡À 0.65-fold (P < 0:05),in comparison to the empty vector.

Human

L

Others

31,885,790

Gene

0

1

0

1

0

TXNIP

10628

protein coding

MEF

Kidney

Aging

Prevent

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

The growth rate of KO MEF cells was dramatically decreased, and the cells almost ceased to proliferate at late passages (P5).KO MEF cells were larger than WT MEF cells. KO MEF cells were stained with SA©\¦Â©\gal and ¦Ã©\H2AX at Passage 5 (P5).The expression levels of g p53, p21, p16,PAI©\1, and PML are upregulated by western blotting and statistical analysis or quantitative real©\time PCR in KO MEF cells in comparision to WT MEF cells.

--

Akt

Downregulation

Co-IP//Western blot

TXNIP was found to interact with AKT, which was also confirmed by immunoprecipitation assays in 293T cells.The expression of TXNIP inhibited the activity of AKT in a dose dependent manner in western blotting with phospho AKT antibodies and in vitro kinase assays.

Human

L

Others

30,168,649

Gene

0

1

0

1

0

1

0

1

0

1

0

AKT1

207

protein coding

MEF

Embryo

Aging

Accelerate

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

WT MEFs began senescing after passage, whereas Akt1/2 DKO MEFs began senescing after passage.This was also confirmed by the cells¡¯enlarged and flattened cell morphology (data not shown),by SA-¦Â-Gal staining,and by BrdU incorporation.

ROS//FOXO//Sestrin 3

Upregulation//Downregulation//--

Immunostaining//qRT-PCR

We found that Akt1/2 DKO MEFs had significantly lower ROS levels compared with WT MEFs, whereas cells expressing activated Akt and Pten-/- cells had significantly higher levels of ROS.FoxO transcriptional activity was elevated in Akt1/2 DKO MEFs.We found that sestrin 3 (Sesn3) expression was highly induced by activated FoxO.Activated Akt induces ROS by increasing oxygen consumption combined with the inhibition of FoxO transcription factors.

--

Human

L

Others

19,061,837

Gene

0

1

0

1

0

1

0

EPHA3

2042

protein coding

hTERT-RPE1

--

Aging

Prevent

SA--gal activity assay

Our results showed that senescence was uniquely detected after knockdown of EPHA3 and not measurably apparent after knockdown of other EPHA RTKs expressed in hTERT-RPE1 cells .

--

p16//p53

--//--

qRT-PCR//Immunostainings//Knockdown

Characterization of the senescence signature showed that siRNA-mediated knockdown of EPHA3 leads to increased p16INK4a and p53 expression .

Human

HL

Others

23,324,396

Gene

0

1

0

MAP2K2

5605

protein coding

IMR-90

--

Aging

Accelerate

SA--gal activity assay//Western blot

MEK induced p53 (about three-fold induction), p21 (aboutfive-fold induction), and p16 (about six-fold induction).Finally, MEK1Q56P-expressing cells exhibited high levels of SA-¦Â-gal activity, with a percentage of positive cells similar to that observed in Ras-expressing or late passage IMR90 cells.

--

Human

L

Others

9,765,203

Gene

0

1

0

1

0

MAPK14

1432

protein coding

WI-38,MRC-5

--

Aging

Accelerate

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

The growth rate of senescent cells was very low in the absence of SB203580.?However, in the presence of SB203580, a significant increase in growth rate was observed. More than 80% of both SB203580- treated and untreated young cells were labelled with BrdU. In contrast, only 18% of the SB203580-untreated senescent cells were BrdU-positive. SB203580 treatment also resulted in a dramatic decrease in the number of SA-¦Â-gal-positive senescent cells and a morphological change from a flat cytoplasm to a spindleshaped one with a refractile appearance.

--

Human

L

Others

12,581,156

Gene

0

1

0

1

0

1

0

1

0

CDK4

1019

protein coding

HDF

--

Aging

Prevent

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

Forced expression of wtCDK4 generated colonies in which growth was rapid for ~4 weeks then began to slow and finally ceased after ~7 weeks in a state resembling M1. Similar growth kinetics were observed for wtCDK6 colonies with a final average size of 7*103 cells (range: 103 to 4*104;n=25) corresponding to 12.5 PD and for mCDK6 colonies with a final colony size of 8*103 cells (range: 0.6*103 to 2.7*104; n=14)corresponding to ~13 PD. Colonies expressing mCDK4 di?ered slightly in that growth started to slow after 2 to 3 weeks.Cells were large and attened with a granular cytoplasm and could be maintained in a proliferatively quiescent but viable state for prolonged periods of time with occasional refeeding. SA¦Â-gal activity was high (>95% of cells positive by histochemical assay), BrdU LI was very low (<2%) (data not shown), and TdT assay confirmed that apoptotic cells in all MintCDK populations were extremely rare.Western blot analysis confirmed that growth arrest was not due to loss of expression of the exogenous CDK genes.

--

p53

Upregulation

Western blot

p53 was elevated (12 ¡À 37-fold) in all CDK overexpressors compared to the pBABEpuro control.

Human

L

Others

12,082,615

Gene

0

1

0

1

0

1

0

1

0

CDK6

1021

protein coding

HDF

--

Aging

Prevent

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

Forced expression of wtCDK4 generated colonies in which growth was rapid for ~4 weeks then began to slow and finally ceased after ~7 weeks in a state resembling M1. Similar growth kinetics were observed for wtCDK6 colonies with a final average size of 7*103 cells (range: 103 to 4*104;n=25) corresponding to 12.5 PD and for mCDK6 colonies with a final colony size of 8*103 cells (range: 0.6*103 to 2.7*104; n=14)corresponding to ~13 PD. Colonies expressing mCDK4 di?ered slightly in that growth started to slow after 2 to 3 weeks.Cells were large and attened with a granular cytoplasm and could be maintained in a proliferatively quiescent but viable state for prolonged periods of time with occasional refeeding. SA¦Â-gal activity was high (>95% of cells positive by histochemical assay), BrdU LI was very low (<2%) (data not shown), and TdT assay confirmed that apoptotic cells in all MintCDK populations were extremely rare. Western blot analysis confirmed that growth arrest was not due to loss of expression of the exogenous CDK genes.

--

p53

Upregulation

Western blot

p53 was elevated (12 ¡À 37-fold) in all CDK overexpressors compared to the pBABEpuro control.

Human

L

Others

12,082,615

Gene

0

1

0

1

0

1

0

1

0

EWSR1

2130

protein coding

Lin-Sca1+c-Kit+,BM,HSPC

Blood

Ewing sarcoma

Prevent

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

When we assessed senescence-associated ¦Â-galactosidase activity (SA-¦Â-gal), Ews-/- embryos at a late stage of gestation had significantly higher levels of endogenous SA-¦Â-gal activity compared with WT embryos, suggesting that senescence has already begun from prenatal stages.Cell-cycle analysis showed that a substantial number of Lin , Sca-1 , and c-Kit cells (81%) in Ews- /- mice progressed through the G1, S, and G2/M phases, which was in marked contrast with the Ews- /- mice (34% in G1, S, and G2/M).

--

Human

L

Others

21,030,557

Gene

0

1

0

1

0

1

0

MAP3K7

6885

protein coding

IMR-90,MRC-5,GP293

--

Aging

Accelerate

Colony formation assay//qRT-PCR

A total of 33 kinases showed pro-senescence effects (particularly MAP3K7, PRKCD, and MATK).

--

NF-¦ÊB

Activation

Luciferase reporter assay

Kinases with strong pro-senescence effects, induced expression of SASP-component genes (IL1A, IL1B, IL6 and IL8) through activation of an NF-kappaB-dependent transcriptional program.The luciferase activity was strongly induced by the 3 kinases indicating that these prosenescent kinases activate the NF-¦ÊB pathway.

Human

HL

Others

26,583,757

Gene

0

1

0

1

0

PRKCD

5580

protein coding

IMR-90,MRC-5,GP293

--

Aging

Accelerate

Colony formation assay//qRT-PCR

A total of 33 kinases showed pro-senescence effects (particularly MAP3K7, PRKCD, and MATK).

--

NF-¦ÊB

Activation

Luciferase reporter assay

Kinases with strong pro-senescence effects, induced expression of SASP-component genes (IL1A, IL1B, IL6 and IL8) through activation of an NF-kappaB-dependent transcriptional program.The luciferase activity was strongly induced by the 3 kinases indicating that these prosenescent kinases activate the NF-¦ÊB pathway.

Human

HL

Others

26,583,757

Gene

0

1

0

1

0

MATK

4145

protein coding

IMR-90,MRC-5,GP293

--

Aging

Accelerate

Colony formation assay//qRT-PCR

A total of 33 kinases showed pro-senescence effects (particularly MAP3K7, PRKCD, and MATK).

--

NF-¦ÊB

Activation

Luciferase reporter assay

Kinases with strong pro-senescence effects, induced expression of SASP-component genes (IL1A, IL1B, IL6 and IL8) through activation of an NF-kappaB-dependent transcriptional program.The luciferase activity was strongly induced by the 3 kinases indicating that these prosenescent kinases activate the NF-¦ÊB pathway.

Human

HL

Others

26,583,757

Gene

0

1

0

1

0

FOXM1

2305

protein coding

GBC-SD

--

Gallbladder cancer

Prevent

Knockdown//SA--gal activity assay

Positive SA-¦Â-gal staining was observed in approximately 20% of GBC-SD cells treated with FoxM1 siRNA, which was significantly higher than those in the control group and NC-shRNA group (P < 0.05).

--

Human

L

Others

25,071,344

Gene

0

1

0

1

0

IGFBP3

3486

protein coding

WI-38

--

Aging

Accelerate

Knockdown//SA--gal activity assay

Incorporation of [3 H]thymidine was estimated between 48 and 72 h after the last stress.A decreased proliferative potential of HDFs (pb0.001) was observed in t-BHP- and ethanoltreated cells compared to control cells (no siRNA). The presence of IGFBP-3 siRNA strongly attenuated the reduction of the proliferative potential of HDFs observed after treatment with either t-BHP (pb0.01) or ethanol (pb0.001).S-A ¦Â-gal was monitored using microfluidic detection at 72 h after the last stress.The increased proportion of S-A ¦Â-gal-positive HDFs in t-BHP- or ethanol-induced premature senescence was largely attenuated in HDFs transfected with IGFBP-3 siRNA.

TGF-¦Â1

--

Immunofluorescence

We detected an increase of more than 20-fold in the relative transcript level of IGFBP-3 as well as an increase in IGFBP-3 protein level by immunofluorescence semiquantitative confocal microscopy. Moreover, neutralizing antibody against TGF-¦Â1 added in the medium after the last EtOH- or t-BHP stress inducing premature senescence diminished the increased transcript and protein levels of IGFBP-3.

--

Human

HL

Others

18,329,388

Gene

0

1

0

1

0

RSL1D1

26156

protein coding

HEK293,2BS

--

Aging

Prevent

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

p27Kip1 and PTEN protein levels were greatly decreased (4-fold for PTEN and 7-fold for p27Kip1) in cells with CSIG overexpression.As expected, silencing of CSIG in HEK 293 cells led to increased G0/G1 and reduced S compartments£¬cells overexpressing CSIG exhibited markedly elevated proliferation rates, showed increased S and reduced G1 compartments, and displayed a feature of young cells (lower SA-¦Â-gal activity) compared with the empty-vector-transfected cells.

--

PTEN-p27

Downregulation

Knockdown//Western blot//qRT-PCR//RNA-binding protein (RBP) prediction analysis

Knockdown of PTEN in HEK 293 cells greatly diminished the effect of either overexpression or knockdown of CSIG on p27Kip1 expression.To our surprise, neither overexpression nor silencing of CSIG significantly affected the mRNA levels of PTEN although the PTEN protein level was decreased by 10-fold in CSIG overexpression and increased in CSIG silenced cells (4-fold for 20 nM siRNA and 10-fold for 50 nM siRNA) by Western blot analysis.By Western blotting of CSIG in the pulldown materials, PTEN 5 UTR and CR (but not 3 UTR) were targets of CSIG.

Human

HL

Others

18,678,645

Gene

0

1

0

1

0

1

0

1

0

MBP1

841633

protein coding

HFF

--

Aging

Prevent

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

HFF-MBPsi-4 exhibited a significantly slower rate of proliferation as compared with the HFF-control suggesting that depletion of MBP-1 inhibited cell proliferation. Detection of ¦Â-galactosidase activity at pH 6.0 is a known characteristic of senescent cells. After 48 h, cells were fixed and stained with X-gal for the detection of bgalactosidase activity. More than 70% of HFFMBPsi-4 displayed appearance of enlarged blue cells (panel a) in contrast to the control HFF cells, which failed to exhibit adetectable blue appearance.

--

p53-p21

--

Knockdown//Western blot

We have observed p53 acetylation at Lys382 site and phosphorylation at Ser15 in MBP-1 knockdown HFF. These results suggest that MBP-1 knockdown in HFF results in activation of p53. Enhancement of p53 by phosphorylation and/or acetylation results in activation of its target genes. The cyclin dependent kinase inhibitor, p21 is one of the p53 target genes. Therefore, we examined whether an increase in p21 expression in HFF-MBPsi-4 was due to activation of p53 transcriptional activity. Our results demonstrated a significant increase in p21 in the HFF-MBPsi-4 as compared to HFF-control.

Human

HL

Others

18,852,884

Gene

0

1

0

1

0

1

0

IFNG

3458

protein coding

HUVEC,MEF

--

Atherosclerosis

Accelerate

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

Cell proliferation decreased slightly with IFN-g treatment in time- and dose-dependent manners. A decrease in cell proliferation by IFN-g treatment (more than 1000 U/ml) for 3 days was statistically significant (p < 0.05).The induction of cellular senescence by prolonged treatment with IFN-g was confirmed by an increase in SA-¦Â-gal-positive cells in the treated cells compared to the control cell. IFN-¦Ã treatment increased the G0/G1 cell population and decreased the S and G2/M cell population, suggesting that IFN-¦Ã inhibited cell proliferation through G0/G1 arrest in the cell cycle. No difference in cell cycle status was observed in IFN-a treated cells for 6 days.

--

p53

--

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

Prolonged stimulation of IFN-g in the p16-knockdown (p16sh) cells increased SA-¦Â-gal activity staining to a level similar to that in control cells. In contrast, no increase in SA-¦Â-gal staining was observed in the p53-knockdown (p53sh) cells following prolonged stimulation with IFN-g. To obtain more evidence that IFN-g-induced senescence involves a p53 signaling pathway, we measured the effects of IFN-g on cellular senescence in wild, p16-/-, or p53-/- MEFs. We reproducibly noticed an increase in the number of SA-b-galpositive cells in p16-/- MEFs, but not in IFN-g-treated p53-/- MEFs, after prolonged stimulation with IFN-g. Therefore, these results suggest that IFN-g-induced cellular senescence is mediated through a p53-dependent pathway.

Human

L

Others

19,071,156

Gene

0

1

0

1

0

1

0

CPEB1

64506

protein coding

HFF,WI-38

--

Aging

Prevent

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

After 68 population doublings, the mock-infected and shTETR-infected cells stopped dividing and assumed a flat senescent-like morphology. These cells also stained for ¦Â-galactosidase activity at acidic pH, a common marker for senescence. The shCPEB-infected cells, however, continued to grow, did not undergo a morphology change, and did not stain for ¦Â-galactosidase activity. Moreover, while mock-infected and shTETR-infected cells expressed high levels of p21CIP1 and p16INK4A, which is consistent with entry into senescence, the cells infected with shCPEB did not.

p53

--

Western blot

Two days later, the cells were infected with a virus expressing CPEB; the cells were then analyzed for growth and p21, a target gene of p53.While CPEB induced senescence in cells lacking GSE-22, they were unable to do so if they contained the inhibitory peptide. Moreover, GSE-22 prevented p21 expression, thus demonstrating that it indeed inhibited p53 activity. These results indicate that CPEB-induced senescence requires p53 in human cells.

--

Human

L

Others

19,141,477

Gene

0

1

0

1

0

1

0

PLA2R1

22925

protein coding

HDF

--

Aging

Accelerate

Growth curve assay//SA--gal activity assay

We assessed the growth of control and PLA2R overexpressing WI38 cells. Cell growth blockade was observed in growth curve analysis and colony formation assay when PLA2R was overexpressed, and was mainly due to senescence induction, as PLA2R-overexpressing cells showed a strong SA-¦Â-gal activity.

--

p53

Activation

Knockdown//Western blot

In shPLA2R-infected HDFs, p53, and its targets p21 and human double minute 2 (HDM2), decreased when compared with control cells. Phospho-Rb increased, suggesting that the cells were proliferating. Conversely, when PLA2R was ectopically expressed, the levels of p53, p21 and HDM2 increased when compared with control senescing cells and phospho-Rb was found to decrease.

Human

HL

Others

19,197,340

Gene

0

1

0

1

0

SUPT5H

6829

protein coding

HeLa

--

Aging

Prevent

Flow cytometry

Tetracycline addition resulted in a gradual decrease in the populations corresponding to S and G2/M phases and a concomitant increase in the population corresponding to G1 phase.

--

p53

--

Knockdown//Western blot

We found that Spt5 depletion resulted in a significant elevation of the p53 protein level and a concomitant increase in the CDNK1A/p21 protein level.

Human

HL

Others

19,210,550

Gene

0

1

0

PRKCA

5578

protein coding

TIG-1,Human normal diploid cell

--

Aging

Accelerate

Growth curve assay//SA--gal activity assay

Adenoviral transduction of PKC-¦Ä induced growth inhibition in TIG-1 cells. Bistratene A, a PKC-d activator, also strongly inhibited the growth of TIG-1 cells . Furthermore, PKC-¦Ä transduction and activation caused enlarged and flattened cell morphology and augmented SA-¦Â-Gal activity in TIG-1 cells, as was observed in the senescent TIG-1 cells.

hTERT

Downregulation

qRT-PCR

We investigated whether PKC-¦Ä when activated in senescent cells functions in repression of the hTERT gene. The results showed that hTERT repression in senescent TIG-1 cells was relieved following infection with PKC-¦Ä-KN, demonstrating that PKC-¦Ä functions in the repression of the hTERT gene in senescent cells.

--

Human

L

Others

19,279,193

Gene

0

1

0

1

0

TOP1

7150

protein coding

WI-38,IMR-90,U2OS,HDF

--

Aging

Accelerate

Colony formation assay//Growth curve assay//Knockdown//SA--gal activity assay

Colony formation by Top1-depleted HDFs correlated with a loss of senescence markers: the formation of senescence-associated heterochromatin foci and the appearance of senescence-associated ¦Â-galactosidase activity.Top1 knockdown extended the life span of WI38 cells to f10 more population doublings,Growth of U2OS or WI38 cells constitutively expressing GFPTop1 was much slower than that of control-transfected U2OS cells.

--

p53

Activation

Western blot

Furthermore, measurement of p53 activity in the presence of an increasing amount of Top1 with a constant amount of a p53 activity reporter revealed dose-dependent activation of p53 by Top1. To further confirm the involvement of the p53 pathway, we examined the effect of p53 inhibition by a dominant negative form of p53 (p53DN) over the proliferation arrest induced by GFPTop1. Interestingly, p53 pathway inhibition reverted efficiently the proliferation arrest induced by GFPTop1.

Human

HL

Others

19,435,923

Gene

0

1

0

1

0

1

0

1

0

RUNX1

861

protein coding

Hs68

--

Aging

Accelerate

SA--gal activity assay

We found that ectopic RUNX1 expression in these cells induced growth arrest with early onset and a senescence-like morphology with a flatter appearance than that seen with H-RASV12.

p53//p38MAPK

--//Activation

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

In p53-null MEFs, RUNX1-ETO did not induce senescence-like morphology or SA-¦Â-gal staining (not shown), although a more marked growth delay was induced compared with that in RUNX1. These results suggest that both RUNX1 and RUNX1- ETO also have cell cycle inhibitory effects that are independent of p53, although it is clearly required for the full expression of the senescent phenotype. The abilities of RUNX1 and RUNX1-ETO to activate p38MAPK were examined using a specific antibody that recognizes two phosphorylation sites responsible for p38MAPK activation (Thr180 and Tyr182).Indeed, western blot analysis on day 7 of the culture period showed that both RUNX1 and RUNX1-ETO induced elevated levels of phospho-p38, with RUNX1-ETO being the more potent agonist, inducing phosphop38 to a similar degree as does H-RASV12.Notably, the levels of total p38MAPK remained unaffected.

--

Human

L

Others

19,448,675

Gene

0

1

0

PPM1D

8493

protein coding

mesenchymal-stem-cell

--

Aging

Prevent

Growth curve assay//XTT assay

Early-passage hMSCs (p5, open circle) and late-passage Wip1-hMSC (p12, filled square) proliferated normally, whereas late-passage Cont-hMSCs (p12, open square) underwent distinct cell growth arrest(To define ¡®¡®early-passage¡¯¡¯ and ¡®¡®late-passage¡¯¡¯ cells, we referred to the previous report that elucidated the growth kinetics of hMSCs [6]. According to those results, the growth of hMSCs is significantly retarded after passage number 10; thus, we used the term late passage for hMSCs grown more than 10 passages. This difference in cell growth rate was reproduced using the XTT assay: late-passage hMSCs (p13) barely proliferated after 1 week, whereas Wip1-hMSCs at the same passage level proliferated in a fashion similar to early-passage hMSCs.We therefore concluded that Wip1-hMSCs bypassed the cell growth arrest that occurs in late-passage hMSCs.

p16//p38

Downregulation//Downregulation

qRT-PCR//Western blot

Consistently, p16 mRNA levels determined by semiquantitative real-time PCR were upregulated in late-passage hMSCs, compared with early-passage hMSCs that had not experienced growth arrest. Interestingly, p16 mRNA levels in later passage Wip1-hMSCs were significantly lower than in the late-passage hMSCs.As expected, p38 activity determined by p38 in vitro kinase assay using ATF-2 as a substrate was increased by 50%, whereas p38 activity in Wip1-expressing hMSCs was decreased by 50%.

--

Human

L

Others

19,544,416

Gene

0

1

0

1

0

RPS6KA6

27330

protein coding

HCT116,IMR-90

Colon

Colorectal cancer

Accelerate

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

We tested RSK4 expression levels in IMR90 after inducing senescence by cumulative passages. Cells stopped proliferating, acquired the enlarged, flattened morphology typical of senescent cells, and were positive for SA-¦Â-gal staining (data not shown). The senescent cells showed increased RSK4 expression on quantitative real-time PCR and Western blot .

--

Human

L

Others

19,584,160

Gene

0

1

0

1

0

1

0

SIN3B

23309

protein coding

MEF

--

Aging

Accelerate

Knockdown//SA--gal activity assay

The percentage of Sin3B-/-MEFs that were positive for SA-¦Â-gal staining was significantly lower than that of their wild-type counterparts at passage 6. This observation corroborates the noticeable differences in cell morphology after six to eight passages between Sin3B-/-and Sin3B+/+ MEFs, which appeared significantly more flat and enlarged, irrespective of the cell density.

E2F

--

CHIP

To showthe direct role of Sin3B in mediating E2F transcriptional control, we performed chromatin immunoprecipitation experiments. We found that Sin3B is strongly and specifically enriched at E2F target promoters upon RasV12 overexpression , but not at nonrelevant promoters such as oct4 , we showed that this enrichment for heterochromatin marks at E2F target promoters upon oncogenic stress requires the presence of Sin3B.

p19-p53

--

Western blot//Knockdown

As previously reported, expression of p19ARF was induced upon transduction of activated Ras into wild-type cells . Surprisingly, in Sin3B / cells overexpressing activated Ras, the level of p19ARF protein was comparable with that detected in Sin3B+/+ cells. However, the amount of p19ARF detected in the absence of oncogenic stress was significantly higher in Sin3B -/-cells compared with Sin3B+/+ cells .Our observation that Sin3B-/- fibroblasts do not senesce upon RasV12 expression, in spite of high levels of p19ARF, strongly suggests that Sin3B functions downstream of p19ARF in oncogene-induced senescence. These results showthat, although Sin3B participates in the transcriptional repression of p19ARF under normal culture conditions, it is required for the induction of senescence upon activation of the p19ARF/p53 pathway.

Human

L

Others

19,654,306

Gene

0

1

0

1

0

PSMD14

10213

protein coding

H1299,HeLa,hTERT-HMEC1

--

Aging

Prevent

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

The cell cycle was arrested at the G0-G1 phase in PSMD14 knockdown cells. A significant increase of SA-¦Â-Gal positive cells was observed in PSMD14 knockdown cells (52%).

CDK1//Cyclin B//CDC25C

Downregulation//Downregulation//Downregulation

Western blot

Three days post-transfection with PSMD14 siRNA,the levels of cyclin B1, CDK1 and CDC25C were decreased to 6%, 33% and 30% of the control level(NT siRNA knockdown sample), respectively (from 30%, 64% and 62% of NT siRNA control, respectively at 2 days of knockdown).

--

Human

HL

Others

19,732,767

Gene

0

1

0

1

0

1

0

MECP2

4204

protein coding

mononuclear cell

Bone

Aging

Prevent

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

We observed a significant reduction of S-phase cells, along with an increase of G1 cells in samples with partially silenced MECP2.The decrement of S-phase cells resulted in a decrease of cell proliferation as evaluated by cell count.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-siRNACTRL.

--

Human

L

Others

20,065,105

Gene

0

1

0

1

0

1

0

1

0

NFE2L2

4780

protein coding

HFL-1

--

Aging

Prevent

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

Knockdown of Nrf2 resulted in inhibition of proliferation and induction of premature senescence in HFL-1 fibroblasts.The cells acquired the typical senescent morphology, exhibited a higher percentage of ¦Â-galactosidase positive staining and revealed increased levels of p16 protein,as compared with the HFL-1 cells transfected with scrambled siRNA.

18¦ÁGA

--

qRT-PCR//Western blot//Immunoblotting

Specifically, treatment of HFL-1 young cells with 2 ¦Ìg/ml of 18¦ÁGA for 2 h (optimal conditions, see next paragraph) induced both the RNA and protein expression levels of Nrf2. In support, we have observed reduced levels of Keap1 as well as increased levels of its modified isoform (a protein greater than 150 kDa). Moreover, cell fractionation experiments as well as immunofluorescence analysis under the same conditions revealed that treatment with 18¦ÁGA promoted Nrf2 translocation to the nucleus, a feature that concurs with its activation.

--

Human

L

Others

20,068,043

Gene

0

1

0

1

0

1

0

1

0

TRX1

824267

protein coding

BJ

--

Aging

Prevent

Knockdown//SA--gal activity assay

As demonstrated by cessation of population doublings and by upregulated senescence-associated beta-galactosidase (SA-beta-gal) activity.Quantification of the SA-beta-gal staining showed that the percentage of cells with positive activity was significantly higher in cells infected with either of the shTRX1 constructs than for cells infected with the control shRNA construct against GFP.

--

p53//p16

--

Western blot

TRX1 suppression in BJ cells led to persistent upregulation of p53/p21 and p16INK4a proteins .TRX1 suppression in the BJELT cells prevented the proliferation defect observed in normal BJ cells with intact p53 and p16INK4a pathways , verifying that either one or both pathways have functional roles in establishing the shTRX1-induced senescent phenotype as opposed to the proliferation defect arising from quiescence or another non-senescence-related mechanism.

Human

L

Others

20,074,557

Gene

0

1

0

1

0

CENPA

1058

protein coding

TIG-3,HeLa

--

Aging

Prevent

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

A BrdU incorporation assay (BrdU is a marker of DNA synthesis) demonstrated that CENP-A depletion in TIG3 cells reduced the proportion of cells transiting S phase of the cell cycle. The proportion of M-phase cells was also reduced in the CENP-A-depleted TIG3 cells, as determined by immunostaining for S10-phosphorylated histone H3 (a marker of late G2 and mitotic cells),immunoblotting analysis demonstrated that CENP-A depletion in TIG3 cells led to a reduction in the levels of cyclin B and CDC2, which are essential for cell cycle progression, and an elevation of the levels of the CDK inhibitors, p16INK4a and p21CIP1.TIG3 cells depleted of CENP-A also exhibited the increase in SA- ¦Â-Gal activity that is the cytological marker of senescent cells.

--

p53

--

Knockdown//Proliferation assay

P53 appeared to be essential for this proliferation arrest; CENP-A depletion in p53-depleted TIG3 cells did not stop proliferating.

Human

L

Others

20,160,010

Gene

0

1

0

1

0

1

0

1

0

ENDOG

2021

protein coding

HUVEC

Umbilical vein

Aging

Prevent

Knockdown//SA--gal activity assay

We found that knocking down EndoG in both early passage and late passage cells led to a significant delay in cell proliferation.This was accompanied by an increase in the percentage of cells staining positive for senescence-associated ?-galactosidase, suggesting that depletion of EndoG from human endothelial cells induces premature senescence.

--

Human

L

Others

20,211,237

Gene

0

1

0

1

0

DDB2

1643

protein coding

MEF

Embryo

Aging

Accelerate

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

WT and DDB2 -/- MEFs at various passages were compared for the levels of p19Arf, which is critical for senescence in the MEFs. We consistently found a decrease in the levels of p19Arf mRNA in the DDB2 -/-MEFs.To measure oxidative stress-induced premature senescence, the cells were assayed for SA-¦Â-Gal. Clearly, there was a significantly lower expression of SA-¦Â-Gal in the DDB22-/-MEFs .

--

Human

L

Others

20,351,176

Gene

0

1

0

1

0

1

0

snail

778762

protein coding

LNCaP,PC-3

--

Prostate cancer

Prevent

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

There was a significant 30¨C79% reduction in the relative ATP amount of SNAI1-siRNA-treated cells compared to cells treated with IR-siRNA after long-term knockdown of Snail for 5 days. In IRsiRNA-treated LNCaP cells, less than 5% stained positive for SA-¦Â-gal, as compared to up to 30% of SNAI1-siRNA-treated LNCaP cells in some wells (p< 0.05£©. As expected from the morphology, only a few PC-3 cells were positive for SA-¦Â-gal,independent of siRNA treatment (data not shown).

--

Human

L

Others

20,397,042

Gene

0

1

0

1

0

1

0

PTTG1

9232

protein coding

IMR-90,BJ-1,WI-38

--

Aging

Accelerate

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

The cell number of PTTG1- expressing cells did not increase after infection, indicating that PTTG1 inhibited proliferation of normal cells. Flow cytometry analysis revealed that the proportion of G1 cells was reduced in PTTG1-expressing cells.Our results clearly indicated that IMR90 cells expressing PTTG1 showed an induction of SA-¦Â -gal activity.

p53

Upregulation

SA-¦Â-gal activity assay//Cell proliferation assay//Knockdown

The level of PTTG1-activated p53 was decreased upon shRNA treatments. Significantly, the growth of PTTG1- expressing cells was increased upon knocking down p53 expression. The results indicated that reduced p53 function could restore PTTG1-induced growth inhibition in normal cells. The SA-¦Â-gal activity was then checked to evaluate the effect of a lower p53 level on PTTG1-induced senescence. In the p53 knockdown cells, the number of SA-¦Â-gal-positive cells was also significantly lower than that of the control cells.

--

Human

L

Others

20,452,981

Gene

0

1

0

1

0

1

0

Vdup1

38023

protein coding

2BS

--

Aging

Accelerate

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

The results showed that cells expressing VDUP1 arrested cell cycle progression at the G0/G1 phase,displayed the morphology of flat cells, stained positive for SA-¦Â-gal, and formed SAHF 9 days after retroviral infection (5 days post-selection); moreover, molecular markers of senescence, such as increased p53, p21, p16, and decreased p-Rb levels, were also detected after VDUP1 overexpression.

FOXO3a//miR-17-5p

Binding//--

Luciferase reporter assay//qRT-PCR

Mutation of the FOXO-binding element decreased about 2.2- fold luciferase activity in senescent cells as compared with the wild-type construct. In contrast, the luciferase activity from the mutant construct slightly decreased as compared with the wildtype construct in young cells.To demonstrate that miR-17-5p interacts with specific target sequence localized in this region of VDUP1 3-UTR, an additional reporter construct was generated in which the 7-bp ¡°seed¡± sequence (CGUGAAA) of two putative miR-17-5p target sites were mutated using PCR.The resulting construct, pVDUP1¨C3UTR/ miR-17- 5p, was transfected into young and senescent cells; this mutation dramatically increased luciferase activity in young cells, whereas only a slight elevation was observed in senescent cells.

--

Human

L

Others

20,656,682

Gene

0

1

0

1

0

1

0

1

0

TNFSF15

9966

protein coding

CEP

Peripheral blood

Aging

Accelerate

Knockdown//SA--gal activity assay

The efficiency of the two knockdown constructs to downregulate endogenous TL1A versus control. TL1A knockdown cells were stained for SA-¦Â-gal 7 and 9 days after transduction, and the amount of stained cells was quantified.For both TL1A-specific shRNAs, the amount of SA-¦Â-gal¨Cpositive cells was decreased compared with control.

--

Human

HL

Others

20,675,618

Gene

0

1

0

1

0

BRG1

834543

protein coding

MSC

Bone marrow

Aging

Prevent

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

BRG1 silencing reduced the percentage of S-phase cells and induced a decrease in apoptosis,We observed a small increase of G1 in MSCs with silenced BRG1 compared with controls (67.61 versus 58.33%). It is noteworthy that MSCs with silenced BRG1 had a significant lower percentage of S-phase cells (Po0.05; 29.11 versus 40.23%) and an increase of G2/M cells (3.28 versus 1.43%).We observed signs of senescence in cells treated with Ad-siRNA-2405, as detected by acid-¦Â-galactosidase, compared with cells transduced with control Ad-siRNA.

--

Rb-p53

--

Western blot//RT-PCR

In MSCs transduced with Ad-siRNA2405 or with control virus, we inhibited P53 with Ad-CMVE1A(YH47-928) and RB family proteins with AdCMV-E1A(RG2). Both RB and P53 seemed to have a role in BRG1-mediated senescence. In situ acid-bgalactosidase staining showed that in cells with silenced BRG1 the inhibition of P53 and of RB reduced the percentage of senescent cells . In contrast, protection from apoptosis seemed to rely mainly upon the RB family, because inactivation of these proteins significantly increased the percentage of apoptotic cells, reaching a percentage higher than that observed when BRG1 was silenced.

Human

L

Others

20,697,355

Gene

0

1

0

1

0

1

0

CHK1

852577

protein coding

HCT116

--

Colorectal cancer

Accelerate

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

A reduction in the cell numbers in the G1 or G2/M phases of HCT116 wt or p53¨C/¨Cat 72 hrs following Chk1 siRNA transfection caused enhanced cell death reflected by increased Pre-G1 cell populations.cyclin B1 was found in the nucleus in 90% of p53¨C/¨Ccells, allowing for the conclusion that p53¨C/¨C cells accumulated in late G2 just before mitosis .We found that wt cells establish a G1 arrest following Chk1-involved G2 arrest, which was associated with a senescent phenotype as shown by staining for ¦Â-galactosidase activity.In addition, wt cells showed the characteristic flattened and enlarged morphology in the majority of cells together with prolonged expression of p53 and p21WAF1.

--

Human

L

Others

20,716,119

Gene

0

1

0

1

0

1

0

1

0

NEK6

10783

protein coding

IMR-90,EJ

--

Aging

Prevent

SA--gal activity assay

While EJ-vector control cells became flattened and enlarged after p53 expression, EJ-Nek6 cells maintained normal morphology and grew continuously.In addition, whereas SA ¦Â-gal activity was significantly increased in EJ control cells starting 4 days after p53 expression, and a majority of EJ control cells showed SA ¦Â-gal activity at 6 day later, SA ¦Â-gal activity was significantly reduced in EJ-Nek6 cells indicating that the onset of p53- induced senescence was suppressed by Nek6 overexpression.

--

Human

L

Others

21,099,361

Gene

0

1

0

VENTX

27287

protein coding

293,IMR-90

--

Aging

Accelerate

Cell proliferation assay//SA--gal activity assay

Consistent with its role as an inhibitor of cell proliferation, induction of VentX expression is associated with significant inhibition of U2OS/VentXTet cell growth. Interestingly, VentX expression caused a striking morphological change with U2OS/VentXTet cells appeared to be enlarged and flattened. These cells displayed positive staining for SA ¦Â-galactosidase, a characteristic marker of cellular senescence.

--

p53-p21//p16-Rb

Activation//Activation

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

We found that VentX expression led to a significant increase in the protein levels of p53 and two CDK inhibitors: p21 and p16ink4a ,Consistently, we showed that ectopic expression of VentX trans-activated the human p53 and p21 promoter-luciferase reporters in a dose-dependent manner.A ChIP assay therefore was performed to examine a potential direct interaction between VentX and the p53 promoter. The ChIP assay revealed a specific binding of VentX to the p53 promoter. The potential transcriptional activation of p16ink4a by VentX was suggested by the increased p16ink4a mRNA levels upon ectopic expression of VentX . Using p16ink4a promoterluciferase reporter assay, we further demonstrated that VentX promoted p16ink4a transactivation in a dose depend ent manner. Human p16 promoter also contains putative homeodomain binding sites.Consistently, the ChIP assay demonstrated a specific binding of VentX to the p16ink4a promoter.

Human

L

Others

21,325,273

Gene

0

1

0

1

0

YPEL3

83719

protein coding

MCF-7

Mammary Gland

Breast cancer

Accelerate

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

When compared to MCF-7 cells, shYPEL3-expressing MCF-7 cells showed a statistically significant increase in cell numbers when grown over 10 days. Using an MTT-based assay, YPEL3 knockdown for six days led to an increase in cell numbers while YPEL3 induction over the same period resulted in a decrease in cell number.MCF7 cells stably selected for the loss of YPEL3 grown in charcoalstripped serum did not demonstrate an elevation in cellular senescence.

--

Human

L

Others

21,671,470

Gene

0

1

0

1

0

1

0

IGFBP6

3489

protein coding

HDF

--

Aging

Prevent

BrdU assay//Growth curve assay//SA--gal activity assay

At the end of the lifespan, overexpression of IGFBP-6 enabled the cells to perform 4¨C 5 additional population doublings.This was accompanied by a significant increase in the cell proliferation rate as measured by BrdU incorporation and a significant decrease in apoptotic cell death determined by Annexin V-staining and propidium iodide staining at 90¨C95% of lifespan completed. 120 days after infection (ca. 95% of lifespan completed), nearly all the cells in the controls stained SA-¦Â-gal positive, whereas roughly 50% of the IGFBP-6 expressing cells stained still SA-¦Â-gal negative and displayed reduced p21Waf1/Cip1 protein expression relative to controls.

--

Human

L

Others

21,820,463

Gene

0

1

0

1

0

1

0

RUVBL2

10856

protein coding

A498,KRC/Y

The tumor specimens and corresponding tumor adjacent renal tissue

Renal carcinoma

Prevent

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

The results showed that the percentage of ¦Â-Gal- positive in control RCC cells was significantly lower than that in reptin siRNA-treated cells,Flow cytometry analysis demonstrated that these reptin siRNA-treated cells underwent growth arrest at the G1 phase.

--

Human

L

Others

22,341,977

Gene

0

1

0

1

0

1

0

IGFBP7

3490

protein coding

MCF-7

--

Breast cancer

Accelerate

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

Addition of CM from IGFBP-rP1-transfected cells to untransfected MCF-7 cells blocked cell proliferation and increased SA-¦Â-gal activity, whereas CM from the control vector (pEGFP-N1)-transfected or untransfected cells failed to inhibit cellular proliferation and induce senescence.A significant increase in the cell population at the G0/G1 phase of the cell cycle was detected in IGFBP-rP1-transfected MCF-7 cells, which is one of the typical phenotypes in cellular senescence.

p21

Upregulation

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

The IGFBP-rP1-transfected MCF-7 cells had increased levels of p21 in comparison with the control cells.The results showed that cell proliferation block and increased SA-¦Â-gal activity in response to IGFBP-rP1 were partially reversed by p21 knockdown. These results suggest that cellular senescence induced by IGFBP-rP1 is mediated at least in part by p21 up-regulation.

--

Human

L

Others

22,392,074

Gene

0

1

0

1

0

1

0

MCL1

4170

protein coding

HCT116,MCF 10A,MCF-7,MEL526,MEF

Tumor tissue

Cancer

Prevent

BrdU assay//Colony formation assay//SA--gal activity assay

As control cells induced to senesce have significant increases in the number of nuclear PML bodies compared to untreated cells. However, cells overexpressing Mcl-1 had significant abrogation of senescent changes after treatment, including reduced SA-¦Â-gal+ and no increase in PML foci compared to HCT116 empty vector cells. In contrast, doxorubicintreated control cells formed significantly fewer colonies compared to those overexpressing Mcl-1. In addition, using the BrdU incorporation assay, we observe that the proliferation of cells growing in drug-free media was equivalent regardless of the level of Mcl-1 expression and that doxorubicin treatment of control cells resulted in a marked decrease in BrdU incorporation.

p53

--

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

We noticed that the accumulation of p53 in cells overexpressing Mcl-1 and treated with doxorubicin is the same as in control cells despite a significant difference in SA¦Â-gal-activity.

--

Human

HL

Others

22,451,485

Gene

0

1

0

1

0

1

0

TRPM8

79054

protein coding

BxPC-3,PANC-1

Pancreas/duct

Pancreatic cancer

Prevent

Knockdown//SA--gal activity assay

Results of the SA ¦Â-gal assay indicate that RNA interferencemediated silencing of TRPM8 induced senescence in the pancreatic cancer cell lines BxPC-3 and PANC-1.

--

Human

L

Others

22,555,807

Gene

0

1

0

1

0