Venkatachalam/aging_reg_dataset · Datasets at Fast360

RBL2

5934

protein coding

MSC

--

Retinoblastoma

Accelerate

Flow cytometry//SA--gal activity assay

Irradiation of wild-type MSCs increased the percentage of senescent cells. A similar pattern was observed in cells lacking RB2 or P107. In detail, the cells with silenced RB2 showed a degree of senescence lower than controls in basal conditions following X-ray treatment.

--

Human

L

apoptosis

27,124,644

Gene

0

1

0

1

0

GGCT

79017

protein coding

PC-3

--

Prostate cancer

Prevent

Knockdown//SA--gal activity assay

Both ¦Âgalactosidase staining and ¦Â-galactosidase activity measurement confirmed that pro-GA induced senescence, consistent with prior findings that GGCT knockdown induces senescence.

--

Human

L

apoptosis

31,519,583

Gene

0

1

0

1

0

RASSF1

11186

protein coding

A549

Tumor tissue

Colorectal cancer

Accelerate

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

Conditional RASSF1A expression arrested A549 cells in the G1 phase of the cell cycle.RASSF1A-mediated cell cycle arrest in A549 human lung cancer cells was accompanied by morphologic and biochemical features of sensescence.Moreover, tumors with conditionally expressed RASSF1A exhibited SA-¦Â-gal activity, which serves as an indicator for senescence occurring in vivo, as well as enhanced p21Cip1/Waf1 expression.

p21/WAF1

Upregulation

Western blot

In agreement with our findings in A549 cells, RASSF1A expression increased endogenous p21Cip1/Waf1 levels in p21Cip1/Waf1-proficient HCT116 cells.

--

Human

L

apoptosis

19,223,555

Gene

0

1

0

1

0

1

0

CLU

1191

protein coding

--

Lung

Idiopathic pulmonary fibrosis

Prevent

Histological staining//Knockdown//Western blot

After 14-days of bleomycin administration, both bleomycin-treated wildtype and CLU?/? mice had increased percent p-H2A.X+ cell clusters, as defined by clusters of 3 or more positive cells, but there were no significant differences between the genotypes. Further, there was no difference in either 8-Oxo-dG intensity or percent p-H2A.X+ cell clusters, 28-days after saline and bleomycin-treatment of wildtype mice. However, there was a significant increase in 8-Oxo-dG staining intensity and p-H2A.X+ cell clusters, in CLU?/? compared to wildtype mice, 28-days after bleomycin-challenge. Finally, exogenously administered Clusterin protein to saline or bleomycin treated wildtype mice did not modulate the number of p-H2A.X+ cell clusters or the percentage of 8-Oxo-dG positive cells in the airways. there was a significant decrease in the expression of msh2, msh6, and ogg1 transcripts in CLU?/? bleomycin-challenged mice compared to CLU?/? saline-control mice. Moreover, there was a significant decrease in msh6 in CLU?/? compared to wildtype mice following bleomycin challenge at Day 28. There was an increase in whole lung p21 and IL1? proteins and KC transcript expression in the bleomycin-challenged CLU?/? compared with their wildtype counterparts at this time point. Finally, there was a significant increase in sftpc transcript expression in wildtype but not CLU?/? mice, 28 days after bleomycin administration.

--

Human

HL

apoptosis

29,133,960

Gene

0

1

0

1

0

1

0

DAXX

1616

protein coding

mOSE

--

Ovarian cancer

Prevent

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

Morphological observations showed that cultured Daxxflox/flox mOSE cells were larger in size with flattened shapes after 15 days in culture, which is a feature of cellular senescence.This showed that 4% of cells hadundergone senescence after 4 days in culture, and >90% of cells were senescent after 15 days in culture. However, senescence (positive staining for SA-¦Â-gal) was barely detectable in immortalized mOSE cells.p16, p21,and p27 mRNAs were highly expressed in Daxxflox/floxmOSE cells after culture for 1 day, whereas these were weakly expressed in immortalized mOSE cells.Senescence occurred rapidly for Daxxflox/floxmOSE cells at 24 h after infection. SA-¦Â-Gal staining showed that about 35% of Daxxflox/floxmOSE cells infected with pAd-GFP-Cre were stained positively vs. about 10% of Daxxflox/floxmOSE cells that were infected with pAd-GFP as a control.

--

p53-p21

--

Western blot//Immunofluorescence//qRT-PCR

p21 and p53 accumulated in DAXX-deleted mOSE cells, which suggested that p21/p53 may play an essential role in DAXX deletion-induced senescence. Immunofluorescent staining showed that p27 expression in DAXX-deleted mOSE cells was significantly increased compared with that in control cells. Western blot and real-time PCR analyses showed that the expression of p27 (a cyclin-dependent kinase inhibitor) wa up-regulated in DAXX-deleted cells. However, NOXA and PUMA (p53 inducible genes) mRNA expression increased significantly in DAXX-deleted cells as shown by real-time PCR.DAXX-induced senescence appeared to be largely p21-dependent.

Human

HL

apoptosis

23,542,781

Gene

0

1

0

1

0

1

0

CSNK2A1

1457

protein coding

A172,U87 MG

--

Glioblastoma

Accelerate

Flow cytometry//SA--gal activity assay

Treatment with Pifithrin-¦Á reversed CK2-I-induced cell cycle arrest. Similar results were obtained in U87 cells also (data not shown). The increase in senescence-positive cells observed in cells treated with CK2-I both in the presence and absence of TNF¦Á, was abrogated in the presence of Pifithrin-¦Á.

p53

Upregulation

Western blot

A dramatic increase in p53 levels accompanied with an increase in phosphorylation (Ser-15) and acetylation (Lys373, 382) of p53 was observed upon treatment with CK2-Is either alone or in the presence of TNF¦Á. CK2-Is either alone or in combination with TNF¦Á increased mRNA levels of p53-induced pro-apoptotic molecules GADD45¦Â and Noxa.

--

Human

HL

apoptosis

22,318,540

Gene

0

1

0

1

0

CSNK2B

1460

protein coding

A172,U87 MG

--

Glioblastoma

Accelerate

Flow cytometry//SA--gal activity assay

Treatment with Pifithrin-¦Á reversed CK2-I-induced cell cycle arrest. Similar results were obtained in U87 cells also (data not shown). The increase in senescence-positive cells observed in cells treated with CK2-I both in the presence and absence of TNF¦Á, was abrogated in the presence of Pifithrin-¦Á.

p53

Upregulation

Western blot

A dramatic increase in p53 levels accompanied with an increase in phosphorylation (Ser-15) and acetylation (Lys373, 382) of p53 was observed upon treatment with CK2-Is either alone or in the presence of TNF¦Á. CK2-Is either alone or in combination with TNF¦Á increased mRNA levels of p53-induced pro-apoptotic molecules GADD45¦Â and Noxa.

--

Human

HL

apoptosis

22,318,540

Gene

0

1

0

1

0

MAP2K7

5609

protein coding

MCF-7

--

Breast cancer

Prevent

SA--gal activity assay

Upon treatment with inhibitors of MEK, or its downstream effector ERK, senescence was induced to the same degree as in replication stress response competent cells, suggesting MEK pathway inhibition effectively recovered the ability of cells to undergo oncogene-induced senescence.

--

Mdm2-p21

--

Western blot

This result was confirmed by western blot analysis, where ERK inhibition blocked MDM2 phosphorylation and increased p21 levels.

Human

HL

apoptosis

29,768,207

Gene

0

1

0

MAPK1

5594

protein coding

MCF-7

--

Breast cancer

Prevent

SA--gal activity assay

Upon treatment with inhibitors of MEK, or its downstream effector ERK, senescence was induced to the same degree as in replication stress response competent cells, suggesting MEK pathway inhibition effectively recovered the ability of cells to undergo oncogene-induced senescence.

--

Mdm2-p21

--

Western blot

This result was confirmed by western blot analysis, where ERK inhibition blocked MDM2 phosphorylation and increased p21 levels.

Human

HL

apoptosis

29,768,207

Gene

0

1

0

BRD4

23476

protein coding

Mouse Fibroblast cell

--

Aging

Prevent

Southern blot

Treatment with OTX015 caused significant telomere shortening in both human HeLa cells, and mouse CAST/EiJ fibroblasts.

--

Human

HL

apoptosis

28,854,735

Gene

0

1

0

HDAC4

9759

protein coding

U87 MG,U251MG

--

Cancer

Prevent

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

RT treatment with 8 Gy significantly and persistently increased SA-¦Â-gal activity,and up-regulated p21WAF1/CIP1but not p27CIP/KIPor p16ink4a protein expression in HDAC4- but not in HDAC6-silenced U87MG and U251MG cells. The role of p21WAF1/CIP1up-regulation in RT-induced HDAC4-silenced GBM cells senescence, was investigated by silencing p21WAF1/CIP1with specific siRNAs. In the presence of silenced HDAC4, p21WAF1/CIP1 knocking-down significantly (p ¡Ü 0.01) counteracted RT-induced senescence and restored the ability of silenced-HDAC4 GBM cells to form colonies after RT-treatment. p21WAF1/CIP1 siRNA efficiency is reported.

--

Human

L

apoptosis

28,342,984

Gene

0

1

0

1

0

1

0

1

0

MYCN

4613

protein coding

HEL,K562

--

Erythroleukemia

Prevent

FCM analysis//Flow cytometry//Knockdown//SA--gal activity assay//qRT-PCR

Suppression of MYCN resulted in increased senescence-associated acidic ¦Â-gal staining. Cell cycle analysis showed an increased percentage of cells with G0/G1 phase and a decreased percentage of cells with S phase in the MYCN-knockdown cells. The results showed that depletion of MYCN elevated the P21 expression in HEL cells analysis showed that etoposide treatment resulted in an increase in P21 expression, and MYCN depletion enhanced the etoposide-induced P21 activation in HEL cells. Further comparison analysis showed that overexpression of MYCN inhibited etoposide-mediated P21 activation in HEL and K562 cells.

EZH2//p21

Upregulation//Downregulation

qPCR//CHIP//Knockdown

The qPCR analysis showed that knockdown of MYCN significantly decreased the expression of EZH2 mRNA . The results showed that knockdown of MYCN led to significantly reduced enrichment of DNA binding in the EZH2 promoter. Further qPCR analysis showed that the expression of MYCN had positive correlation with the expression of EZH2 in the patients with acute erythroleukemia. The results showed that depletion of MYCN elevated the P21 expression in HEL cells. In this study, FCM analysis showed that etoposide treatment resulted in an increase in P21 expression, and MYCN depletion enhanced the etoposide-induced P21 activation in HEL cells . Further comparison analysis showed that overexpression of MYCN inhibited etoposide-mediated P21 activation in HEL and K562 cells.

--

Human

HL

apoptosis

29,022,893

Gene

0

1

0

1

0

1

0

1

0

1

0

CDKN2A

1029

protein coding

NP-9,NP-18,NP-29,NP-31

Tumor tissue tissue

Pancreatic cancer

Accelerate

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

In three of the four cell lines assayed, cells acquired a more refractile cytoplasm and showed a flattened and enlarged morphology, characteristic of arrested or senescent cells.Results from these experiments clearly indicate that in Ad-p16¨Ctransduced NP-9 cells, DNA synthesis was stopped, whereas transduced NP-18 cells did not show variations in the percentage of cells in S phase. NP-29 and NP-31 cells did not show such a clear arrest in their cell cycle profile as NP-9 cells when transduced with Ad-p16. However, significant decreases in S phase percentage were observed in 5-BrdU incorporation experiments . Acidic ¦Â-gal staining indicated that NP-9, NP-29, and NP-31 cells were committed to a senescent phenotype, as assessed by the increase in their SA-¦Â-gal activity. NP-18 cells did not show any increase in SA-¦Â-gal activity, corroborating the different destination of these cells when p16 is overexpressed.

--

Human

L

apoptosis

11,687,897

Gene

0

1

0

1

0

1

0

KRAS

3845

protein coding

DLD1(KRAS wt/mut),DWT7(wt/-)

--

Lung cancer

Accelerate

DAPI staining//SA--gal activity assay

In an isogenic model, KRAS wild-type cells were sensitive to treatment with erlotinib alone , with the induction of premature cellular senescence accounting for at least some of the effect. In contrast, erlotinib-treated KRAS-mutant cells were resistant to senescence induction.

--

Human

L

apoptosis

24,648,348

Gene

0

1

0

1

0

SPRY4

81848

protein coding

SK-MEL-119NRAS*

Tumor tissue tissue

Melanoma

Accelerate

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

In vitro analysis of SK-MEL-119NRAS confirmed the increase in apoptosis and SA-¦Â-galactosidase enzyme activity with SPRY4 induction . Compared to control tumors, SPRY4 tumors also harbored reduced Ki67, enhanced tumor cell apoptosis as shown by TUNEL staining and increased SA-¦Â-gal expression. However, upon induction of iBRAF in SK-MEL-119NRAS, there was a significant arrest, as expected, with the control siNTC.

--

Human

HL

apoptosis

30,651,601

Gene

0

1

0

1

0

1

0

HSPA4

3308

protein coding

TF1-L1FK clone A5

--

Aging

Prevent

MTT assay

We observed that AP20187 exerted a proliferative effect that was about ninefold higher in comparison with untreated and KNK437©\treated cells. In AP20187 plus KNK437©\treated cells, the proliferative response was markedly diminished in comparison with AP20187©\treated cells; however; the proliferative response in these cultures was significantly higher (about fourfold) in comparison with KNK437©\treated cells.

Akt

--

Western blot

We analyzed lysates of A5 cells that were treated with AP20187 and KNK437 and immunoblotted using antibodies to Akt and Erk1/2. Akt was present in abundant levels in AP20187©\stimulated cells. When cells were coincubated with AP20187 and increasing concentrations of KNK437, a dose©\dependent decrease in Akt production was observed. This effect was less acute in comparison with cells treated with KNK437 alone.

--

Human

HL

apoptosis

16,076,962

Gene

0

1

0

HSPH1

10808

protein coding

TF1-L1FK clone A5

--

Aging

Prevent

MTT assay

We observed that AP20187 exerted a proliferative effect that was about ninefold higher in comparison with untreated and KNK437©\treated cells. In AP20187 plus KNK437©\treated cells, the proliferative response was markedly diminished in comparison with AP20187©\treated cells; however; the proliferative response in these cultures was significantly higher (about fourfold) in comparison with KNK437©\treated cells.

Akt

--

Western blot

We analyzed lysates of A5 cells that were treated with AP20187 and KNK437 and immunoblotted using antibodies to Akt and Erk1/2. Akt was present in abundant levels in AP20187©\stimulated cells. When cells were coincubated with AP20187 and increasing concentrations of KNK437, a dose©\dependent decrease in Akt production was observed. This effect was less acute in comparison with cells treated with KNK437 alone.

--

Human

HL

apoptosis

16,076,962

Gene

0

1

0

NBNC

4683

protein coding

Fibroblast

--

Nijmegen Breakage Syndrome

Prevent

SA--gal activity assay

When comparing the telomere attrition rate, we foundthat NBS cells showed a higher telomere shortening rate compared to that in normal cells in vitro. For each replication cycle, the telomere shortening rate of NBS cells is around 30bp faster than that of its respective normal counterparts. Consistent with the accelerated telomere shortening, NBS fibroblasts exhibited a significantly higher percentage of cells undergoing senescence compared to normal cells with the same PDLs.

--

Human

L

apoptosis

22,161,642

Gene

0

1

0

SRF

6722

protein coding

Fibroblast

Prostate

Prostatic Hyperplasia

Prevent

Knockdown//SA--gal activity assay

After the second and third transfections of SRFsiRNA, a 5- to 8-fold increase of senescent cells was observed, whereas siSCR transfection did not increase the relative number of senescent cells.

--

Human

HL

apoptosis

23,308,381

Gene

0

1

0

1

0

TP53

7157

protein coding

SH-EP

--

Neuroblastoma

Accelerate

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

This arrest appeared to be permanent for p53 wild-type cells, the majority of which stayed on the plates during the whole duration of the experiment. Arrested p53 wild-type cells expressed acidic ¦Â-galactosidase, a marker of cellular senescence. In contrast, however, in p53-deficient cells after DNA damage, growth arrest was either temporary (as judged by reappearance of S-phase) or incomplete. Resumption of cell cycle progression was accompanied by gradual accumulation of cells with both sub-G1 and polyploid (>4C) DNA content. Simultaneously, numerous multinucleated cells and cells with multiple micronuclei appeared (data not shown) and the population underwent significant cell loss: By the end of the period of observation, most parts of the plate surface contained no cells. At the same time, in treated populations of p53-deficient cells, we observed formation of multiple colonies of rapidly dividing cells with no traces of senescence-specific ¦Â-galactosidase.

--

Human

HL

apoptosis

17,974,978

Gene

0

1

0

1

0

1

0

KRAS

3845

protein coding

MEF

--

Lung cancer

Accelerate

Knockdown//SA--gal activity assay

Senescence-associated ¦Â-galactosidase staining was twofold higher in B600E/K12D cells compared with cells expressing one oncogene.Knockdown of either Cdkn2a (encoding p16 and p19) or Cdkn2b (encoding p15) expression restored proliferation of B600E/K12D MEFs.

--

Human

L

apoptosis

26,028,035

Gene

0

1

0

1

0

BRAF

673

protein coding

MEF

--

Lung cancer

Accelerate

Knockdown//SA--gal activity assay

Senescence-associated ¦Â-galactosidase staining was twofold higher in B600E/K12D cells compared with cells expressing one oncogene.Knockdown of either Cdkn2a (encoding p16 and p19) or Cdkn2b (encoding p15) expression restored proliferation of B600E/K12D MEFs.

--

Human

L

apoptosis

26,028,035

Gene

0

1

0

1

0

TP53

7157

protein coding

HCT116

--

Colorectal cancer

Accelerate

Knockdown//SA--gal activity assay

HCT116 p53+/+ cells were much more vulnerable to premature senescence induced by ionizing radiation than p53?/? cells.It is apparent that signals secreted by irradiated p53+/+ cells were more effective in inducing senescence in p53?/? bystander cells than were those from p53?/? in their own bystanders.

--

Human

L

apoptosis

26,099,456

Gene

0

1

0

1

0

IGF1

3479

protein coding

MSC

Bone

Aging

Accelerate

SA--gal activity assay

The senescent (SA-¦Â-gal positive) MSCs showed a blight-blue staining in their central cytoplasm.It is noteworthy that the reduced proliferation, accumulative NCD and marker gene expressions, and the increased senescence were also observed in the void vector transfected MSCs after the selection, indicating that the unexpected cellular senescence may be, at least partly, due to a side effect of puromycin.

Ras

Activation

Western blot

In unselected cultures, no significant changes in activities of Ras(GTP-Ras/Total Ras) were observed in both void vector and IGF-1 transfected cells comparing to the parallel culture; but in the puromycin-selected cultures, the Ras activities in both void vector and IGF-I transfected MSCs were significantly increased with a higher level found in the void vector transfected cells.

--

Human

L

apoptosis

19,177,843

Gene

0

1

0

TINF2

26277

protein coding

TIN2+/+,TIN2+/DC-cond,TIN2+/+ mTR-/-,TIN2+/DCmTR-/-MEF

Embryo

Dyskeratosis congenita

Prevent

FISH//Knockdown//SA--gal activity assay

Furthermore, morphological examination and staining for ¦Â-galactosidase showed evidence of senescence in a small fraction of the TIN2+/DC Mtr-/- cells.Q-FISH measurements indicated that at a comparable PD, both TIN2+/DC mTR-/-MEFs had sustained greater telomere shortening than the TIN2+/+ mTR-/- MEFs, with the second TIN2+/DC mTR-/- MEF line showing the greatest reduction in mean telomere length.

--

Human

HL

telomere attrition

24,449,270

Gene

0

1

0

1

0

1

0

NHEJ1

79840

protein coding

CD34+,293T

Blood

Aging

Prevent

Cell proliferation assay//Knockdown//Telomere length assay//qRT-PCR

shNHEJ1.2/ CD34t transduced cells showed an inhibition of growth compared with scramble infected cells, as previously observed in 293T cells. These cells also showed increased expression of p21 (CDKN1A) 3-fold in relation with control cells and telomerase activity was reduced by 30%.Expression of several telomerase genes was also inhibited, mainly TERT (40%), TERC (20%), and also the expression of some of the shelterin genes and shelterinassociated factors: TERF1 (40%), TERF2 (20%) and TINF2 (20%) and also CTC1 (25%) and RTEL (30%).

--

Human

HL

telomere attrition

28,369,633

Gene

0

1

0

1

0

1

0

1

0

TINF2

26277

protein coding

HeLa-S3

--

Aging

Accelerate

SA--gal activity assay

TIN2-15C, much more than TIN2-13, retarded cell proliferation and induced senescence-associated ¦Â-galactosidase.

--

Human

L

telomere attrition

18,443,218

Gene

0

1

0

ZNF365

22891

protein coding

MEF

--

Breast cancer

Prevent

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

Upon knockdown of ZNF365, numerous 53BP1-positive foci appeared, with many localizing to telomeres, and cultures showed robust activation of p53 as well as an early-senescent phenotype. To measure the degree of telomere dysfunction, we determined the anaphase bridge index (ABI) out of total late anaphase. ZNF365-depleted TKO cells exhibited a high ABI, indicating the presence of unseparated sister chromatids or DNA bridges.

p53

--

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

Upon knockdown of ZNF365, numerous 53BP1-positive foci appeared, with many localizing to telomeres, and cultures showed robust activation of p53 as well as an early-senescent phenotype .

--

Human

HL

telomere attrition

23,776,040

Gene

0

1

0

1

0

1

0

1

0

MDC1

9656

protein coding

IMR-90

--

Aging

Prevent

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

We noted that upon more prolonged culturing [>2 wk], primary human fibroblasts expressing MDC1 shRNAs sh1 or sh2 started proliferating more slowly than the control cells and attained a senescence phenotype, even in the absence of TRF2-DN.in human cells the up-regulation of p53 and p21 in response to TRF2-DN was unaltered by MDC1 knockdown, and primary human fibroblasts underwent senescence within a week of TRF2 inhibition regardless of the level of MDC1.

--

ATM

--

Knockdown//RT-PCR//Western blot

We found that this shRNA [sh3] has extensive sequence identity to the mRNA for the ATM kinase and induced a significant reduction in ATM protein levels.

Human

L

telomere attrition

17,158,742

Gene

0

1

0

1

0

1

0

DCLRE1B

64858

protein coding

IMR-90

--

Aging

Prevent

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

The arrested Apollo knockdown cells had a senescent morphology and expressed SA-¦Â-galactosidase, a marker for cellular senescence.In addition, all Apollo shRNAs induced the upregulation of the CDK inhibitor p21, a read-out for p53 activation.Primary human IMR90 fibroblasts with diminished Apollo mRNA levels showed a clear growth defect.Within a week of introduction of Apollo shRNAs, the cells gradually slowed their proliferation and appeared to arrest.

--

Human

L

telomere attrition

16,730,176

Gene

0

1

0

1

0

1

0

1

0

RTEL1

51750

protein coding

LCL

--

Hoyeraal¨CHreidarsson syndrome

Prevent

Southern blot

Transduction of healthy LCL (S1) with RTEL11219 caused significant telomere shortening.

--

Human

HL

telomere attrition

23,959,892

Gene

0

1

0

TERT

7015

protein coding

Fibroblast

Skin

Dyskeratosis congenita

Prevent

Northern blot//TRAP assay//qRT-PCR

Northern analysis also verified that cells transduced with the TER/eGFP construct expressed a mature TER RNA of approximately 450 bases in length.Expression of TER and TERT together in AD DC fibroblasts brought the level of telomerase to approximately the same as what was observed in normal fibroblasts that expressed TERT alone.Telomere signal was clearly much greater in AD DC or normal cells that expressed TER and TERT together.

--

Human

L

telomere attrition

17,381,549

Gene

0

1

0

1

0

1

0

TOP2A

7153

protein coding

U2OS,HeLa

--

Cancer

Prevent

TRF assay

TOP2a or TOP2b downregulated U2OS cells displayed telomere shortening phenotypes compared to control cells.

TIFs

Downregulation

FISH//Immunostaining

Increases in TIFs were observed in shTOP2a and shTOP2b ALT cancer cells but not in telomerase-positive cells.

ALT

--

Survival curve//Southern blot//FISH//Immunostaining

ICRF-193-treated ALT cells displayed an inhibition of proliferation in a dose-dependent manner. Telomere shortening was also observed in ICRF-193-treated ALT cells, but not in telomerase-positive cells. We next measured the numbers of APBs and TIFs to determine whether the ALT pathway is affected in drug-treated ALT cells. ICRF-193 treatment decreased APBs and increased TIFs in ALT cells. These findings suggest that TOP2s participate in telomere¨Ctelomere recombination and that ALT cells are more sensitive to the TOP2 inhibitor than telomerase-positive cancer cells.

Human

L

telomere attrition

25,430,478

Gene

0

1

0

TERF2

7014

protein coding

HEp-2

--

Laryngeal cancer

Prevent

CCK-8 assay//FISH//Knockdown

The telomere length of pEGFP-shTRF2-shRAD51- shNBS1-shTERT treated group was distinctly shorter than saline treated group.HEp-2 cell proliferation was inhibited by pEGFP-shTRF2-shRAD51-shNBS1-shTERT.

--

Human

L

telomere attrition

27,726,944

Gene

0

1

0

1

0

1

0

RAD51

5888

protein coding

HEp-2

--

Laryngeal cancer

Prevent

CCK-8 assay//FISH//Knockdown

The telomere length of pEGFP-shTRF2-shRAD51- shNBS1-shTERT treated group was distinctly shorter than saline treated group.HEp-2 cell proliferation was inhibited by pEGFP-shTRF2-shRAD51-shNBS1-shTERT.

--

Human

L

telomere attrition

27,726,944

Gene

0

1

0

1

0

1

0

TERF2

7014

protein coding

HEp-2

--

Laryngeal cancer

Prevent

CCK-8 assay//FISH

The telomere length of pEGFP-shTRF2-shRAD51- shNBS1-shTERT treated group was distinctly shorter than saline treated group.HEp-2 cell proliferation was inhibited by pEGFP-shTRF2-shRAD51-shNBS1-shTERT.

--

Human

L

telomere attrition

27,726,944

Gene

0

1

0

1

0

TERT

7015

protein coding

HEp-2

--

Laryngeal cancer

Prevent

CCK-8 assay//FISH

The telomere length of pEGFP-shTRF2-shRAD51- shNBS1-shTERT treated group was distinctly shorter than saline treated group.HEp-2 cell proliferation was inhibited by pEGFP-shTRF2-shRAD51-shNBS1-shTERT.

--

Human

L

telomere attrition

27,726,944

Gene

0

1

0

1

0

LMNA

4000

protein coding

Jun-82

--

Hutchinson-Gilford progeria syndrome

Accelerate

Cell counting//qRT-PCR

Interestingly,a fibroblast line overexpressing wild-type lamin A (82-6 +wt) also exhibited a diminished replicative lifespan,comparable to those expressing mutant forms of lamin A.Telomeres of fibroblasts overexpressing lamin A,either wildtype or mutant, exhibited accelerated rates of shortening as compared to the parental control.When the rates of the telomere loss were plotted against the labeling indices, however, fibroblasts overexpressing wt lamin A showed the fastest rate of telomere loss .

--

Human

L

telomere attrition

17,870,066

Gene

0

1

0

1

0

CGGBP1

8545

protein coding

1064Sk

--

Aging

Prevent

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

CGGBP1 depletion by CGGBP1 siRNA resulted in DNA damage as demonstrated by an increase in the number of ¦ÃH2AX foci as compared with control siRNA-treated 1064Sk cells.Proliferation assays starting at 3 wk after first passage posttransfection and stable selection showed strongly reduced cell proliferation in S164A cells .The slow dividing S164A cells acquired the morphology of senescent fibroblasts and a very strong increase in ¦Â-galactosidase positivity. Cell cycle analysis showed an accumulation of cells in S-and G2/M-phases in S164A cells, suggesting a block or slow passage through these phases of cell cycle.

--

Human

L

telomere attrition

24,196,442

Gene

0

1

0

1

0

1

0

1

0

BMP7

655

protein coding

HeLa,PMC42

--

Cervical cancer

Accelerate

Telomerase activity assay//Telomere length assay

Incubation of human cervical cancer HeLa cells with different concentrations of BMP7 for 48 hours resulted in significant inhibition of telomerase activity.BMP7 induced significant inhibition of telomerase activity in a dose-dependent manner in PMC42 cells.Analyzing the mean telomere length in control and BMP7-treated cells showed that the mean telomere length in the BMP7-treated group could be f25% shorter than that in the controls.

hTERT//c-Myc

Downregulation//Downregulation

qRT-PCR

BMP7 induced a significant downregulation of hTERT gene expression in both HeLa and PMC42 cells.We noted that BMP7 also induced a significant down-regulation of c-myc gene expression .

--

Human

L

telomere attrition

19,010,887

Gene

0

1

0

1

0

TERF2

7014

protein coding

AG06814,GM00319,AG00780,AG03141

--

Werner syndrome

Accelerate

Cell morphological analysis//SA--gal activity assay

Notably,greater than 80% of Flag-TRF2¡÷B¡÷M-expressing fibroblasts showed a replicative senescence-like phenotype, exhibiting a flattened morphology, and stained positive for SA-¦Â-gal activity.

WRN

Binding

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

Eight days after transduction, 70% of the wild-type WRN-complemented WS fibroblasts expressing Flag-TRF2¡÷Bstained positive for SA-¦Âgal, demonstrating that TRF2¡÷B-induced cellular senescence was reconstituted by the expression of a functional WRN protein. In contrast, overexpression of Flag-TRF2¡÷B failed to increase the percentage of SA-¦Âgal-positive cells in the lines expressing WRN variants lacking helicase, exonuclase, or both activities.

p53-p21

Upregulation

Western blot

Western blot analysis shows that p53 and its downstream target, p21, are upregulated in normal fibroblasts expressing Flag-TRF2¡÷B¡÷M.

Human

L

telomere attrition

18,212,065

Gene

0

1

0

1

0

PRDX1

5052

protein coding

HCT116

--

Aging

Prevent

Knockdown//Southern blot//Southern hybridization//TRF analysis

The MTH1 knockout clone lost 15 base pairs(bp) of telomeric DNA per population doubling (PD),and the PRDX1/MTH1 double-knockout lost 23 bp/PD.On the contrary, disruption of MTH1 or MTH1 and PRDX1 together reduced the elongation rate in 20% O2to 38 and 9¨C11 bp/PD, respectively. In 40% O2, telomeres shortened in the absence of MTH1, with faster shortening upon codisruption of PRDX1.When grown in the presence of 20% O2, TSQ1 repeat DNA incorporation was reduced in MTH1 knockout cells when compared with wild type, and a further reduction of the TSQ1 repeat DNA was obtained with the MTH1/PRDX1 double-knockout cells.

--

Human

HL

telomere attrition

29,773,556

Gene

0

1

0

1

0

1

0

1

0

NUDT1

4521

protein coding

HCT116

--

Aging

Prevent

Knockdown//Southern blot//Southern hybridization//TRF analysis

The MTH1 knockout clone lost 15 base pairs(bp) of telomeric DNA per population doubling (PD),and the PRDX1/MTH1 double-knockout lost 23 bp/PD .On the contrary, disruption of MTH1 or MTH1 and PRDX1 together reduced the elongation rate in 20% O2to 38 and 9¨C11 bp/PD, respectively. In 40% O2, telomeres shortened in the absence of MTH1, with faster shortening upon codisruption of PRDX1.When grown in the presence of 20% O2, TSQ1 repeat DNA incorporation was reduced in MTH1 knockout cells when compared with wild type, and a further reduction of the TSQ1 repeat DNA was obtained with the MTH1/PRDX1 double-knockout cells.

--

Human

HL

telomere attrition

29,773,556

Gene

0

1

0

1

0

1

0

1

0

SMC5

23137

protein coding

SUSM1

--

Aging

Prevent

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

Knockdown of the SMC5/6 complex in SUSM1 AL T cells caused a progressive increase in the number of SA¨C¦Â-gal¨Cpositive cells over time.This increase correlated with the substantial decrease in telomere lengths in these cells.AL T cells treated with SMC5/6-specific RNAi showed other expected senescence phenotypes, such as morphological changes and an upregulation of the cyclin-dependent kinase inhibitor p21.

--

Human

L

telomere attrition

17,589,526

Gene

0

1

0

1

0

1

0

1

0

SMC6

79677

protein coding

SUSM1

--

Aging

Prevent

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

Knockdown of the SMC5/6 complex in SUSM1 AL T cells caused a progressive increase in the number of SA¨C¦Â-gal¨Cpositive cells over time.This increase correlated with the substantial decrease in telomere lengths in these cells.AL T cells treated with SMC5/6-specific RNAi showed other expected senescence phenotypes, such as morphological changes and an upregulation of the cyclin-dependent kinase inhibitor p21.

--

Human

L

telomere attrition

17,589,526

Gene

0

1

0

1

0

1

0

1

0

BTG2

7832

protein coding

BJ

--

Aging

Accelerate

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

The shBTG2 cells exhibited increased proliferative potential compared with control cells.Similarly, in Hs68?shp53 fibroblasts that had bypassed senescence as a result of p53 repression,ectopic BTG2 resulted in cell cycle arrest in G1 and ¦Â-galactosi-dase staining indicative of senescence.

Pin1

Binding

Co-IP//Western blot

The levels of both Pin1 and BTG2 protein were elevated in senescent cells compared with young cells and these levels were unaffected by serum.BTG2 coimmunoprecipitated with Pin1 in both young and senescent cells, and the binding between these two proteins was greatly enhanced after serum stimulation.

--

Human

L

telomere attrition

20,569,234

Gene

0

1

0

1

0

1

0

TP53

7157

protein coding

Hs68

--

Aging

Accelerate

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

As a consequence of sustained p53 inhibition, both BJ and Hs68 cells escaped senescence and grew an additional 10 MPDs compared with control cells expressing pSuper vector .Ectopic expression of p53RR led to the establishment of senescence as measured by the acidic ¦Â-galactosidase assay and by the accumulation of cells in the G1 phase of the cell cycle.

--

Human

L

telomere attrition

20,569,234

Gene

0

1

0

1

0

1

0

CDKN1A

1026

protein coding

BJ

--

Aging

Accelerate

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

The shp21 cells exhibited increased proliferative potential compared with control cells.Ectopic expression of p21 led to the establishment of senescence as measured by the acidic ¦Â-galactosidase assay and by the accumulation of cells in the G1 phase of the cell cycle.

--

Human

L

telomere attrition

20,569,234

Gene

0

1

0

1

0

1

0

CHEK2

11200

protein coding

HFF,IMR-90

--

Aging

Accelerate

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

Knockdown of endogenous Chk2 protein led to short-term responses equivalent to those seen with the dominant-negative Chk2, namely reversion to a¡®young¡¯ morphology, reduced SA-¦Â-gal expression and increased BrdU incorporation.

p21//Rb//Cyclin A

Downregulation//--//Upregulation

Western blot

We observed a reduction of p21 protein level,and an increase in the hyperphosphorylated form of Rb and in cyclin A expression compared to age-matched control HFF.

--

Human

L

telomere attrition

15,192,702

Gene

0

1

0

1

0

1

0

BRCA1

672

protein coding

HMEC

Mammary Gland

Breast cancer

Prevent

Cell morphological analysis//SA--gal activity assay

This premature growth arrest (M*) was observed across multiple patient-derived BRCA1mut/©HMECs with different BRCA1 mutations and was observed in BRCA1mut/©HMECs well . Cells in M* displayed the senescent phenotype, characterized by enlarged, flattened morphology and positive staining for SA-¦Â-galactosidase.

Cyclin A //SIRT1

--//--

Western blot

Levels of cyclin A were significantly decreased in senescent BRCA1mut/©HMECs compared with WT HMECs.Examination of SIRT1 levels in HMECs from BRCA1-mutation carriers revealed significantly reduced protein expression insenescent cells.

pRb

--

Knockdown

Compared with control, knockdown of pRb in BRCA1mut/©HMECs led to an increase in replicative potential, indicating that pRb activity was mediating premature senescence.

Human

HL

telomere attrition

26,106,036

Gene

0

1

0

1

0

TERT

7015

protein coding

WS,Fibroblast

--

Werner syndrome

Prevent

Immunostaining//Southern Blot//Western blot

The expression of hTERT in WS MSCs appears to rescue senescence through reduction of the p16 level (but not of p53/p21) and the DNA damage marker ¦ÃH2AX.Longer telomere length was found in WS-MSCtert, but not in WS-MSCp53i,suggesting a rescue of the accelerated telomere attrition by telomerase.

--

Human

HL

telomere attrition

24,749,076

Gene

0

1

0

1

0

1

0

TP53

7157

protein coding

WS,Fibroblast

--

Werner syndrome

Accelerate

Knockdown//SA--gal activity assay

As a consequence in MSCs, p53i enhanced their proliferative potential and rescued the premature senescence phenotype without the need for high telomerase activity and long telomere length.

--

Human

HL

telomere attrition

24,749,076

Gene

0

1

0

1

0

POT1

25913

protein coding

HFF,BJ

Foreskin

Aging

Prevent

FISH//Immunostaining//SA--gal activity assay

Suppression of hPOT1 in the HFF and BJ cells that had proliferated for the longest time in culture resulted in a 2- to 4-fold greater increase in doubling time as well as a marked induction in senescence-associated h-galactosidase (SA ¦Â-gal)staining. We found colocalization of ¦Ã-H2AX and telomeric foci in both early- and late-passage cells in which hPOT1 was suppressed.

--

Human

L

telomere attrition

18,922,974

Gene

0

1

0

1

0

1

0

CDK4

1019

protein coding

HMEC,keratinocyte

Mammary Gland

Aging

Prevent

Growth curve assay

Overexpression of Cdk4mock infection in epidermal keratinocytes and HMECs extended their lifespan to 82 and 70 PD, respectively,compared to controls (in the absence of feeder layers 13 and 16 PD.

p16//p53//p21

Upregulation//Upregulation//Upregulation

Western blot

The expression of p16INK4a was greatly elevated in Cdk4-overexpressing cells cultured in the absence of feeder layers.However, higher levels of p53 and upregulation of p21WAF1 was seen in cells overexpressing Cdk4 compared to uninfected controls.

--

Human

L

telomere attrition

12,545,164

Gene

0

1

0

HLX

3142

protein coding

IMR-90

--

Aging

Prevent

Flow cyotmetry//Knockdown//qRT-PCR

Depletion of INK4a expression rescued the cell cycle arrest triggered by HLX1 knockdown almost completely, indicating that INK4a is a key mediator of the effects of HLX1 on senescence. However, the knockdown of HLX1 still caused a small decline in BrdU incorporation in cells lacking p16INK4a expression, suggesting that HLX1 could also control senescence by additional mechanisms.

INK4a

--

Knockdown//qRT-PCR

Depletion of INK4a expression rescued the cell cycle arrest triggered by HLX1 knockdown almost completely, indicating that INK4a is a key mediator of the effects of HLX1 on senescence.

--

Human

L

telomere attrition

24,067,365

Gene

0

1

0

1

0

1

0

TERT

7015

protein coding

DC,Fibroblast

Skin

Dyskeratosis congenita

Prevent

Flow cytometry

TERT expressing DC-1 and normal cells had much higher telomerase activity.TERT-expressing DC cells exhibited a significant 50% decrease in both DHE and MitoSOX oxidation, relative to empty vector treated cells, demonstrating that overexpression of TERT in DC cells decreased steady-state levels of superoxide.

--

p53-p21

Downregulation

Western blot

TERT expression also reduced levels of phospho-p53 in DC cells.

Human

L

telomere attrition

21,087,144

Gene

0

1

0

FUNDC1

139341

protein coding

HeLa

--

Aging

Accelerate

Knockdown//SA--gal activity assay

To test the effect of FUNDC1-/HSC70-mediated pathway on cell fate, we established a model of senescence by treating HeLa cells with MG132 for 8 h and then incubating them in normal complete medium for a further 3 days. More than 20% of the cells displayed SA-¦Â-gal-positive staining.

AMPK

Activation

Western blot

Immunoblotting analysis showed that the AMPK activity and the levels of oxidized proteins in MG132-pulse-treated cells were consistently higher than those in control cells until 3 days after treatment withdrawal.

--

Human

L

mitochondrial dysfunction

30,591,555

Gene

0

1

0

1

0

STAT3

6774

protein coding

Fibroblast

Lung

Idiopathic pulmonary fibrosis

Accelerate

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

We found that treatment with STA-21 supressed levels of p21, as measured by immunoblot confirmed by graphed densitometry, and reduced staining of p21 in the nuclei of H2O2-treated fibroblasts.Reduced IL-6 secretion suggests an attenuated secretory profile, and decreased SA-¦Â-Gal content measured using a cytochemical assay.

--

Human

L

mitochondrial dysfunction

30,608,861

Gene

0

1

0

1

0

1

0

1

0

CLPP

8192

protein coding

HepG2,HuH-7

--

Decompensated cirrhosis

Prevent

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

Both HepG2 and Huh7 cells were transfected with either CLPP-GFP or GFP alone and stable cell clones were selected showing more than 90% GFP positivity.Unlike the control cells which showed a pan GFP expression, the CLPP over-expressing cells showed a punctate pattern in the cytoplasm which colocalized with Mitotracker, thereby confirming its mitochondrial localization.The growth kinetics of CLPP over-expressing cells was similar to their control counterpart. When treated with doxorubicin, the CLPP-GFP cells showed almost 50% reduction in SA-¦Â-Gal staining when compared to control GFP cells. This was also accompanied with increased levels of LaminB1 and decreased levels of ¦ÃH2AX and H3K9Me3 in doxorubicin treated CLPP cells.Senescence is often accompanied by a secretory phenotype which was much subdued in CLPP expressing cells when exposed to doxorubicin.

--

Human

L

mitochondrial dysfunction

30,878,663

Gene

0

1

0

1

0

1

0

1

0

ALOX15B

247

protein coding

Pca

--

Aging

Accelerate

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

We followed PC3 cells that had been stably transfected with 15-LOX2 or 15-LOX2sv-b for multiple passages. These cells were derived from clonal cultures and enough cells generally became available for analysis only at P4¨CP5. To our surprise, these cells also showed passage-related phenotypic changes resembling replicative senescence in NHP cells. For instance, most of the early-passage cells were generally small, Activately proliferating, and SA-¦Âgal-negative (not shown). By P6, 10¨C15% of the stably transfected cells became big and flat, some of which were also SA-¦Âgal + and most of which were BrdU (not shown). With increasing passage, the percentage of big/flat cells also increased in both 15-LOX2- and 15-LOX2sv-b-expressing PC3 cells.

--

Human

L

deregulated nutrient sensing

15,750,631

Gene

0

1

0

1

0

1

0

BCL6

604

protein coding

WI-38,MEF

--

Aging

Accelerate

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

Consistent with the ability of p53 to induce a G1 arrest, BCL6-transduced WI-38 cells exhibited an increase in the G1 phase fraction from 74 versus 42% in control cells; and a progressive reduction in their proliferative rate that could not be overcome by increasing serum concentration or passage in culture (data not shown).Also, certain morphological features, such as cytoplasmic vacuoles and enlarged cell size and flattening were observed. These changes were suggestive of cellular senescence, which was further confirmed by detection of pH-dependent -galactosidase levels. Altogether,within 8 days, 37.5% of BCL6-transduced WI-38 cells displayed evidence of senescence as compared with 6% of GFP transduced or untransduced cells.

--

p53

Upregulation

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

Transduction of BCL6 in Tp53-/- MEFs failed to provoke growth arrest. Senescence was largely p53-dependent because no more than 10% of Tp53- /- MEFs became positive for ¦Â-galactosidase. Like WI-38 cells, Tp53+/+ MEFs displayed upregulation of Tp53 upon transduction of BCL6. Not only did BCL6-transduced Tp53 -/- MEFs maintain their proliferative potential, but also acquired the ability to form colonies on tissue culture plates in contrast to control transduced cells.

Human

L

deregulated nutrient sensing

18,524,763

Gene

0

1

0

1

0

1

0

TPP2

7174

protein coding

Fibroblast,PBMC

Skin

Evans syndrome

Prevent

Knockdown//SA--gal activity assay

Finally, as described in murine cells,fibroblasts of P1 also showed increased staining with ¦Â-galactosidase indicating cellular senescence. proliferation of control T cells was diminished after pretreatment with butabindide.

--

Human

HL

immunosenescence

25,414,442

Gene

0

1

0

1

0

TLR8

51311

protein coding

MCF-7,M628,PC-3

--

Aging

Prevent

Knockdown//SA--gal activity assay

Knockdown of TLR8,MyD88, and IRAK4 in tumor cells significantly blocked the Poly-G3-mediated reversal of tumor-induced CD4+T-cell senescence. These results confirmed that TLR8 signaling can prevent the induction of T-cell senescence mediated by tumor cells.

cAMP

Downregulation

ELISA

Indeed, Poly-G3 treatment significantly decreased the cAMP levels in tumor cells. Notably, pretreatment of tumor cells with Poly-G3 also markedly decreased the cAMP levels in T cells co-cultured with tumor cells.

--

Human

L

immunosenescence

25,231,413

Gene

0

1

0

1

0

TERT

7015

protein coding

HCT116

--

Colorectal cancer

Prevent

Knockdown//SA--gal activity assay

In both p53+ and p53? HCT116 cells, RNAi-mediated knockdown of TERT substantially increased the number of cells that stained positively for senescence-associated ¦Â-galactosidase activity, indicative of senescence induction.

--

p53

--

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

The level of senescence was higher in p53+ HCT116 TERT knockdown cells than in p53? HCT116 TERT knockdown cells, as expected, because p53-directed pathways contribute to senescence [1].In addition, knockdown of TERT increased the percentage of p53? HCT116 cells but not p53+ HCT116 cells in G2/M.

Human

HL

epigenetic alterations

23,284,306

Gene

0

1

0

1

0

ETV1

2115

protein coding

HCT116

--

Colorectal cancer

Prevent

Knockdown//SA--gal activity assay

Significantly, RNAi-mediated knockdown of ETV1 or ATR also induced senescence.

p53

--

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

However, following knockdown of ETV1 or ATR, the induction of senescence was much greater in p53? HCT116 cells compared to p53+ HCT116 cells ,consistent with the difference in TERT levels .Notably, a similar preferential increase in the percentage of p53? HCT116 cells in G2/M occurred following knockdown of ETV1 or ATR.

--

Human

HL

epigenetic alterations

23,284,306

Gene

0

1

0

1

0

ATR

545

protein coding

HCT116

--

Colorectal cancer

Prevent

Knockdown//SA--gal activity assay

Significantly, RNAi-mediated knockdown of ETV1 or ATR also induced senescence.

p53

--

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

However, following knockdown of ETV1 or ATR, the induction of senescence was much greater in p53? HCT116 cells compared to p53+ HCT116 cells, consistent with the difference in TERT levels.Notably, a similar preferential increase in the percentage of p53? HCT116 cells in G2/M occurred following knockdown of ETV1 or ATR .

--

Human

HL

epigenetic alterations

23,284,306

Gene

0

1

0

1

0

SKP2

6502

protein coding

PC-3

--

Prostate cancer

Prevent

SA--gal activity assay

As predicted, a striking increase of ¦Â-galactosidase was found in PC3-shSKP2 cells, as compared to that in PC3-scrambled cells, in agreement with literature that SKP2 deficiency induces cellular senescence.

JARID1B

--

Western blot

SKP2 ablation resulted in a striking increase of JARID1B compared to the control. Quantification analysis revealed that JARID1B levels in PC3-shSKP2 cells were 4-fold higher than that in control cells.

--

Human

HL

epigenetic alterations

25,596,733

Gene

0

1

0

TGFB1

7040

protein coding

MEF

Embryo

Aging

Accelerate

Immunofluorescence//SA--gal activity assay

Disruption of TGF-¦Â signaling by E-616452 restored H4K20me3 levels, attenuated SA-¦Â-gal staining and enhanced the Mki67 signals in Suv4-20h-depleted cells.

miR-29//H4K20me3

Upregulation//Downregulation

Western blot//qRT-PCR

Activation of TGF-¦Â signaling by oxidative stress was accompanied by increased miR-29 accumulation and accelerated reduction in the abundance of total H4K20me3 .Furthermore, inhibition of TGF-¦Â signaling by inhibitor treatment and Smad4 depletion diminished miR-29 expression, attenuated the reduction in Suv4-20h1 and Suv4-20h2 expression and produced partial recovery of H4K20me3.

--

Human

L

epigenetic alterations

29,967,491

Gene

0

1

0

1

0

MYSM1

114803

protein coding

C4-2,22Rv1

--

Prostate cancer

Accelerate

Flow cytometry//SA--gal activity assay

Similarly, a significant change of cell cycle distribution was detected in MYSM1-deficient cells. There was a decrease in the percentage of G1-phase cells and an increase in that of S-phase cells, indicating an accelerated progression of cell cycle. Our results showed that MYSM1 downregulation led to decreased proportion of ¦Â-gal positive cells.

--

Human

HL

epigenetic alterations

31,761,786

Gene

0

1

0

1

0

MYC

4609

protein coding

AG00780,AG03141,Fibroblast

--

Werner syndrome

Accelerate

Flow cytometry//SA--gal activity assay

However, within 2¨C3 passages, ¡«30%¨C70% of the WRN¨C/¨C cells expressed senescence-associated (SA-) ¦Â-galactosidase and lost proliferative capacity. The senescent phenotype in c-myc-transduced WRN¨C/¨C cells was also confirmed at the gene expression level by microarray analysis, which demonstrated elevated expression of several genes characteristic of replicative senescence, such as the matrix proteases. WRN¨C/¨C cells that were senescent by virtue of MYC overexpression were largely G1-arrested with a small G2 fraction (data not shown).

WRN

Binding

Flow cytometry

WRN¨C/¨C cells that were senescent by virtue of MYC overexpression were largely G1-arrested with a small G2 fraction (data not shown).

--

Human

HL

genomic instability

12,842,909

Gene

0

1

0

1

0

WRN

7486

protein coding

MEF

Embryo

Werner syndrome

Accelerate

Giemsa staining

After ¡«2 mo in culture, several foci emerged among 8 ¡Á 106 senescent G5 mTerc-/- Wrn-/- MEFs at a frequency of ¡«1.4 ¡Á 10-5.Under identical culture conditions, foci were not detected in senescent cultures of ¡«5 ¡Á 107 G5 mTerc-/- Wrn+/+ and ¡«2.5 ¡Á 107 G5 mTerc-/- Wrn+/- MEFs.

--

Human

L

genomic instability

16,264,192

Gene

0

1

0

TERT

7015

protein coding

BJ

--

Aging

Prevent

Flow cytometry//SA--gal activity assay

Despite little induction of senescence-associated protein immediately after metal exposure, there was a progressive and dose-dependent increase of ¦Â-galactosidase stained cells up to 30 days after metal treatment of hTERT? cells .Preliminary experiments with higher doses of Cr (0.8?¦Ì M) and V (10?¦Ì M) revealed an increase in G2/M in hTERT? cells, but again there was no change in the hTERT+ cells (data not shown).

--

Human

L

genomic instability

16,449,970

Gene

0

1

0

1

0

TWIST1

7291

protein coding

NPTX,HPr-1

--

Aging

Prevent

SA--gal activity assay

Higher percentage of SA-¦Â-gal-positive cells was found in the Sh-TWIST transfectants (filled columns) compared with the controls (open columns) in response to both agents in a dose-dependent manner.

p14

Downregulation

Western blot

Reverse transcriptase¨Cpolymerase chain reaction analysis showed that the expression of TWIST was also up-regulated at transcription level which was associated with down-regulation of p14 ARF (right panel). We also found that the p14 ARF expression levels were much lower in the TWIST over-expressing cells, either before or after exposure to H 2 O 2 and CP (right panels), compared with the vector control, confirming the negative effect of TWIST on p14 ARF.

--

Human

L

genomic instability

17,690,110

Gene

0

1

0

BCL6

604

protein coding

WI-38

--

Aging

Accelerate

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

Consistent with the ability of p53 to induce a G1 arrest, BCL6-transduced WI-38 cells exhibited an increase in the G1 phase fraction from 74 versus 42% in control cells; and a progressive reduction in their proliferative rate that could not be overcome by increasing serum concentration or passage in culture (data not shown). Also, certain morphological features, such as cytoplasmic vacuoles and enlarged cell size and flattening were observed. These changes were suggestive of cellular senescence, which was further confirmed by detection of pH-dependent ¦Â-galactosidase levels . Altogether, within 8 days, 37.5% of BCL6-transduced WI-38 cells displayed evidence of senescence as compared with 6% of GFP transduced or untransduced cells.

p53

Upregulation

qPCR//Western blot

However, just as in fibroblasts, BCL6 transduction powerfully induced TP53 mRNA and protein up-regulation as well as P21.p53 protein levels were correspondingly increased in the presence of BCL6.

--

Human

L

genomic instability

18,524,763

Gene

0

1

0

1

0

1

0

TERT

7015

protein coding

RPTEC

Kidney

Aging

Prevent

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

RPTEC/TERT1 cells were propagated as a mass culture because of poor cloning efficiency. They showed growth rates of 0.25 PD/day and a population doubling time of ¡«96 h, comparable to the normal counterpart, and have now accomplished >90 PDs, corresponding to more than threefold life span extension since normal RPTECs enter growth arrest after 24 ¡À 3 PDLs.RPTEC/TERT1 cells retained morphology very similar to early-passage cells as well as few SA-¦Â-Gal-positive cells. Interestingly, a considerable fraction of ¡«20% positive cells was observed also in early-passage cells.¦Ã-H2AX focus formation in RPTEC/TERT1 cells was not reduced to early-passage cell levels, with a number of 7.1 foci per cell.

--

Human

L

genomic instability

18,715,936

Gene

0

1

0

1

0

1

0

SIRT1

23411

protein coding

ES

--

Aging

Prevent

FISH

Repair by NHEJ, the prominent DSB repair pathway in G1 and post-mitotic cells, was also reduced in this assay system, but to a lesser extent.H2O2 treatment caused a significant increase in chromosomal aberrations specifically in SIRT1 deficient cells. The frequency of chromatid breaks was comparable between knock-down and control ES cells, but the number of chromosomal fusions, in particular dicentric chromosomes and Robertsonian translocations, was significantly higher in the absence of SIRT1 .

--

Human

HL

genomic instability

19,041,753

Gene

0

1

0

BRCA1

672

protein coding

MCF-7,HCC1937,HCC+BR,HBL-100

--

Aging

Prevent

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

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.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.

--

Human

L

genomic instability

19,372,557

Gene

0

1

0

1

0

1

0

1

0

LIN9

286826

protein coding

BJ,MEF

Embryo

Aging

Prevent

Cell morphological analysis//SA--gal activity assay

After 10¨C13 days in culture, LIN9-depleted cells exhibited a large and flat morphology, a phenotype that is commonly observed in senescent cells.Indeed, staining for senescence-associated ¦Â-galactosidase activity, a marker of senescent cells, confirmed senescence after loss of LIN9.Two weeks after deletion of LIN9, MEFs became senescent, as previously described .

p16//p21

--//--

Western blot

Immunoblot analysis showed induction of the cell cycle inhibitors p16INK4a and p21Waf1 in LIN9-depleted cells.Similar to what was observed in human cells, loss of LIN9 in two different MEF clones was also accompanied by induction of p16INK4a and p21Waf1.

--

Human

L

genomic instability

21,860,417

Gene

0

1

0

1

0

CTSL

1514

protein coding

MEF

--

Aging

Accelerate

Western blot

Altogether, our data demonstrate that the upregulation of CTSL activity upon loss of A-type lamins leads to degradation of 53BP1 and defective repair of DNA DSBs by NHEJ.Whereas Lmna-/-fibroblasts presented defects in the fast phase of repair corresponding to NHEJ, depletion of CTSL was sufficient to restore the repair of IR-induced DSBs to a degree that was indistinguishable from that of WT cells.

A-type lamins//53BP1//NHEJ

--//Downregulation//--

Western blot

Western blots showing that acute depletion of CTSL by lentiviral transduction with an shRNA leads to stabilization of 53BP1 protein in Lmna-/-MEFs.CTSL overexpression led to increased levels of the Activate form of the enzyme and increased CTSL activity, as well as a decrease in 53BP1 levels.Altogether, our data demonstrate that the upregulation of CTSL activity upon loss of A-type lamins leads to degradation of 53BP1 and defective repair of DNA DSBs by NHEJ.

--

Human

L

genomic instability

21,750,527

Gene

0

1

0

SIRT1

23411

protein coding

HCT116

--

Aging

Prevent

Immunofluorescence

SIRT1 depletion led to an increase in partially condensed, disorganized mass of chromosomes that failed to align properly on metaphaseplate in the presence of relatively normal spindles.

histone 1(H1)//condensin I complex

--//--

Western blot

Protein gel blot analysis from asynchronous cell culture shows the reduction of the chromatin-bound condensin component CAp-D2 and histone H1 in cells treated with SIRt1 siRNA but not control siRNA.The levels of chromatin-bound histione H1 were significantly reduced in cells transfected with anti-SIRT1 siRNA. However, the levels of chromatin-bound CAP-D2, which is a condensin I component, were significantly reduced in cells transfected with anti-SIRT1 siRNA despite unchanged levels in nuclear soluble fraction.

--

Human

L

genomic instability

21,636,977

Gene

0

1

0

MAD2L1

4085

protein coding

IMR-90

--

Aging

Prevent

Cell morphological analysis//SA--gal activity assay

Consistent with this observation we scored in IMR90 human fibroblasts with MAD2 depletion roughly 70% of metaphase spreads with prematurely separated sister chromatids.At this time the majority of the human fibroblasts showed enlarged and flattened morphology a typical feature of cellular senescence.Moreover, these IMR90-siMAD2 cells showed nuclei alterations such as binucleate or multinucleated nuclei in 48% of the cells.

p21//p14

--//--

RT-PCR//Western blot

Real time RT-PCR revealed a high increase (seven fold) of p21waf1 mRNA in primary human fibroblasts with MAD2 haploinsufficiency when compared to IMR90-siGFP control cells. Also MCF10A-siMAD2 cells showed p21waf1 increased levels but to a lesser extent than those found in IMR90-siMAD2 cells.As expected western blotting confirmed that p21waf1 protein was significantly higher in siMAD2 than in IMR90-siGFP transfected cells. As expected western blotting confirmed that p21waf1 protein was significantly higher in siMAD2 than in IMR90-siGFP transfected cells.At 72 hours post-transfection, p14ARF mRNA was highly increased in IMR90 human fibroblasts .Similarly, western-blotting experiments showed a high increase of p14ARF protein levels in IMR90-siMAD2 cells.

p53-p21

--

RT-PCR

When p53 was post-transcriptionally silenced in IMR90-siMAD2 cells, expression level of the p21waf1 gene resulted similar to that present in IMR90-siGFP control cells, as assessed by Real time RT-PCR analysis.

Human

L

genomic instability

22,170,163

Gene

0

1

0

1

0

MLH1

4292

protein coding

CD34+

--

Aging

Prevent

Immunocytochemistry//qRT-PCR

A 10-fold decrease in the RQ of MLH1 expression relative to the normalized sample was identified in 2 of the 5 adult CD34+samples.The percentage of CD34+cells with positive staining for MLH1 declined as a function of donor age. Across the donors analyzed, the percentage of CFC with MLH1 expression declined significantly as a function of age.

--

Human

HL

genomic instability

22,740,444

Gene

0

1

0

1

0

SIRT6

51548

protein coding

Fibroblast,HCA2-HR

--

Aging

Prevent

Immunocytochemistry

SIRT6 overexpression reduces the number of ¦ÃH2AX foci in presenescent cells.

PARP1//Rad51//Rad51C//Rad52//NBS1//SIRT6//CtIP

Activation//Upregulation//Upregulation//Upregulation//Upregulation//Upregulation//Upregulation

Western blot

In the presence of PJ34, SIRT6 overexpression had no effect on HR,indicating that SIRT6-mediated rescue of HR in aging cells is dependent on PARP1.Western blot showing overexpression of HR-related proteins in HCA2-HR cells.

--

Human

L

genomic instability

22,753,495

Gene

0

1

0

ATM

472

protein coding

HPr-1

--

Aging

Accelerate

Western blot

Examination of cH2AX expression revealed that knockdown of ATM or ATR both suppressed the induction of cH2AX by androgen treatment, suggesting that the androgen-induced DNA damage response was significantly suppressed by ATM/ATR knockdown.

CDC25A

Downregulation

Western blot

Knockdown of ATM, but not ATR, partially recovered the CDC25A protein expression in LNCaP cells, suggesting that downregulation of CDC25A by androgen requires the activation of ATM.

--

Human

L

genomic instability

23,272,087

Gene

0

1

0

RTEL1

51750

protein coding

Lymphoma cell line

Blood

Aging

Prevent

Southern blot

Growth curves showing the population doublings of the LCLs over time. All LCLs carrying RTEL1 mutations reached a stage of growth arrest (indicated by red ¡°X¡±). Interestingly, telomeres in LCLs derived from the parents, each carrying a single heterozygous RTEL1 mutation, were also shorter than those of the noncarrier S1 at a PDL of about 35.

TRF1

Binding

IP

In addition, in a reciprocal experiment using FLAG-tagged TRF1 and TRF2, only FLAG-TRF1 was found to immunoprecipitate RTEL1.None of the mutations significantly affected the interaction of RTEL1 with TRF1.

--

Human

HL

genomic instability

23,959,892

Gene

0

1

0

UHRF1

29128

protein coding

Hepatocyte

Liver

Hepatocellular carcinoma

Accelerate

RNA-seq//SA--gal activity assay//qRT-PCR

However, senescence associated ¦Â-galactosidase (SA-¦Â-gal) staining was detected throughout the liver of most UHRF1-GFP High 5 dpf larvae, but not other transgenic lines. Additionally, the DNA in control hepatocytes was evenly distributed throughout the uniformly sized nuclei compared to the large nuclei where DNA resembled senescence associated heterochromatic foci. RNAseq revealed some pro-proliferative genes(ccnd1 and myc) downregulated.

tp53//CDKN1A

Upregulation//Upregulation

RNA-seq//qPCR

RNAseq and qPCR analysis show that tp53 and its target genes, especially cdkn1a, are significantly induced in the liver of 5 dpf UHRF1-GFP High larvae.

--

Human

HL

genomic instability

24,486,181

Gene

0

1

0

1

0

1

0

SUV39H1

6839

protein coding

Fibroblast

--

Aging

Prevent

SA--gal activity assay//Western blot

Among these, SUV39H1, a histone methyltransferase that specifically methylates H3K9 and was previously associated with functions in aging or senescence (S4), was downregulated in senescent cells (PD 54), as confirmed by Western blot analysis. Further, the surviving cells in PD 47 cultures treated with increasing concentrations of chaetocin became senescent, suggesting that the inhibition of SUV39H1 was sufficient to induce senescence in low PD cells.

p21

--

Western blot//CHIP

p21 protein levels were increased in senescent cells and correlated with the reduced expression levels of SUV39H1.Further, H3K9me3 was less abundant in the promoter region of IL6 in senescent cells when compared to dividing cells,as determined by ChIP, suggesting that reduced SUV39H1 expression levels affect the gene expression profiles of senescent cells.

--

Human

L

genomic instability

25,063,769

Gene

0

1

0

1

0

SPRTN

83932

protein coding

LCL

--

Hepatocellular carcinoma

Prevent

DNA fiber assay//RNA-seq//Western blot

To further confirm that mutations in SPRTN are the cause of the DNA replication defect, we transfected patients¡¯ cells with WT SPRTN, which almost completely corrected the replication defect and restored cellular proliferation. siRNA-mediated SPRTN depletion severely affected the progression of DNA replication and induced an increased number of stalled forks, newly fired origins and formation of DSBs in the S phase of the cell cycle. Further, short interfering RNA (siRNA)-mediated depletion of SPRTN in HEK293T and U2OS human cell lines also caused chromosomal instability and severe proliferation defects, respectively. sprtn silencing, verified by a GFP reporter assay, led to a substantial increase of ¦Ã-H2AX foci, indicating an evolutionarily conserved functional role of SPRTN in the DNA damage response.

--

Human

HL

genomic instability

25,261,934

Gene

0

1

0

1

0

1

0

SDHD

6392

protein coding

Sdh4

--

Paraganglioma

Prevent

Flow cytometry//Immunochemical staining

It was observed that the lifespan of the sdh4D¦¤ and sdh4D¦¤ shh4D¦¤ mutants was reduced compared with the wild-type yeast at day 7. Our results revealed that cellular ROS in the sdh4D¦¤ shh4D¦¤ strain was higher than in the sdh4D¦¤ strain.In our results, the sdh4D¦¤ shh4D¦¤ strain showed lower mtDNA integrity and higher mtDNA mutability .

--

Human

L

genomic instability

25,328,978

Gene

0

1

0

1

0

SSX2

6757

protein coding

MCF-7-TETSSX2,A375-TET-SSX2,HEK293-TET-SSX2(H293-TET-SSX2)

--

Melanoma

Accelerate

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

HEK293-TET-SSX2 cells exhibited very high background levels of SA-¦Â-gal activity, and no difference was observed upon induction of SSX2 expression.Immunocytochemical analysis of MCF7-TET-SSX2 cells 7 days after induction of SSX2 expression further substantiated their senescent phenotype, as they exhibited a clear reduction in expression of the proliferation marker Ki67 and presence of g-H2AX DSB foci. Long-term growth (more than 7 days) of MCF7-TET-SSX2 cells with SSX2 expression induced a dramatic change in the morphology of the cells, which adapted an irregular and enlarged shape.

--

Human

L

genomic instability

25,363,656

Gene

0

1

0

1

0

1

0

BRCA1

672

protein coding

HMEC

Mammary Gland

Aging

Accelerate

SA--gal activity assay//Western blot

Western blot analysis of p16INK4a, total p53, p53 (Ser15) and p21 levels in WT and BRCA1mut/tHMECs at indicated PDs. M0, stasis, Ag, agonescence(WT HMECs), M*, premature growth arrest (BRCA1mut/tHMECs). Cells in both M* and Ag displayed the senescent phenotype, characterized by enlarged, flattened morphology and positive staining for SA-¦Â-galactosidase.

SIRT1

--

SA-¦Â-gal activity assay

Furthermore, knockdown of SIRT1 in WT HMECs resulted in cell-cycle arrest and morphological changes associated with senescence.

p53//pRb

--//--

Western blot

We found that this proliferative barrier was associated with elevated levels of all components of the p53 signalling pathway (phosphorylated p53 (Ser15), total p53, p21, p27). Collectively, these data indicate that activation of the pRb pathway is the primary mediator of HIS in BRCA1mut/t epithelial cells, and when bypass of HIS is forced (via down-regulation of pRb), it results in the activation of the p53 pathway and accumulation of additional genomic abnormalities.

Human

HL

genomic instability

26,106,036

Gene

0

1

0

1

0

PML

5371

protein coding

MRC-5,WI-38

--

Aging

Prevent

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

Strikingly,PML knockdown caused a dose-dependent decrease in cell proliferation, morphological changes consistent with a senescent phenotype and an increased expression of the senescence-related ¦Â-galactosidase enzyme.Moreover, a senescence molecular pathway was activated, as shown by an increased expression of the p53, p16INK4aand p21WAF1proteins in PML-depleted cells with respect to control cells.

--

Human

L

genomic instability

26,119,943

Gene

0

1

0

1

0

1

0

CDH1

999

protein coding

Htertrpe

--

Aging

Prevent

SA--gal activity assay

We then hypothesized that this E3-Ubiquitin ligase could be activated in S-phase after severe replication stress. Addition of the APC/C inhibitor proTAME (64) during HU treatment restored Cyclin A2 and Cyclin B1 levels, proving the role of APC/C in this process.

--

Human

L

genomic instability

26,939,887

Gene

0

1

0

SIRT6

51548

protein coding

U2OS

--

Aging

Prevent

SA--gal activity assay

We detected significantly increased numbers of senescent U2OS cells (over ~20-fold higher than those of control cells) within a week of SIRT6 depletion by lentiviral short hairpin RNAs (shRNAs).

H3K18ac

--

Western blot

SIRT6 also promoted H3K18ac deacetylation when overexpressed in cells, whereas the mutant SIRT6 H133Y protein did not.Together, these data identify H3K18ac as a new chromatin substrate of SIRT6 and demonstrate a physiologic role of SIRT6 in maintaining total cellular levels of this histone mark.

--

Human

HL

genomic instability

27,043,296

Gene

0

1

0

WSB1

26118

protein coding

MEF

Embryo

Aging

Prevent

SA--gal activity assay//Western blot

Consistent with previous publications, expressing H-RasV12 in MEFs (H-RasV12-MEFs) caused cellular senescence, characterized by enhanced senescence-associated ¦Â-galactosidase (SA-¦Â-gal) staining, flattened cell morphology, and upregulation of p53, p21, and p16.

ATM

Downregulation

SA-¦Â-gal activity assay//Colony formation assay

Interestingly, we observed decreased ATM staining and levels in cells overexpressing WSB1.These results led us to hypothesize that WSB1 might negatively regulate A TM levels and that ATM might be a major target of WSB1 in overcoming OIS.We also obtained similar results using ATM+/+ and ATM?/? immortal MEFs. These results suggest that A TM is a major target of WSB1 in inducing cell transformation.

--

Human

L

genomic instability

27,958,289

Gene

0

1

0

1

0

DUSP3

1845

protein coding

HeLa,MEWO

--

Aging

Prevent

SA--gal activity assay//Western blot

The results indicated that decreasing endogenous DUSP3 activity per se caused increased cellular senescence of both cell lines after 72 h (20-30%). As expected, cells exposed to 5 Gy of gamma radiation exhibited increased SA-¦Â-Gal staining, which was further significantly increased in the cells subjected to the combined gamma radiation/DUSP3 inhibitor treatment and reached 40-60% in the HeLa and MeWo cells, indicating that these cells tended to undergo senescence in response to pharmacological DUSP3 inhibition.

--

Human

L

genomic instability

28,389,334

Gene

0

1

0

1

0

PIM1

5292

protein coding

2BS,IMR-90

--

Aging

Accelerate

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

In the meanwhile, 2BS cells and IMR90 cells with UHRF1 knockdown exhibited lower cell proliferation rates and reduced EdU incorporation when compared with corresponding control groups. Furthermore, UHRF1 depletion also increased senescenceassociated ¦Â-galactosidase (SA-¦Â-gal) activity in both cell lines. All of these data point to the same notion that knockdown of UHRF1 may induce premature senescence in human diploid fibroblasts.

UHRF1

Downregulation

LC¨CMS/MS analysis//IP//Western blot

The eluted protein complex was then resolved on sodium dodecyl sulfate¨Cpolyacrylamide gel electrophoresis, silver stained and subjected to LC¨CMS/MS analysis.The identified proteins were listed,including UHRF1, as well as other known interacting proteins of PIM1 such as Hsp9066and SND1,67suggesting that UHRF1 is a potential target of PIM1.Co-immunoprecipitation of UHRF1 followed by western analysis indicated that PIM1 associated with UHRF1 irrespective of whether the PIM1 kinase was wild-type or inActivate mutant.

--

Human

HL

genomic instability

28,394,343

Gene

0

1

0

1

0

SIRT7

51547

protein coding

WI-38,hTRT-WI38

--

Aging

Prevent

SA--gal activity assay//Western blot

We found that shRNA depletion of SIRT7 from WI38 primary human fibroblasts led to substantial accumulation of senescent cells within days after SIRT7 depletion.Levels of the senescence marker p16 were also increased in the SIRT7-deficient cell cultures (data not shown).

SNF2H

Upregulation

IP

This analysis revealed specific binding of SIRT7 to both the SNF2H and TIP5 subunits of NoRC.Intriguingly, these experiment salso revealed that overexpression of SIRT7 leads to increased SNF2H protein levels.

--

Human

L

genomic instability

29,728,458

Gene

0

1

0

1

0

TIMELESS

8914

protein coding

2BS

--

Aging

Prevent

SA--gal activity assay//Western blot

The percentage of senescent cells identified by SA-¦Â-gal staining is significantly increased in response to RasG12Vin 2BS human diploid fibroblasts and the percentage of cells with EdU incorporated is decreased.The cellular senescence model was further confirmed by Western Blot analysis of increased expression of p53, p21 and p16.

E2F1

Binding

IP

Chromatin immunoprecipitation (ChIP) assay was utilized to confirm the binding of E2F1 to the endogenous TIMELESS promoter. Interaction between E2F1 and TIMELESS promoter region was detected by qRT-PCR. As expected, interaction between E2F1 and TIMELESS promoter was observed in young 2BS cells.

--

Human

HL

genomic instability

30,100,061

Gene

0

1

0

1

0

TET1

80312

protein coding

H1299,H1975,H226

Lung

Lung cancer

Prevent

SA--gal activity assay

Cell morphology (enlarged size and flattened shape) suggested that these cells were undergoing cellular senescence and this was confirmed by analyzing the cells for ¦Â-galactosidase activity, a metabolic marker of senescence.

p53

Binding

IP

Thus, the hypothesis that WT p53 negatively regulates TET1 transcription via direct binding to its promoter was tested with ChIP using a p53-specific antibody. As expected, no enrichment of p53 at its predicted binding site within the TET1 promoter region was detected in H1299 cells characterized by a p53-null mutation. Conversely, 23,800- and 13,500-fold enrichments of p53 at the TET1 promoter were found in the WT p53 cell lines A549 and H2228, respectively.

--

Human

HL

genomic instability

30,622,117

Gene

0

1

0