Venkatachalam/aging_reg_dataset · Datasets at Fast360

FLT1

2321

protein coding

HBMEC

--

Aging

Accelerate

Knockdown//SA--gal activity assay

We then performed siRNA mediated VEGFR-1 knockdown as before and confirmed that the siRNA mediated VEGFR-1 knockdown reduced senescence in the A¦Â1¨C42 oligomertreated HBMECs as measured by ¦Â-galactosidase staining.

--

Human

L

cellular senescence

31,513,781

Gene

0

1

0

1

0

JUN

3725

protein coding

MCF-7

--

Aging

Prevent

SA--gal activity assay

However, for MCF7-c-Jun cells maintained in the absence of doxycycline to induce c-Jun expression, the percentage of cells staining positive for ¦Â-galactosidase activity was much lower and was not significantly increased after vinblastine treatment.

--

Human

L

cellular senescence

17,126,817

Gene

0

1

0

CXCL8

3576

protein coding

PMSC

--

Aging

Prevent

CCK-8 assay//Cell morphological analysis//Flow cytometry//SA--gal activity assay

The enlarged and flattened PMSCs stained blue, indicating they have become senescent. The percentage of SA-¦Â-gal-positive PMSCs was significantly increased on day9 after IL-8 knockdown.A flow cytometric analysis indicated that the cellular G2/M phase ratio was significantly increased, indicating that the G2-M phase was prolonged in the IL-8-silenced PMSCs when compared with their controls.The IL-8-silenced PMSCs showed significant growth retardation.

--

Akt-FOXO3a//CXCR2

--//--

qRT-PCR//Western blot

Increased p-AKT expression and decreased FOXO3a protein expression in IL-8-silenced PMSCs were verified by western blot analysis.We found that in IL-8-silenced PMSCs, the expression levels of CXCR2 ligands were time-dependent: most of the CXCR2 ligands were present at reduced levels in pre-senescent shIL-8 PMSCs,while their expression later increased along with the cellular senescence process.

Human

L

cellular senescence

28,418,782

Gene

0

1

0

1

0

1

0

1

0

PRKDC

5591

protein coding

Fibroblast

--

Idiopathic Pulmonary Fibrosis

Accelerate

qRT-PCR

Transcriptomic analysis of Nu7441(DNA-PKcs inhibitor)-treated and untreated fibroblasts showed that this treatment caused a significant increase in the senescence-associated CDKN1A, CDKN1B, CDKN2A, NOX4.

--

Human

L

cellular senescence

31,464,599

Gene

0

1

0

TNF

7124

protein coding

ECFC

--

Aging

Prevent

SA--gal activity assay

We incubated ECFCs with either recombinant TACE or an anti-TNFR2 neutralizing antibody for 6 days and determined development of premature senescence by staining for senescence-associated ¦Â-galactosidase.

p38

Activation

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

We examined the effect of p38 inhibition on the expression of tmTNF and found that blocking p38 completely prevented the loss of tmTNF and subsequent development of premature senescence.

tmTNF-TNFR2

--

Western blot

We treated ECFCs with either TACE or anti-TNFR2 neutralizing antibody and detected the presence of p16ink, a senescence-associated cell cycle regulating protein by Western blot and found that p16ink levels increased dramatically during the course of the 6-day treatment, further confirming that loss of the tmTNF/TNFR2 signaling axis results in premature senescence of ECFCs.

Human

L

cellular senescence

27,076,598

Gene

0

1

0

EGF

1950

protein coding

SK-ES-1,RD-ES

--

Ewing sarcoma

Prevent

Cell counting//Colony formation assay

These assays showed that EGF significantly increased both the proliferation rate and survival in SK-ES-1 and RD-ES cells;Evidence of senescence induction was evaluated with a colorimetric assay after exposure of ES cells to AG1478. The percentage of senescent cells was increased in cells treated with any drug dose compared to controls.

--

Human

L

cellular senescence

29,539,615

Gene

0

1

0

1

0

SOX1

6656

protein coding

Hep3B

--

Hepatocellular carcinoma

Accelerate

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

Our data showed that ectopic expression of SOX1 increased number of cells in G1 while decreasing number of cells in S phase in Hep3B cells.In Hep3B cells,SOX1 expression significantly enhanced the protein level of p21 and p27 but suppressed expression of CDK4 and CDK6 as compared with the control cells.We found that expression of SOX1 in Hep3B cells could enhance the signal of SA-¦Â-gal staining.

p21//p27

Upregulation//Upregulation

Western blot//Flow cytometry

In Hep3B cells, SOX1 expression significantly enhanced the protein level of p21 and p27 but suppressed expression of CDK4 and CDK6 as compared with the control cells. In the SOX1-expressing HepG2 cells, p21 and p27 were also dramatically upregulated. However, there, was no significant difference in the protein levels of CDK4 and CDK6.

--

Human

HL

cellular senescence

22,767,186

Gene

0

1

0

1

0

1

0

SPAG9

9043

protein coding

MDA-MB-231

--

Breast cancer

Prevent

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

Knockdown of SPAG9 by SPAG9 shRNA1(67.03 %) and shRNA2 (65.14 %) resulted in accumulation of most of the cells in G1 phase as compared to NC shRNA (63.50 %) transfected cells. Also, the percentage of G2-M phase cells showed decrease in SPAG9 shRNA [shRNA1 (23.08 %), shRNA2 (26.42 %)]-transfected cells as compared to NC shRNA (28.26 %)-transfected cells.The result showed that there was a significant decrease in cyclins and cyclin-dependent kinases such as cyclin B1, cyclin D1, cyclin E,CDK1, CDK4, and CDK6. Also, upregulation was observed in case of tumor suppressor protein, p21. The percentage of senescent cells was significantly higher(p < 0.0001), 52.6 % and 67.0 %, when transfected with SPAG9 shRNA1 and shRNA2, respectively, as compared to 7.4 % when transfected with NC shRNA.

p21//Cyclin B1//Cyclin D1//Cyclin E//CDK4//CDK6

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

Immunostaining//Western blot

IHC analysis of tumor serial sections revealed significantly enhanced immunoreactivity of p21 and decreased immunoreactivity of cyclin B1, cyclin D1,cyclin E, CDK4, and CDK6 in SPAG9 shRNA2-treated mice as compared NC shRNA-treated mice.Western blotting was carried out to study the various molecules in different phases of cell cycle, which showed that there was a significant decrease in cyclins and cyclindependent kinases such as cyclin B1, cyclin D1, cyclin E,CDK1, CDK4, and CDK6. Also, upregulation was observed in case of tumor suppressor protein, p21.

--

Human

L

cellular senescence

27,449,044

Gene

0

1

0

1

0

1

0

1

0

GFPT2

9945

protein coding

HBEC3-KT

--

Aging

Prevent

Knockdown//SA--gal activity assay

Genetic knockdown of GFPT2 and UAP1 in NSCLC cells revealed increased markers of senescence and reduced clonogenic potential.

--

Human

HL

cellular senescence

30,130,254

Gene

0

1

0

1

0

UAP1

6675

protein coding

HBEC3-KT

--

Aging

Prevent

Knockdown//SA--gal activity assay

Genetic knockdown of GFPT2 and UAP1 in NSCLC cells revealed increased markers of senescence and reduced clonogenic potential.

--

Human

HL

cellular senescence

30,130,254

Gene

0

1

0

1

0

TP53

7157

protein coding

HCA2

--

Aging

Accelerate

FACS analysis//SA--gal activity assay//Western blot

Transient expression of p53 in cells released to G2 phase for 4 hr from a thymidine block resulted in loss of mitotic regulators, cessation of cell proliferation, and an increase in the SA-¦Â-gal-positive fraction.

--

Human

L

cellular senescence

24,910,096

Gene

0

1

0

1

0

1

0

RB1

5925

protein coding

HCA2

--

Aging

Accelerate

FACS analysis//SA--gal activity assay//Western blot

Transient expression (24 hr) of pRb7LP by the addition of doxycycline to G2 cells, in the presence of a Cdk2 inhibitor to prematurely activate Cdh1, resulted in impaired cell proliferation and an increase in the population of cells staining positive for SA-¦Â gal.The resulting senescence phenotype of the cells was further confirmed by the induction of p16 and senescence-associated cytokines such as IL-6 and IL-8.

--

Human

L

cellular senescence

24,910,096

Gene

0

1

0

1

0

1

0

TXNRD1

7296

protein coding

NIH-3T3

--

Aging

Prevent

SA--gal activity assay//Western blot

We show that expression of F-A-TrxR1-HA inhibited SIPS in fibroblasts, as shown by the lesser number of senescence-associated ¦Â-galactosidase-positive cells after oxidative stress as compared with wt-TrxR1-HA-expressing cells.We found that expression of the constitutively Activate F-A-TrxR1-HA inhibited ROS-induced upregulation of p53, expression of p21Waf1/Cip1protein and activation of a p53 responsive element 24 h after oxidative stress,as compared with wt-TrxR1-HA.

Caveolin-1

Downregulation

Western blot

We found that caveolin 1 inhibits TrxR1 activity by preventing the formation of TrxR1 homodimers, as shown by increased levels of dimeric TrxR1 in cells in which endogenous caveolin 1 expression was reduced by short interfering RNA.

--

Human

L

cellular senescence

19,820,694

Gene

0

1

0

1

0

CDKN2A

1029

protein coding

MCF-7

--

Aging

Accelerate

Cell morphological analysis//Flow cytometry

Microscopic analysis revealed that control cells formed a stably proliferating population, whereas cells transfected with the cdk inhibitors, p16 stopped proliferation and adopted a senescent morphology. Flow cytometry data confirmed proliferation of control cells and induction of a G1-phase arrest in cells expressing exogenous p16.

--

Human

L

cellular senescence

19,648,966

Gene

0

1

0

1

0

CDKN1A

1026

protein coding

MCF-7

--

Aging

Accelerate

Cell morphological analysis//Flow cytometry

Microscopic analysis revealed that control cells formed a stably proliferating population, whereas cells transfected with the cdk inhibitors, p21 stopped proliferation and adopted a senescent morphology. Flow cytometry data confirmed proliferation of control cells and induction of a G1-phase arrest in cells expressing exogenous p21.

--

Human

L

cellular senescence

19,648,966

Gene

0

1

0

1

0

CCN1

3491

protein coding

HSC

Liver

Liver disease

Accelerate

Immunostaining//SA--gal activity assay

We stained liver sections for senescence-associated markers, Ki67 and SA-¦Â-gal. Immunohistochemistry for Ki67 revealed that Mdr2-/- livers contained nearly 2-fold more proliferating cells compared with control and Dko mice.

--

Human

HL

cellular senescence

29,105,104

Gene

0

1

0

1

0

EIF4EBP1

1978

protein coding

A549,AD32

--

Aging

Prevent

SA--gal activity assay

AD32 cells had a higher basal level of senescence-associated ¦Â-galactosidase (SA-¦Â-gal)¨Cpositive cells than A549¡ª0.40% and 0.07%, respectively.£¨AD32 cells have decreased levels of 4E-BP1 mRNA and protein, relative to the parental discodermolide-sensitive A549 cells£©.

p53

Downregulation

Western blot

AD324E-BP1cells had decreased levels of p53 protein, indicating that p53 levels may be decreased by overexpression of 4E-BP1 in AD32 cells.

--

Human

L

cellular senescence

21,173,253

Gene

0

1

0

TP73

7161

protein coding

HCT116

--

Aging

Prevent

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

The senescence-associated ¦Â-galactosidase (SA-¦Â-gal) staining showed that p73 knockdown significantly increased the proportion of positive cells (~89.1% in p73i-1 and 79.7% in p73i-2 transfected cells) at 5 dpi, compared to the irradiated controls (~41.2%).However, the proportion of cells in the G2 phase increased significantly in irradiated p73-knockdown cells (30.2% in p73i-1 and 27.2% in p73i-2 transfected cells),compared to that in irradiated control cells (17.8%).

¦¤133p53

Binding

Western blot//Co-IP

The western blot revealed that ¦¤133p53,but not full-length p53,co-immunoprecipitated with p73 at 12 hpi.

--

Human

HL

cellular senescence

29,511,339

Gene

0

1

0

1

0

1

0

ERBB2

2064

protein coding

MCF 10A

--

Cancer

Accelerate

Cell morphological analysis//Growth curve assay//SA--gal activity assay

We expressed HER2 in HSF1-depleted cells, a significant growth inhibition was observed, associated with a significant change in cells¡¯ appearance, including enlarged, flattened morphology and extensive vacuolization reminiscent of senescence.HER2 expression in control MCF-10A cells led to a significant increase in ¦Â-gal-positive population (B30%).

p21//Survivin

Upregulation//Downregulation

Western blot//Co-IP

P21 was mildly upregulated by HER2 expression in control cells.Upon HER2 expression, survivin levels were dramatically decreased in shHSF1 cells.

--

Human

L

cellular senescence

20,622,894

Gene

0

1

0

1

0

1

0

HSF1

3297

protein coding

MCF 10A

--

Cancer

Prevent

SA--gal activity assay

HER2 expression in control MCF-10A cells led to a significant increase in ¦Â-gal-positive population (B30%). Importantly, expression of HER2 in shHSF1 MCF10A cells resulted in about 70% of ¦Â-gal-positive cells.

--

Human

L

cellular senescence

20,622,894

Gene

0

1

0

PTEN

5728

protein coding

BJ-T

--

Aging

Prevent

Cell morphological analysis//SA--gal activity assay

Cells expressing PTEN shRNA exhibited a significant increase in cell size and senescence associated ¦Â-galactosidase activity (SA¦ÂGAL).

--

PI3K-Akt

Activation

Western blot

Cells with depleted PTEN protein levels or exhibited increased levels of phospho-AKT and phosphorylation of the AKT substrate PRAS40.

Human

L

cellular senescence

21,909,130

Gene

0

1

0

1

0

PIK3CA

5290

protein coding

BJ-T

--

Aging

Accelerate

Cell morphological analysis//SA--gal activity assay

Cells expressing PIK3CAE545K exhibited a significant increase in cell size and senescence associated ¦Â-galactosidase activity (SA¦ÂGAL).

--

PI3K-Akt

Activation

Western blot

Cells with expressing PIK3CAE545K exhibited increased levels of phospho-AKT and phosphorylation of the AKT substrate PRAS40.

Human

L

cellular senescence

21,909,130

Gene

0

1

0

1

0

AKT1

207

protein coding

BJ-T

--

Aging

Accelerate

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

Cells expressing activated AKT showed a significant increase in SA¦ÂGAL and cell size.AKT-induced senescence is characterised by an SASP, with increased secretion of IL-1a, IL-1b, IL-6 and IL-8.

--

p53//mTORC1

Upregulation//--

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

SA¦ÂGAL positivity was significantly reduced in BJ-T-myr-AKT/p53 stable knockdown cells as compared with control BJ-T-myr-AKT cells. Acute p53 knockdown, confirmed by immunoblot analysis, rescued the reduced proliferation of myr-AKT1-expressing cells to that of control cells .Upon treatment with rapamycin,the percentage of AKT cells positive for SA¦ÂGAL was significantly reduced .Rapamycin treatment also dramatically reduced AKT-induced effects on cell size, and the SASP, indicating that mTORC1 activity is critical for PI3K/AKT-driven senescence.

Human

L

cellular senescence

21,909,130

Gene

0

1

0

1

0

1

0

HRAS

3265

protein coding

BJ-T

--

Aging

Accelerate

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

Cells expressing activated AKT isoforms and H-RASV12showed a significant increase in SA¦ÂGAL and cell size.The robust induction of SAHF formationwith was observed when RAS was activated.

--

Human

L

cellular senescence

21,909,130

Gene

0

1

0

1

0

1

0

PIK3CA

5290

protein coding

EC

--

Vascular malformations

Accelerate

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

The expression of Activate PI3K evidently modified EC morphology by dramatically increasing average cell size.Indeed the expression of Activate PI3K, both H1047R and E545K mutants, increased the amount of ¦Â-galactosidase positive cells.We measured DNA replication rates by means of EdU incorporation assay. EC-H1047R and EC-E545K showed higher DNA replication rates, which were particularly elevated when EC were stimulated with VEGF-A.

--

Human

L

cellular senescence

29,352,118

Gene

0

1

0

1

0

1

0

CDK4

1019

protein coding

IMR-90

--

Aging

Prevent

Cell proliferation assay

We found that these kinases efficiently blocked PML-induced growth arrest and senescence in normal cells.

--

Human

HL

cellular senescence

27,206,849

Gene

0

1

0

CDK6

1021

protein coding

IMR-90

--

Aging

Prevent

Cell proliferation assay

We found that these kinases efficiently blocked PML-induced growth arrest and senescence in normal cells.

--

Human

HL

cellular senescence

27,206,849

Gene

0

1

0

SIRT6

51548

protein coding

Primary human keratinocyte

--

Aging

Prevent

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

SIRT6 knockdown significantly increased senescence of primary keratinocytes,and this effect was reversed by depletion of RELA by RNAi.

H3K9

--

CHIP//Western blot

ChIP analysis revealed that H3K9 acetylation is induced following TNF-a treatment in SIRT6-proficient control cells at the promoters of multiple NF-kB target genes, consistent with transcriptional induction. In cells depleted of SIRT6, H3K9 was hyperacetylated at these promoters in response to TNF-a.

NF-¦ÊB

Downregulation

Luciferase reporter assay//Knockdown

SIRT6 depletion led to constitutive NF-kB reporter gene activity, which, upon TNF-a treatment,was further enhanced to levels considerably higher than in SIRT6-proficient control cells .

Human

HL

cellular senescence

19,135,889

Gene

0

1

0

1

0

1

0

PNPT1

87178

protein coding

HeLa

--

Aging

Accelerate

Flow cytometry//Western blot

Overexpression of hPNPaseold-35 induces a senescence-like growth arrest and also generates ROS.

--

NF-¦ÊB

Activation

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

However,infection with Ad.hPNPaseold -35 resulted in a 10- to 12-fold induction in relative luciferase activity in comparison with control or Ad.vec-infected cells.On Ad.hPNPaseold-35 infection, the binding pattern changed, with the p50/p50 homodimer disappearing, and the binding of the p50/p65 heterodimer increasing markedly.Expressions of mRNAs and secreted proteins of IL-6 and IL-8, two NF-kB target genes, were analyzed by RT-PCR and ELISA, respectively,after Ad.hPNPaseold-35infection.

Human

HL

delay aging

15,492,272

Gene

0

1

0

1

0

BLVRA

644

protein coding

HDF

--

Aging

Prevent

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

Morphological analysis indicated that HDF cells became enlarged and flattened after BLVRA shRNA treatment.Moreover,knockdown of BLVRA led to induce the expression of the senescence marker SA-¦Â-gal.BLVRA knockdown cells were arrested in the G0-G1 phase of the cell cycle to approximately 78% of the cells whereas 53% of random shRNA-treated cells were arrested in the G0-G1 phase of the cell cycle.Transfection of the cells with shRNA- BLVRA induced the expres-sion levels of p53, 16, and p21 significantly.

--

Human

L

delay aging

21,099,244

Gene

0

1

0

1

0

1

0

1

0

1

0

WRN

7486

protein coding

WI-38

--

Werner syndrome

Prevent

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

We found that upon withdrawal of WRN, the morphology of untransformed primary fibroblasts changed progressively within 5 days posttransfection. The cells became enlarged, flattened and developed the SA-¦Â-Gal activity with a concomitant moderate decline of cumulative cell number.The significant hypophosphorylation of Rb was accompanied by decreased proliferation of the WRN-deficient cells, as evidenced by an average 40% reduction of bromodeoxyuridine incorporation (data not shown) and a similar decrease in S-phase cells with an increased proportion of G0-G1 cells by FACS analysis.

p16

--

Knockdown//Western blot

Untransformed fibroblasts responded to acute knockdown of WRN with a minimal elevation of the levels of the p16 cell-cycle inhibitors within the time frame of 5 days.

p53//Rb

--//--

Knockdown//Western blot

Untransformed fibroblasts responded to acute knockdown of WRN with a minimal elevation of the levels of p21,the downstream p53 target within the time frame of 5 days. However,an almost complete disappearance of retinoblastoma (Rb) phosphorylation and, to a lesser degree, the dephosphorylated Rb protein was evident .

Human

HL

delay aging

16,287,861

Gene

0

1

0

1

0

1

0

1

0

BLM

641

protein coding

WI-38

--

Werner syndrome

Prevent

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

RNAi directed against BLM resulted in a similar or somewhat greater reduction in cell number and appearance of SA-¦Â-Gal activity, albeit with less evident flattening morphology of the cells.

p16

--

Knockdown//Western blot

Untransformed fibroblasts responded to acute knockdown of BLM with a minimal elevation of the levels of the p16 cell-cycle inhibitors within the time frame of 5 days.

p53//Rb

--//--

Knockdown//Western blot

Untransformed fibroblasts responded to acute knockdown of BLM with a minimal elevation of the levels of p21,the downstream p53 target within the time frame of 5 days .Acutely BLM-depleted cells also showed marked dephosphorylation of Rb protein.

Human

HL

delay aging

16,287,861

Gene

0

1

0

1

0

1

0

GCG

2641

protein coding

HUVEC

--

Diabetes

Prevent

SA--gal activity assay

Treatment of HUVECs with GLP-1 attenuated the increase of senescent cells in a dose-responsive manner.

DPP-4//CREB

Upregulation//Activation

Western blot

Treatment of the ZDF animals with vildagliptin resulted in a significant reduction of DPP-4 activity and an almost 6-fold increase of GLP-1 plasma levels.The vildagliptin treatment did, however, decrease cellular senescence in these animals, to levels almost comparable to those of lean rats (2.6¡À0.6% versus 2.3¡À0.5%). This suggests that increased GLP-1 levels by DPP-4 inhibition have a protective effect on the vasculature.Western blot analysis using an anti phosphorylated CREB antibody showed that GLP-1 treatment increased relative phosphorylated CREB levels by 52% compared with the control.

cAMP-PKA

--

Premature senescence assay

PKA inhibition by H89 was also sufficient to block the GLP-1-mediated protective effect on HUVECs. H89 inhibition showed a dose response effect on cellular senescence in combination with GLP-1, with a complete abolishment of the GLP-1 protective effect at a concentration of 1 umol/L H89. Similar results were obtained with another PKA inhibitor KT5720.

Human

L

delay aging

20,448,207

Gene

0

1

0

SRC

6714

protein coding

Human endothelial cell

--

Aging

Accelerate

SA--gal activity assay

Incubation with H2O2 significantly increased acidic ¦Â-galac-tosidase¨Cpositive cells. Interestingly, coincubation with the Src kinase inhibitor PP2 (500 nmol/L) completely blocked the induction of premature senescence indicating that Src kinase activation contributes to endothelial cell senescence.

--

Human

L

delay aging

14,963,003

Gene

0

1

0

SIRT1

23411

protein coding

PAEC

--

Aging

Prevent

Flow cytometry//SA--gal activity assay

Results from SA-¦Â-gal staining, as well as flow cytometric analysis, suggested that SIRT1 alleviated cellular senescence.

--

LKB1-AMPK//Akt

Activation//Activation

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

Importantly, the amount of LKB1 and phosphoAMPK(T172) were significantly lower in SIRT1 transgenic mice. In addition, decreased SIRT1 expression and elevated LKB1/AMPK levels were also observed in the aorta tissues of old mice by comparing to those in young mice.SIRT1 and resveratrol treatment increased Akt(Ser473) phosphorylation in normal cultures,Inhibition of Akt by either Akt inhibitor or kinase-dead Akt could induce senescence only when the experiment was performed under serum containing conditions .These results largely mirrored the effects of Akt inhibition on AMPK activation.

Human

L

delay aging

20,203,304

Gene

0

1

0

1

0

BMI1

648

protein coding

Renal cell

Kidney

Renal tubulointerstitial injury

Prevent

Immunostaining//SA--gal activity assay//TUNEL assay

Results showed that the percentage of Ki67-positive cells was decreased dramatically, whereas the percentage of TUNEL-positive cells, SA-¦Â-gal positive areas, 8-OHdG-positive cells, CD3-positive and F4/80-positive inflammatory cells were significantly increased in Bmi-1-/-mice compared with WT mice.

--

Human

HL

delay aging

28,790,310

Gene

0

1

0

1

0

1

0

CDKN2A

1029

protein coding

Renal cell

Kidney

Renal tubulointerstitial injury

Accelerate

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

P16 deletion was significantly rescued the abnormalities in renal cell proliferation, apoptosis and senescence, DNA damage and inflammatory cell infiltration observed in Bmi-1 -/- mice.However,they were reduced significantly in Bmi-1-/- p16 -/- mice compared with Bmi-1-/- mice.These results demonstrated that p16 deletion ameliorated the proinflammatory secretory phenotype caused by Bmi-1 deficiency.

--

Human

HL

delay aging

28,790,310

Gene

0

1

0

1

0

1

0

1

0

1

0

PLA2R1

22925

protein coding

MRC-5

--

Hutchinson¨CGilford progeria syndrome

Accelerate

Cell counting//Crystal violet assay//Knockdown//SA--gal activity assay//qRT-PCR

Constitutive expression of progerin resulted in proliferation arrest as judged by reduced number of cells observed using crystal violet staining and growth curves and reduced expression of the proliferation marker Ki67, and increased frequency of SA©\¦Â©\Gal©\positive cells and increased expression of p21 (CDKN1A) and the SASP component IL©\8.Knockdown of PLA2R1 with two independent shRNAs abolished all these hallmarks of cellular senescence.

--

p53-FDPS

--

qRT-PCR//Immunostaining

As expected,progerin increased P©\ATM and ¦ÃH2AX DNA damage marks, p53 phosphorylation, the p53 transcriptional target p21, and FDPS, and these inductions were abolished upon PLA2R1 knockdown.

Human

L

delay aging

30,216,637

Gene

0

1

0

1

0

1

0

1

0

1

0

CXCR4

7852

protein coding

SH-SY5Y

--

Alzheimer's disease

Prevent

CCK-8 assay//Knockdown//MTT assay

Moreover, the data of CCK8 assay demonstrated that the cell activity was decreased in siCXCR4 cells. Meanwhile,siCXCR4 cells have significant deficient effects on cell proliferation and activity compared with normal and control groups.

Akt//CREB//p53

Activation//Activation//Activation

Western blot//Immunostaining//Knockdown

Using Western Blotting assay, we observed that the phosphorylation at 308 of AKT in siCXCR4 group was robustly inhibited.Using a pixelby-pixel colocalization analysis, the results illustrated that fluorescent signals of AKT were clearly merged with CXCR4 on plasma membrane. Meanwhile, the phosphorylation of CREB was significantly decreased, while the level of P53 was increased in CXCR4 knock down cells compared with normal or control groups.

CXCL12-CXCR4

Activation

Immunostaining

We firstly found a strong colocalization of CXCR4 and AKT on plasma membrane stimulating by 100ng/ml CXCL12.

Human

L

delay aging

30,080,220

Gene

0

1

0

1

0

1

0

TP53

7157

protein coding

HSC

Spine,tibias,Skin

Aging

Accelerate

BrdU assay//Flow cytometry//Histological staining

P72 mice showed a delayed development of all these aging-related changes compared with R72 mice, with the most obvious differences observed at the age of 18 months.R72 mice showed more significant decreases in both skin dermal thickness and subcutaneous adipose thickness compared with P72 mice.R72 mice showed a more rapid increase in the numbers of LT-HSCs than P72 mice during aging.While there was no significant difference in LT-HSC numbers between young 129SVslR72 and P72 mice, much higher LT-HSC numbers were observed in R72 mice than P72 mice at the age of both 12 and 18 months.The decrease of proliferation HSCs was more rapid in R72 mice than P72 mice during aging .R72 mice displayed a more obvious sign of osteoporosis than P72 mice.However, 12- and 18-month-old R72 mice showed a more pronounced decrease in the wound healing ability than age-matched P72 mice.Notably, 18-month-old R72 mice developed more pronounced lordokyphosis compared with age-matched P72 mice.

p21

Upregulation

RT-PCR//Western blot

p21 mRNA expression levels in the bone marrow were slightly higher in P72 mice compared with R72 mice as determined by real-time PCR assays with this difference being more obvious in older mice than young mice. This difference in p21 expression levels was confirmed at the protein level as determined by Western-blot assays.

--

Human

L

delay aging

29,557,783

Gene

0

1

0

1

0

1

0

COL17A1

1308

protein coding

HFSC

Hair

Aging

Prevent

Histological images analysis

We generated HFSC-specificCol17a1-deficient mice (Col17a1cKO). We found that those mice also show thinning hair and graying hair.The heat map for the global transcriptome of activated HFSC fractions (aHFSCs) revealed a significantly close relationship between Col17a1cKO HFSCs and aged HFSCs. Most of those mice showed significantly fewer miniaturized HFs and an apparent retardation of hair loss even in mice surving for 24 months (N = 3 mice) and 32 months (N = 2 mice).

ELANE

--

Western blot

Indeed, primary keratinocytes treated with ELANE showed that both the 180-kD COL17A1 and its shed form of the 120 kD ectodomain are quickly degraded by ELANE in vitro.

--

Human

HL

delay aging

26,912,707

Gene

0

1

0

CDKN2A

1029

protein coding

HDF

--

Aging

Accelerate

Cell morphological analysis//Knockdown//qRT-PCR

This study showed that senescent HDFs transfected with p16INK4asiRNA showed changes of morphology from senescent morphology to morphology of young cells with the presence of small and spindle-shaped fibroblasts.The data showed that, in senescent HDFs, p16INK4amRNA was significantly upregulated (p < 0.05) compared to young HDF cells.

Cyclin D1

--

Western blot

Cells transfected with p16INK4a siRNA showed downregulation (p < 0.05) of cyclin D1 compared to untreated senescent cells.In senescent cells, cyclin D1 was upregulated significantly (p < 0.05) when compared to young cells.

--

Human

L

delay aging

27,743,340

Gene

0

1

0

1

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 control cells) within a week of SIRT6 depletion by lentiviral shRNAs, and increased senescence was observed as early as 72 hours after transient transfection of SIRT6 siRNAs.

H3K18Ac

--

Western blot

Upon extending this analysis to new peptides, we found that SIRT6 robustly deacetylated lysine K18 on histone H3 peptides (H3K18Ac).SIRT6 also promoted H3K18Ac deacetylation when over-expressed in cells, while the mutant SIRT6 H133Y protein did not.

--

Human

HL

delay aging

27,043,296

Gene

0

1

0

ITGB3

3690

protein coding

BF

--

Aging

Accelerate

BrdU assay//Immunostaining//SA--gal activity assay//qRT-PCR

Indeed, expression of a retroviral vector encoding ITGB3 in BFs reduces their proliferation rate, quantified by measuring the percentage of cells incorporating bromodeoxyuridine (BrdU).Consistent with the activation of senescence, ITGB3 expression led to an increase in the number of cells staining positive for senescence-associated ¦Â-galactosidase (SA-¦Â-Gal) activity, an accumulation of reActivate oxygen species (ROS), and a mild increase in the mRNA levels of different SASPs.

CBX7

--

Western blot//Immunostaining//Knockdown//CHIP

Importantly,we observed that shCBX7 increases the number of cells presenting av¦Â3-stained FA complexes by IF. The regulation of ¦Â3 protein levels by CBX7 was also confirmed in IMR-90 fibroblasts.We checked the levels of the integrin heterodimer avb3 by immunofluorescence (IF)and the ¦Â3 subunit by immunoblot upon CBX7 knockdown or Cbx7 ectopic expression.Our data show a reduced binding of CBX7 to the ITGB3 TSS during OIS, suggesting that the endogenous upregulation of ITGB3 during OIS is due to the transcriptional deregulation of the locus by the loss of CBX7 binding.

p53-p21//TGF-¦Â

Activation//Activation

SA-¦Â-gal activity assay//qRT-PCR

Using a previously characterized shRNA targeting TP53 (shp53) , we impaired not only the proliferation arrest induced by ITGB3 but also the increase in SA-¦Â-Gal activity. The use of a short interfering RNA (siRNA) targeting TP53 (sip53) also impaired the growth arrest induced by ITGB3 expression. Our data demonstrate that both siRNAs against TGFBR2 overcome senescence induced by the overexpression of ITGB3, as shown by measuring the relative cell number and p21CIPlevels by IF. Indeed, qPCR analyses of a range of regulators implicated in the TGF-¦Â pathway are upregulated in BFs expressing ITGB3.

Human

L

delay aging

28,273,461

Gene

0

1

0

1

0

1

0

1

0

DLX3

1747

protein coding

B-MSC

--

Aging

Accelerate

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

The number of SA-¦Â -gal+ staining cells 38% in TDO-BMSCs and 55% in CON-BMSCs suggested that BMSCs with DLX3 mutation remained a younger status while the normal BMSCs entered a premature senescence.After 72 h osteoinduction, aging-related markers p16INK4a and p15INK4b mRNA expression detected by real-time PCR and p16INK4a and GLB1 protein expression examined by western blot were significantly increased in WT-DLX3 but decreased in MT-DLX3 and TR-DLX3 when compared with EGFP-EV . Stemness markers Oct4 and Nanog mRNA expression detected by real-time PCR were significantly decreased in WT-DLX3 but increased in MT-DLX3 and TR-DLX3 when compared with EGFP-EV .The results showed that percentage of SA-¦Â -gal-positive cells was significantly higher in WT-DLX3, but much lower in MT-DLX3 and TR-DLX3.

--

Human

HL

delay aging

27,924,851

Gene

0

1

0

1

0

1

0

CIZ1

25792

protein coding

MEF

Brain

Neurodegenerative disease

Prevent

Behavioral assessment//Comet assay//Flow cytometry//Immunostaining

Using flow cytometry, MEFs from null mice showed cell-cycle defects and increased apoptosis (two-tailed t(8) = 3.14, P = 0.014) in comparsion with MEFs from WT littermates. For this purpose, we investigated DNA damage in the cerebellum and hippocampus of aged WT and null mice with comet assays and immunohistochemistry. We found increased % tail DNA in the cerebellum (two-tailed t(10) = 12.14, P <0.0001) and hippocampus (two-tailed t(10) = 14.07,P <0.0001) of aged Ciz1?/ ? mice as compared with WT littermates .Behaviorally, male and female Ciz1?/ ? mice were more aggressive than their WT littermates in the dominance tube (P < 0.0001). Similar to motor and behavioral findings, we also observed cognitive dysfunction in Ciz1?/ ? mice as assessed by cross maze and Morris water maze testing.

--

NF-¦ÊB

--

Western blot

Consistent with our gene expression data, we observed increased protein levels of NF-¦ÊB (p65) in the nuclear extracts from the cerebellae and hippocampi.

Human

L

delay aging

29,154,038

Gene

0

1

0

1

0

1

0

1

0

JAG1

182

protein coding

MSC

--

Aging

Prevent

Flow cytometry//SA--gal activity assay

As we expected,activation of Notch signaling by JAG1 led to a reduced cell senescence in both 1- and 5-day sheet cultures, as demonstrated by decreased frequencies of SA-¦Â-gal positive cells (8.25¡À1.5% and 20.5¡À4.2%, respectively) when compared with MSCs from lgG control groups .By comparison, the G0 phase cells in JAG1 groups was 45.2¡À2.8% and 57.5¡À8.2% in the 1-day and 5-day cultures respectively, and significantly lower than those in the lgG group.

p16//CCND1

Downregulation//Upregulation

RT-PCR//Western blot

In lgG control group,relative quantification by RT -PCR revealed an ~ 5-fold up-regulation of p16 RNA and a 2.7-fold up-regulation of p21 RNA in 5-day cultures compared with 1-day cultures, which suggests that these two factors act synergistically to induce MSC senescence. Finally, protein expression of p16 in western blot confirmed our PCR results by showing thinner band in JAG1 treated cells and thicker band in Hes1 deficient cells, and no significant change was observed in protein expression of p21 .Consistent with our previous finding, CCND1 expression was induced by JAG1 in 1-day cultures and no significant changes were observed in 5-day cultures compared with lgG controls.

JAG1-Notch-Hes1

Activation

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

Therefore, we further measured Notch target Hes1 expression in 1- and 5-day sheet cultures. RT -PCR analysis of total RNA revealed a decreased expression of Hes1from 1-day to 5-day cultures in control lgG-coated plates, while Hes1 expression at both time points was significantly increased by JAG1.Western blot analysis using protein from 5-day sheet cultures further confirmed these knockdown results by showing a significantly reduced Hes1 protein expression in shRNA lentivirus-infected MSCs with or without JAG1 treatment.More importantly, although the cellular senescence in 5-day cultures was significantly inhibited by JAG1-mediated Notch activ ation, this inhibitory effect was almost completely abrogated after knocking down Hes1 expression in these MSCs.

Human

L

delay aging

28,151,468

Gene

0

1

0

1

0

CD9

928

protein coding

A549

Bone,Muscle,Adipose,Hair,Lung

Chronic obstructive pulmonary disease

Prevent

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

At 80 weeks of age, CD9/CD81 DKO mice were smaller and had less hair of a brownish color than WT mice, although DKO and WT mice could not be distinguished at 3 wk of age.Moreover, DKO mice developed progressive kyphosis and decreased bone mineral density. Muscle and visceral adipose tissue were significantly reduced in volume, as determined by CT quantitation.Consequently, DKO mice had remarkably shorter survival than WT mice. Histological examination revealed that DKO mice developed emphysema and osteoporosis at 80 wk. Moreover, Although the CD4/CD8 ratio, one of markers in immunosenesence, was not altered in younger mice, it was reduced in aged DKO mice in comparison with WT mice.Double knock-down (DKD) of CD9/CD81 in epithelial cells resulted in cells with a large flattened morphology, and the proportion of SA-¦Â-Gal-positive cells increased.

--

SIRT1

--

Western blot//ELISA//Knockdown

Notably, knockdown of CD9 and CD81 in epithelial cells additively downregulated the expression of SIRT1, whereas knockdown of CD151 did not. The reduced expression of SIRT1 was further verified by immunocytochemistry and ELISA .

Human

HL

delay aging

29,572,511

Gene

0

1

0

1

0

1

0

1

0

CD81

975

protein coding

A549

Bone,Muscle,Adipose,Hair,Lung

Chronic obstructive pulmonary disease

Prevent

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

At 80 weeks of age, CD9/CD81 DKO mice were smaller and had less hair of a brownish color than WT mice, although DKO and WT mice could not be distinguished at 3 wk of age .Moreover, DKO mice developed progressive kyphosis and decreased bone mineral density. Muscle and visceral adipose tissue were significantly reduced in volume, as determined by CT quantitation.Consequently, DKO mice had remarkably shorter survival than WT mice. Histological examination revealed that DKO mice developed emphysema and osteoporosis at 80 wk. Moreover, Although the CD4/CD8 ratio, one of markers in immunosenesence, was not altered in younger mice, it was reduced in aged DKO mice in comparison with WT mice.Double knock-down (DKD) of CD9/CD81 in epithelial cells resulted in cells with a large flattened morphology, and the proportion of SA-¦Â-Gal-positive cells increased.

--

SIRT1

--

Western blot//ELISA//Knockdown

Notably, knockdown of CD9 and CD81 in epithelial cells additively downregulated the expression of SIRT1, whereas knockdown of CD151 did not. The reduced expression of SIRT1 was further verified by immunocytochemistry and ELISA.

Human

HL

delay aging

29,572,511

Gene

0

1

0

1

0

1

0

1

0

FLT1

2321

protein coding

HUVEC

--

Alzheimer's disease

Accelerate

SA--Gal activity assay//Western blot

We show that overexpression of chimeric EGLT-VEGFR-1 in HUVECs for 72 h resulted in robust induction of the senescent phenotype as measured by ¦Â-galactosidase staining, with or without stimulation with EGF.

--

p21-p53

Activation

Western blot//qRT-PCR

Western blotting analysis from chimeric receptor EGLT-transfected lysates confirmed the increased expression of the VEGFR-1 protein and increased p21 proteins levels.qRT-PCR analysis showed increased mRNA levels of VEGFR-1, p21, and p53.

Human

L

delay aging

30,576,228

Gene

0

1

0

1

0

LMNA

4000

protein coding

--

Mice

Aging

Prevent

Immunostaining//Knockdown

The body weight of Lmna?/?mice began to decline at the 10th week of age, while the weight of wild mice continued to increase, showing that, at that stage, the Lmna?/? mice started to lose weight, indicating a decline in the metabolic state of their body.A signifcant increase in the number of p16INK4aexpressing cells was observed in the BAT of Lmna?/?mice (64.33¡À2.333% versus 50.33¡À2.603,P = 0.0161).

UCP1//beta3-AR//PRDM16

--// --// --

Western blot

UCP1 and beta3-AR protein levels in Lmna?/?mice were significantly lower than those in WT mice at 14 weeks of age (UCP1: 0.04740.0089versus 1.000¡À0.0666, P =0.0001; beta3-AR: 3143¡À0.0329 versus 1.000¡À0.0445, P = 0.0002).Both the protein and mRNA levels of PRDM16 were decreased at the age of 14 weeks in Lmna?/?mice.

--

Human

L

delay aging

30,116,163

Gene

0

1

0

1

0

DLX2

1746

protein coding

BJ

--

Aging

Accelerate

SA--gal activity assay

We also confirmed the senescence bypass phenotype by staining for senescence-associated ¦Â-galactosidase (SA-¦Â-Gal).

--

p53-p21//ATM-p53

Downregulation//Downregulation

Western blot

We examined the status of the phosphorylation mark of p53 activation on Ser15 and p21 expression level in both young and senescent cells. We found that DLX2 expression led to reduced p53 Ser15 phosphorylation and p21 expression. We found that 2 wk after Ras virus infection, hTERT immortalized BJ cells expressing DLX2 showed reduced activation of p53 and the DNA damage marker ¦ÃH2AX . Consistent with our results with replicative senescence, DLX2 expression also led to reduced levels of ATM and DNA-PKcs protein .

Human

HL

delay aging

26,833,729

Gene

0

1

0

SIRT1

23411

protein coding

SH-SY5Y

--

Alzheimer's disease

Prevent

CCK-8 assay//MTT assay

We further found that siSIRT1 treatment resulted in inhibited cell proliferation as indicated by the MTT assay and the CCK8 assay in a time course monitoring days 1 to 7.

p53//CREB

--//--

Western blot

We further found that siSIRT1 also inhibited CREB phosphorylation and enhanced p53 expression.

PI3K-Akt

--

Western blot//Immunofluorescence

Using the Western blotting assay, we observed that SIRT1 expression was absent in the siSIRT1-treated cells, but the PI3K levels were similar regardless of the treatment. AKT phosphorylation at the 308 site was inhibited by siSIRT1 treatment while the phosphorylation at the 473 site was similar among all of the three groups, indicating that SIRT1 regulates AKT phosphorylation, specifically at the 308 site.We next examined the subcellular localization of SIRT1 and AKT using immunostaining analysis, and the results showed that AKT was activated in these cells as indicated by plasma membrane localization.

Human

L

delay aging

28,962,864

Gene

0

1

0

1

0

SIRT1

23411

protein coding

VSMC

--

Abdominal aortic aneurysms

Prevent

SA--gal activity assay

As expected, the SA-¦Â-gal activity assay also confirmed the antiaging effect of SIRT1 overexpression.

p21

Downregulation

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

These results suggest that SIRT1 inhibited Ang II¨Cinduced p21 expression by deacetylation of p53 in AAAs. The increased p21 expression caused by Ang II was significantly greater in the aortas of aged mice than that in young mice. Similarly, increased p21 expression was found in human AAA samples.The results of SA-¦Â-gal staining indicated that p21 knock-down not only blocked Ang II¨Cinduced VSMC senescence but also eradicated the promotional effect of SIRT1 knockout.

--

Human

HL

delay aging

27,650,558

Gene

0

1

0

CAV1

857

protein coding

WI-38,IMR-90

--

Aging

Prevent

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

We found that down-regulation of caveolin-1 by shRNA was sufficient to induce cellular senescence in both WI-38 and IMR-90 cells, as quantified by senescence-associated ¦Â-galactosidase activity (SA-¦Â-gal) staining and immunoblot analysis for p21, a marker of cellular senescence.

IFT88//AURKA

--//--

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

More specifically, because down-regulation of IFT88 is known to inhibit ciliogenesis, IFT88 protein expression was downregulated by siRNA in caveoli n-1¨Clacking WI-38 cells. We found that, when primary cilia formation was prevented by IFT88 down-regulation in caveolin-1¨Clacking cells, cellular senescence was dramatically inhibited .To this end, caveolin-1 deficiency was achieved by shRNA in both WI-38 and IMR-90 cells, and the protein levels of AURKA were quantified by immunoblotting analysis. AURKA was virtually lost in caveolin-1 lacking cells.

--

Human

L

delay aging

30,596,512

Gene

0

1

0

1

0

1

0

NBR1

4077

protein coding

MCF-7

--

Aging

Prevent

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

In MCF-7 cells, cellular senescence was induced after NBR1 siRNA transfection, as determined by cellular morphologies, cell counts, SA-¦Â-gal staining, BrdU incorp- oration,and expression levels of p53 and p21WAF1/CIP1(p21) proteins and IL-6 and -8 mRNAs.

p53//p21//p38

--//--//Downregulation

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

Cellular senescence induced by NBR1 abrogation p53- and p21-dependent. Inhibition of p53 by RNAi or pifithrin-a treatment prevented senescence. In addition, NBR1 abrogation induced senescence in p53 wild-type (WT) HCT-116 cells, but not in isogenic p53-null HCT-116 or p53-null PC3 cells. Knockdown of p21 also prevented cellular senescence .NBR1 down-regulated p38 activity in that basal and anisomycin-induced activation of p38 was enhanced by NBR1 abrogation, but was reduced by overexpression of NBR1.

ERstress-ATF6a

--

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

Knockdown of ATF6a suppressed cellular senescence, as determined by cellular morphologies, cell counts, SA-¦Â-gal staining, and expression levels of p53 and p21 proteins .In addition, transcription by ATF6a increased after NBR1 abrogation as demonstrated by a reporter assay. BiP expression, ATF6a cleavage, eIF2a phosphorylation and X-box binding protein (XBP)1 splicing were all upregulated .Moreover, treatment with the ER stress inhibitor, salubrinal,attenuated cellular senescence. Oxidative stress triggered NBR1 abrogation-induced ER stress because the ER stress was prevented by an antioxidant (Tiron) or inhibitors of NOX (DPI and AEBSF), or by knockdown of NOX2 and -4.

Human

L

delay aging

30,260,700

Gene

0

1

0

1

0

1

0

1

0

SIRT1

23411

protein coding

VSMC

--

Diabetes

Prevent

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

Senescence was evident at day 4 with an inverse correlation (p < 0.0359) (r2 = 0.2469) between SIRT1 levels and the presence of senescent cells.Furthermore, there was a significant increase in the level of cellular senescence under osteogenic conditions compared to untreated control cells (p < 0.0417), and a further increase in the numbers of SA-¦Âgal positive cells when exposed to hyperglycaemic osteogenic conditions, compared to the osteogenic conditions (p < 0.0094).Upstream of p21, p53 mRNA was also increased in all treatments where SIRT1 is inhibited (p < 0.0232).

--

RUNX2

Downregulation

ChIP//Western blot//qRT-PCR

The acetylation profile of the RUNX2 promotor was measured via ChIP qPCR. RUNX2 promotor acetylation was significantly reduced in the hyperglycaemic conditions with the addition of SRT1720, suggesting a decrease in RUNX2 transcription was a direct result of SIRT1 activation . Furthermore, SRT1720 activation of SIRT1 activity resulted in a reduction in RUNX2 mRNA under osteogenic conditions, with a further significant reduction in RUNX2 expression under hyperglycaemic conditions (p < 0.0231), compared to the untreated cells. The decrease in RUNX2 mRNA expression after SIRT1 activation correlates with a decrease in RUNX2 protein expression in both osteogenic and hyperglycaemic conditions at day 4. Conversely, RUNX2 protein was increased following inhibition of SIRT1.

Human

L

delay aging

30,696,833

Gene

0

1

0

1

0

1

0

SCN9A

6335

protein coding

HEC

--

Aging

Accelerate

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

As expected, inducing the oncogenic stress (+4-OHT) resulted in proliferation arrest, as demonstrated by their reduced ability to form colonies and by the decrease in the level of the proliferation marker KI67,while it led to an increase in the SA-¦Â-Gal activity and in the expression of two SASP components IL8 and IL6,both major hall-marks of senescence. Strikingly, the knockdown of SCN9A in HEC-TM cells overcame all of the hallmarks of senescence induced by an oncogenic stress.

NF-kB

--

qRT-PCR//Immunofluorescence

We examined whether or not the inhibition of NF-KB transcription factors, either by constitutively expressing a stabilized version of IKBA (mIKBA), a well-known inhibitor of NF-KB, or by knocking down the expression of RELA, the main subunit of the NF-KB transcription factors, blocked the induction of SCN9A during OIS. Both approaches significantly reduced the induction of SCN9A following an oncogenic stress at the mRNA, as well as at the protein levels.

Ca-Rb-E2F

--

qRT-PCR//Live calcium imaging

The inhibition of Rb by E7 prevented the repression of mitotic genes induced by plasma membrane depolarization and blocked plasma membrane depolarization-induced senescence.We observed increased calcium after plasma membrane depolarization in HEC-T cells.

Human

HL

delay aging

29,446,526

Gene

0

1

0

1

0

1

0

CST1

1469

protein coding

MDA-MB-231,SW480

--

Aging

Prevent

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

Following CST1 knockdown, cell populations exhibiting SA-¦Â-gal-positive staining increased to 70¨C80% and 55¨C90% in MDA-MB-231 and SW480 cells, respectively. Notably, the gene expression of representative SASP genes, including interleukin-6 (IL-6) and chemokine C-C motif ligand 20 (CCL20), was induced by CST1 knockdown in MDA-MB-231 and SW480 cells.To confirm that the G0/G1-phase cell cycle arrest was caused by CST1 knockdown, we conducted western blotting and found that CST1 knockdown suppressed cyclin D1 and phospho-retinoblastoma (p-Rb) and induced p21.

CatB

--

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

CST3 knockdown rescued extracellular CatB activity and significantly inhibited SA-¦Â-gal activity in CST1 knockdown MDA-MB-231 cells.We found that CST1 knockdown suppressed extracellular, but not intracellular, CatB activity.

GSK3¦Â

--

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

The increased GSK3¦Â phosphorylation caused by CST1 knockdown was inhibited by the addition of rCys-SN. Although SA-¦Â-gal activity induced by CST1 knockdown was unaltered in mock vector and wild-type GSK3¦Â-expressing cells, the ectopic expression of GSK3¦Â-S9A (Activate form) significantly suppressed the SA-¦Â-gal activity induced by CST1 knockdown . CatB knockdown also induced the inhibitory phosphorylation of GSK3¦Â at serine 9.

Human

L

delay aging

28,383,558

Gene

0

1

0

1

0

1

0

1

0

ATF6

22926

protein coding

NHDF

--

Aging

Accelerate

Knockdown//SA--gal activity assay

Only ATF6¦Á knockdown significantly reduced the number of SA-¦Â-Gal positive-cells upon DTT treatment.

--

COX-2-PGE2

Activation

ELISA//qRT-PCR//Knockdown//Western blot

ELISA assays showed that the increase in PGE2 synthesis and secretion induced by DTT was totally abolished upon ATF6 knocked-down cells .Knockdown of ATF6 and IRE1 but not PERK, reduced COX2 mRNA and protein levels at senescence, as well as the production and secretion of PGE2.

Human

L

delay aging

28,803,844

Gene

0

1

0

1

0

SIRT1

23411

protein coding

VSMC

Plaques and normal vessel

Atherosclerosis

Prevent

Western blot//qPCR

SIRT1 mRNA was significantly decreased in the media of plaques versus normal vessels, associated with significantly increased p16ink4.SIRT1 mRNA and protein expression were reduced in plaque VSMCs and senescent aortic VSMCs versus early-passage normal human VSMCs.

--

Human

L

delay aging

23,224,247

Gene

0

1

0

1

0

CEBPG

1054

protein coding

MEF

--

Aging

Prevent

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

MEF cultures contained many cells with a flattened morphology and vacuolated cytoplasm, indicative of premature entry into senescence. This observation was confirmed by SA¨C¦Â-Gal staining assays, which showed that mutant MEFs contain ¡«4-fold more senescent cells than WT MEFs.We used qPCR to evaluate expression of candidate SASP genes (GRO¦Á/Cxcl-1, Cxcl2, Ccr-1, Il6, Il1a, and Il1b)in Cebpg-/-MEFs and RasV12-expressing Cebpb-/-MEFs. Each gene was induced in C/EBP¦Ã-deficient MEFs compared to WT cells.

C/EBP¦Â

--

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

C/EBP¦Â knockdown with two independent shRNAs increased the proliferative capacity of Cebpg-/-MEFs, as judged by cell densities 7 days after plating. C/EBP¦Â ablation also significantly reduced the proportion of senescent cells and reversed the aberrant expression of SASP genes Cxcl1 and Cxcl2.

--

Human

HL

delay aging

23,775,115

Gene

0

1

0

1

0

1

0

1

0

SIRT1

23411

protein coding

HUC-F2

--

Aging

Prevent

SA--gal activity assay

Results clearly showed that SIRT1 significantly promoted proliferation and prevented replicative senescence HUC-F2 cells.

c-Myc//hTERT

--//--

qRT-PCR//CHIP//Luciferase reporter assay

The results indicated that SIRT1, as well as starvation conditions, increased the promoter activity of c-MYC [11] and the transcription of c-MYC gene. Further, we observed the increased amount of c-MYC recruited to the hTERT promoter. The results showed that SIRT1 significantly increased the transcriptional activation ability of c-MYC.Results showed that SIRT1, but not SIRT1-HY, increased hTERT transcription, as evidenced by the promoter assay and quantitativeRT-PCR (qRT-PCR).

--

Human

L

delay aging

22,197,555

Gene

0

1

0

FOXQ1

94234

protein coding

2BS

--

Esophageal cancer

Prevent

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

Meanwhile, miR30-FOXQ1 also resulted in emerging the morphological features of senescence, characterized by enlarged and flattened cell size, increased senescenceassociated heterochromatin foci, elevated activity of senescence-associated ¦Â-galactosidase (SA-¦Â-gal), a biomarker for senescent cells, and reduced S and increased G1 compartments compared with scramble control vector.Conversely, LPC-FOXQ1 induced much lower SA-¦Â-gal activity and less cell cycle progression than the infection with its corresponding empty control vector.

SIRT1//IL-6//IL-8//p16

Binding//Downregulation//Downregulation//Downregulation

Western blot//qRT-PCR//CHIP-qPCR

Western blot analysis revealed that FOXQ1 overexpression significantly increased the protein level of SIRT1 in HEK293T cells.In parallel with the findings from western blot analysis, qRT-PCR analysis also showed a positive regulation of SIRT1 mRNA expression by FOXQ1,indicating that FOXQ1 could activate transcription of SIRT1.The ChIP-qPCR results indicated a significant enrichment of FOXQ1 in the promoter of SIRT1 in the FOXQ1 overexpressed cells compared with the control cells. LPC-FOXQ1 markedly decreased the mRNA abundance of IL-6 and IL-8 compared with control transfection . Besides, increased FOXQ1 expression resulted in a decreased level of p16INK4aprotein, while removal of FOXQ1 exhibited an opposite effect on p16INK4aprotein level.

NF-¦ÊB

Upregulation

Western blot

We found that the protein level of I¦ÊB¦Á, an inhibitor of NF- ¦ÊB, positively correlated with FOXQ1 and SIRT1.Western blot results revealed that the FOXQ1-induced upregulation of I¦ÊB¦Á level was abolished by addition of EX-527 treatment.These results suggested that the FOXQ1-mediated regulation of SASP factors was dependent on SIRT1-NF-¦ÊB pathway.

Human

L

delay aging

28,726,780

Gene

0

1

0

1

0

1

0

SIRT1

23411

protein coding

HUVEC

--

Diabetic vascular complication

Prevent

Knockdown//SA--gal activity assay

Moreover, Knockdown of SIRT1 not only suppressed the deacetylase activity of SIRT1,but also increased the percentage of SA ¦Â-Gal staining in both RSV- and MET-treated cells.

--

Human

L

delay aging

26,629,991

Gene

0

1

0

1

0

PRKAA1

5562

protein coding

HUVEC

--

Diabetic vascular complication

Prevent

SA--gal activity assay//Western blot

It was shown that silencing of AMPK expression diminished SIRT1 activation and its downstream signaling.Moreover, it abolished the protective effects of RSV and MET against enhanced oxidative stress and accelerated cellular aging.

SIRT1

--

Western blot

It was shown that silencing of AMPK expression diminished SIRT1 activation and its downstream signaling.

--

Human

L

delay aging

26,629,991

Gene

0

1

0

1

0

NRAS

4893

protein coding

Melanocyte

--

Aging

Accelerate

Cell morphological analysis//DAPI staining//Immunostaining//SA--gal activity assay//SAHF

As expected, 15 days post-transduction the majority of N-RASQ61K transduced melanocytes displayed several markers of oncogene-driven senescence, namely cell flattening, increase in cellular size, significantly reduced Ki67 expression, increased SA-¦Â-Gal activity and the formation of SAHF.

--

MAPK//AKT//p16INK4a-pRb//p53-p21Waf1

Activation//Activation//Activation//Activation

Western blot

N-RASQ61K induced melanocyte senescence was also associated with activation of the MAPK and AKT pathways, as shown by the increased phosphorylation of ERK (p-ERK), and AKT (p-AKT) at 5,10 and 15 days post infection.In addition, expression of oncogenic N-RAS led to p53 induction, increased expression of the p16INK4a and p21Waf1 cyclin dependent kinase inhibitors and reduced accumulation of pRb phosphorylated at serine residues -807 and -811 (p-pRb).These data suggest that the activation of pRb is the dominant effector of oncogene-induced melanocyte senescence.

Human

L

delay aging

20,157,537

Gene

0

1

0

1

0

1

0

1

0

1

0

TGFB1

7040

protein coding

U937

--

Cancer

Accelerate

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

While SAHF and increased p16 expression was observed already after 3 days, SA-¦Â-Gal was not apparent until 6 days of treatment. No signs of increased proliferation was observed during continued culture during a couple of weeks.

Mad1//Myc

Upregulation//Downregulation

Western blot

To investigate the effects of TGF-¦Â1 on Myc/Max/Mad network protein expression, the respective proteins were immunoprecipitated from 35S-labeled U-937-myc-6 cell extracts . The synthesis of Mad1 increased substantially;meanwhile, the synthesis of c-Myc decreased after TGF-¦Â1 treatment. A slight decrease in v-Myc synthesis was also observed while no major change in the expression of Max occurred.

--

Human

L

delay aging

19,766,114

Gene

0

1

0

1

0

1

0

BAZ1A

11177

protein coding

A549,U2OS

--

Cancer

Prevent

CCK-8 assay//EdU assay//Flow cytometry//Knockdown//SA--gal activity assay

CCK-8 assay showed BAZ1A-KD cells had decreased proliferation rate compared to control cells.In addition,cell cycle was arrested at G1 phase in BAZ1A-KD cells. Noteworthy, all of the five BAZ1A-KD cell lines showed increased percentage of positive SA-¦Â-Gal stained cells. Moreover, reduced EdU incorporation rate was also observed in BAZ1A-KD cells , reflecting decreased level of DNA synthesis, which is also a well-known molecular phenotype of senescent cells.

SMARCA5

--

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

To examine whether SMARCA5 can regulate the abundance of BAZ1A and then influence senescence, we stably knocked down SMARCA5 by lentivirus mediated short hairpin RNA in A549 and U2OS cells, and reduced BAZ1A protein levels were found in SMARCA5-KD cells, which also exhibited senescenceassociated phenotypes, including decreased proliferation rate, cell cycle arrest at G1 phase, and increased percentage of positive SA-¦Â-Gal stained cells.

Smad3-p21

--

ChIP-qPCR//qRT-PCR//Western blot

The results showed enriched signal of BAZ1A binding to the promoter region of SMAD3 compared to non-specific IgG binding control, which was further validated by Chromatin Immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) .Our data showed the upregulation of both SMAD3 and CDKN1A in BAZ1A-KD cells,indicating upregulated SMAD3 activated the expression of CDKN1A and led to slower cell proliferation and ultimately cellular senescence.

Human

HL

delay aging

31,085,244

Gene

0

1

0

1

0

1

0

1

0

1

0

RRM2B

50484

protein coding

IMR-90

--

Aging

Prevent

SA--gal activity assay//Western blot

Unexpectedly, the silencing of RRM2B triggered premature senescence in young IMR90 cells. The specific silencing of RRM2B led to the progressive accumulation of multiple senescent regulators, including p53, p21CIP1 and p16INK4A, from day 7 to 19.

--

p38 MAPK//p53

--//--

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

p38MAPK phosphorylation was profoundly increased when RRM2B was silenced compared to shRRM2Bmut-expressing cells. Downstream targets of p38MAPK were also activated, as indicated by the significant elevation of phosphorylated MAPKAPK-2, a substrate of p38MAPK, and the phosphorylation of HSP27, a substrate of MAPKAPK-236.Interestingly, the silencing of p53 by shRNA in shRRM2Bexpressing cells was sufficient to rescue premature senescence, as indicated by the significant reduction in SA-¦Â-gal activity and the increase in the replication index .?Cells expressing both shRRM2B and shTP53 showed reduced expression of p21CIP1 and increased levels of RRM1 and RRM2 , whereas p16INK4A was unchanged compared to cells expressing shRRM2B alone.

Human

HL

delay aging

23,139,867

Gene

0

1

0

1

0

NR2E1

7101

protein coding

LNCaP,DU 145

--

Prostate cancer

Prevent

SA--gal activity assay

SA-¦Â-Gal analysis revealed that there was a significant reduction of SA-¦Â-Gal-positive cells in LNCaPTLX infectants compared to LNCaP-pBABE infectants.Cytochemical analysis on SA-¦Â-Gal activity revealed that there was a dramatic increase of SA-¦Â-Gal-positive cells in both LNCaP-shTLX and DU145-shTLX infectants as compared to Scramble-shRNA infectants.

p21//SIRT1

Downregulation//Upregulation

Western blot//CHIP

Immunoblot analysis of senescence-associated markers showed that the expression level of cyclin-dependent kinase inhibitor p21WAF1/CIP1 (hereafter as p21) was markedly suppressed or undetected in LNCaP-/PC-3-TLX infectants but became upregulated in shTLX-infectants of both LNCaP and DU145 cells.ChIP analysis performed in prostate cancer cells identified that there was a significant enrichment of TLX at one consensus TLX-binding motif (TTCAGT) located at - 656 -650 bp and also a palindromic sequence (ACTGAA) at -616 -610 bp in the proximal CDKN1A (p21) promoter.Interestingly, we also detected a significant elevation of protein expression of an NAD- dependent protein deacetylase Sirtuin-1 (SIRT1) in LNCaP-TLX infectants but a reduction in shTLX-infectants by immunoblot analysis.ChIP assay showed that a proximal region of human SIRT1 gene promoter (-129 ? +32 bp) could be immunoprecipitated in TLX- and vector-transfected prostate cancer cells.

--

Human

L

delay aging

25,557,355

Gene

0

1

0

ALPL

249

protein coding

MSC

Adipose

Aging

Prevent

SA--gal activity assay//Western blot

Specifically, the number of SA-¦Â-gal+ cells and the expression of p16 were obviously increased in the Alpl+/- BM, but TERT expression was decreased relative to that in the WT controls.

ATP

--

RadioActivate ATP assay

After the induction, the Alpl+/- MSCs, but not the WT MSCs, released a large amount of ATP. More strikingly, the downregulation of Alpl led to increased extracellular ATP in the WT MSCs, whereas enforcing expression in the Alpl+/- MSCs significantly reduced the extracellular ATP level, suggesting that Alpl in MSCs probably regulates ATP release.

AMPK¦Á

--

Western blot

The supernatant of the Alpl+/- MSCs suppressed the expression levels of p-AMPK¦Á and p-ACC in 293T cell lines, indicating that the higher extracellular ATP levels due to the Alpl deficiency could inactivate the AMPK¦Á pathway.

Human

HL

delay aging

30,210,899

Gene

0

1

0

1

0

CAV1

857

protein coding

A549

--

Aging

Prevent

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

Unexpectedly, Cav-1 knockdown increased ¦Â-gal positivity and changed the cellular morphology from a normal epithelial shape to a fried egg-like shape.Cav-1 knockdown significantly decreased cellular proliferation and colony-forming capacity compared with the si-control-treated cells.

SIRT1

--

Western blot//Knockdown

Therefore, we measured SIRT1 activity by measuring acetylated p53 expression after Cav-1 knockdown in A549 cells using immunoblotting. The acetylation of p53 was gradually increased with time after Cav-1 knockdown.

p53-p21

--

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

Analyses of cell numbers, ¦Â-gal staining positivity, and p-pRb, p53, and p21 expression levels showed that p53 or p21 knockdown prevented Cav-1 knockdown-induced cellular senescence.

Human

L

delay aging

28,514,055

Gene

0

1

0

1

0

1

0

1

0

1

0

TBX3

6926

protein coding

MEF

--

Ulnar-mammary syndrome

Prevent

Histological staining

TBX3 is able to immortalize MEF cells, suggesting inhibition of senescence.Cells infected with pFB-Neo-TBX3 have passed 15 passages without senescence.

--

Human

HL

delay aging

15,289,316

Gene

0

1

0

TBX3

6926

protein coding

MEF

--

Ulnar-mammary syndrome

Accelerate

Histological staining

In contrast, in cells harboring TBX3+2a, acceleration of senescence occurs, and growth is slower than in the control (pFB-Neo). Cells infected with pFBNeo and pFB-Neo-TBX3+2a stopped growing at eight and six passages, respectively.

--

Human

HL

delay aging

15,289,316

Gene

0

1

0

TAT

6898

protein coding

MSC

--

AIDS

Accelerate

SA--gal activity assay//Western blot

In control cells, the percentage of senescent cells X-gal stained was 8.6¡À2.2% and 9.3 ¡À 2.2%, after 10 and 20 days of culture, respectively. After 10 days, senescence was increased in cells treated with Tat+Nef, and after 20 days, its level was of 17.1 ¡À 1.6%, 18.0 ¡À 4.1% and 20.2 ¡À 3.7% in Tat-, Nef- and Tat+Nef-treated cells, respectively.

--

NF-¦ÊB

Activation

Western blot

Treatment with Tat but not Nef resulted in an increased nuclear translocation of the activated pro-inflammatory and pro-senescent transcription factor NF-¦ÊB, as shown by the accumulation of the activated phospho-Ser 536 form of p-65 in the nucleus .

Human

L

delay aging

25,847,297

Gene

0

1

0

1

0

nef

156110

protein coding

MSC

--

AIDS

Accelerate

SA--gal activity assay//Western blot

In control cells, the percentage of senescent cells X-gal stained was 8.6¡À2.2% and 9.3 ¡À 2.2%, after 10 and 20 days of culture, respectively. After 10 days, senescence was increased in cells treated with Tat+Nef, and after 20 days, its level was of 17.1 ¡À 1.6%, 18.0 ¡À 4.1% and 20.2 ¡À 3.7% in Tat-, Nef- and Tat+Nef-treated cells, respectively.

Beclin1

Downregulation

Immunostaining//Co-IP

The cellular oxidase activity, was unchanged after 10 days of treatment with the HIV proteins, but increased after 20 days by 1.3- to 1.4-fold by Tat and/or Nef, when compared to control cells along with an increased superoxide dismutase activity (SOD).Moreover, we observed a direct interaction between Nefand Beclin-1, which were co-immunoprecipitated, suggesting that Nef could inhibit autophagy through a direct interaction with Beclin-1.

--

Human

L

delay aging

25,847,297

Gene

0

1

0

1

0

GDF15

9518

protein coding

Aortic endothelial cell

--

Atherosclerosis

Accelerate

SA--gal activity assay

Upregulation of GDF15 caused a decrease in cell proliferation and an increase in SA-¦Â-gal staining compared with the control lentivirus-transduced cells.

ERK

Activation

Western blot//Immunostaining

We noted that ERK phosphorylation was increased following the rhGDF15 protein treatment. In addition, IR-induced ERK activation was controlled by the downregulation of GDF15.ROS generation was increased in GDF15-tranduced cells compared to control virus-transduced cells.

p16-Rb

Upregulation

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

On the contrary, the overexpression of GDF15 had no significant effects on cell proliferation in the p16 knockdown cells . The measurement of SA-¦Â-gal activity indicated that p16 knockdown inhibited GDF15-induced cellular senescence, but p53 knockdown did not.Increased expression of GDF15 induced p16 expression and treatment with GDF15 recombinant protein increased p16 mRNA by approximately 2.5 fold. Both endogenous and exogenous GDF15 protein increased p16 protein and decreased the phosphorylation of Rb, which causes its detachment from E2F transcription factor.

Human

HL

delay aging

26,909,594

Gene

0

1

0

PAK4

10298

protein coding

Hs 578T

--

Breast cancer

Prevent

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

After transient transfection of two independent small interfering RNAs (siRNAs) targeting the human PAK4 gene, Hs 578T breast cancer cells adopted a flatter and larger senescenceassociated morphology and exhibited elevated SA-¦Â-gal activity (as measured with the two substrates X-Gal29and MUG30) that was accompanied by a significant decrease in BrdU-incorporation.Genes involved in cell cycle arrest, DNA damage/ repair, and SASP factors are typically upregulated in senescent cells. PAK4 knockdown also increased protein expression of the known senescence-regulators p53 and p21.

RELB

Downregulation

Western blot

This inverse correlation was also observed at the protein level in Hs 578T breast cancer cells where PAK4 knockdown upregulated RELB. Considering expression as continuous vari- ables, the expression of PAK4 and RELB displayed the strongest significant inverse association .

NF-¦ÊB

Downregulation

qRT-PCR

PAK4 inhibits NF-¦ÊB signaling.Upregulation of several NF-¦ÊB target genes upon PAK4 knockdown was validated by RT-qPCR in Hs 578T cells, including the NF-¦ÊB subunits NFKB1, NFKB2, and RELB as well as the previously characterized NF-¦ÊB response genes CD82, S100A4, TIMP2, CDKN1C, PRKCD, TWIST1, SPP1, TP53, and TRAF2.

Human

HL

delay aging

31,399,573

Gene

0

1

0

1

0

1

0

1

0

CCND1

595

protein coding

MCF-7

--

Breast cancer

Prevent

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

Cyclin D1-depleted cells appeared flattened with large volumes of cytoplasm and were positive for SA-¦Â-Gal staining. Cyclin D1-depleted cells also showed a significant increase in senescence-associated heterochromatin foci (SAHF), another putative marker for cellular senescence (Narita et al., 2003).

--

p38-FOXO3a-p27

--

Western blot//Immunostaining//Cell counting

We detected upregulation of phospho-p38 (Thr180/Tyr182) and JNK-mediated c-JUN phosphorylation at Ser73 in cyclin D1- depleted cells around 48-72 h after cyclin D1 depletion, but not in control cells.We detected an upregulation of FOXO3a protein in associatio nwith increased FOXO3a Ser7 phosphorylation, which is involved in the stress-induced activation of FOXO3a by p38 and translocalization of FOXO3a from the cytoplasm to the nucleus after cyclin D1 depletion. We found that p27 protein level was also upregulated in cyclin D1-depleted cells, whereas GADD45a and SOD2 were not upregulated at the protein level .

Human

L

delay aging

29,880,532

Gene

0

1

0

1

0

1

0

1

0

1

0

FBXO46

23403

protein coding

MCF-7

--

Aging

Prevent

Knockdown//SA--gal activity assay

It was observed that depletion of FBXO46 significantly increased the population of senescent cells, which was inhibited upon co-depletion with FBXO31.

FBXO31

Downregulation

Western blot//Co-IP

Immunoblot analysis revealed the presence of FBXO46 in the FBXO31 immunoprecipitates. In a reciprocal co-immunoprecipitation assay, FBXO31 was found to be present in the FBXO46 immunoprecipitates, suggesting that FBXO46 and FBXO31 interact with each other.The results showed that FBXO46 significantly decreased FBXO31 levels in a dose- dependent manner.

--

Human

L

delay aging

30,171,069

Gene

0

1

0

1

0

POT1

25913

protein coding

MRC-5,WI-38,NHF

--

Aging

Prevent

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

The shRNA knockdown of endogenous POT1v1 or POT1v5 induced cellular senescence, characterized by cell growth arrest and SA-¦Â-Gal activity, in normal human fibroblast strains MRC-5, NHF, and WI-38.The significant decrease in bromodeoxyuridine incorporation was associated with sh-v1 induced shsenescence(1.9% ,compared with 43.9% in control cells£©.

--

p53//p16

--//--

Western blot

The p53 dependence of sh-v5¨Cinduced senescence in this experiment was consistent with the Western blot results of cellular senescenceregulatory factors in normal human fibroblasts. Both sh-v1 and sh-v5 led to the increase in Ser15-phosphorylated p53 and the up-regulation of p21WAF1, an effector of p53-mediated cellular senescence.In contrast, only sh-v1 induced the expression of p16INK4A, another major effector for cellular senescence in human cells.

Human

L

delay aging

18,089,797

Gene

0

1

0

1

0

1

0

POT1

25913

protein coding

MRC-5,WI-38,NHF

--

Aging

Prevent

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

The shRNA knockdown of endogenous v1 or v5 induced cellular senescence, characterized by cell growth arrest and SA-¦Â-Gal activity, in normal human fibroblast strains MRC-5, NHF, and WI-38.The significant decrease in bromodeoxyuridine incorporation was associated with sh-v5¨Cinduced senescence (2.0%,compared with 43.9% in control cells£©.

--

p53

--

Western blot

The p53 dependence of sh-v5¨Cinduced senescence in this experiment was consistent with the Western blot results of cellular senescenceregulatory factors in normal human fibroblasts. Both sh-v1 and sh-v5 led to the increase in Ser15-phosphorylated p53 and the up-regulation of p21WAF1, an effector of p53-mediated cellular senescence.

Human

L

delay aging

18,089,797

Gene

0

1

0

1

0

1

0

MYC

4609

protein coding

IMR-90

--

Aging

Accelerate

BrdU assay//Crystal violet assay//GSEA analysis//SA--gal activity assay//qRT-PCR

Expression of the reprogramming factors (OSKM) in IMR90 human fibroblasts causes a senescence-like growth arrest that constitutes an intrinsic barrier to reprogramming (Banito et al. 2009). Similar to oncogenic RASG12V, the expression of OSKM induces the cyclin-dependent kinase (CDK) inhibitors (CDKIs) p15INK4b, p16INK4a, and p21CIP1, which are involved in implementing the stable growth arrest associated with senescence. Gene set enrichment analysis (GSEA) found signatures for senescence and the SASP significantly enriched in the transcriptome of cells expressing OSKM.

CDKN1A//MYOT//mTOR//UBE2E1

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

Crystal violet staining//SA-¦Â-gal activity assay//BrdU assay

The ability of shRNAs targeting CDKN1A, MYOT, MTOR, and UBE2E1 to prevent OSKM-induced senescence was confirmed by increased proliferation, a higher percentage of cells incorporating BrdU, and a decrease in the percentage of senescence-associated ¦Âgalactosidase (SA-¦Â-Gal)-positive cells when compared with IMR90 cells infected with OSKM and a control vector .

TGF¦Â

--

BrdU assay//Follow-up analysis

Moreover, inhibition of TGFBRI signaling blunted the growth arrest triggered by OSKM. In this regard, the scRNA-seq data and the follow-up analysis highlighted that TGF-¦Â signaling was induced by OSKM .

Human

HL

delay aging

29,138,277

Gene

0

1

0

1

0

1

0

1

0

1

0

BRD4

23476

protein coding

MKN28

--

Gastric cancer

Prevent

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

Depletion of Brd4, but not Brd2 and Brd3, increased the number of SA-¦Â-Gal-positive cells.In line with increased SA-¦Â-Gal activity, the levels of p21 were enhanced in Brd4 knockdown cells.

--

E2F-miR-106b-p21

--

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

Depletion of Brd4 or treatment of MKN28 cells with JQ1 up-regulated p21 mRNA levels with 2 3 folds induction in Brd4 knockdown cells and less than 2 folds induction in JQ1-treated cells.Consistently, depletion of Brd4 also increased the activity of 3 -UTR of p21 reporter. In contrast, overexpression of BRD4 in MKN28 cells decreased the luciferase activity of p21 3 -UTR luci- ferase reporter. When miR- 106b-5p and miR-519d-3p mimics were transfected into MKN28 cells followed by JQ1 treatment, miR-106b-5p but not miR-519d-3p mimics reduced the JQ1-induced cellular levels of p21, indicating that miR-106b targets p21 mRNA in MKN28 cells. Treatment of MKN28 cells with HLM006474 efficiently inhibited the binding of BRD4 to the promoter of miR-106b-5p,suggesting that E2F regulates the recruitment of BRD4 to the promoter of miR-106b-5p. Importantly, HLM006474 also down-regulated the expression of MCM7 and the expression of miR-106b-5p.

Human

L

delay aging

29,434,197

Gene

0

1

0

1

0

1

0

EZH2

2146

protein coding

SGC-7901

--

Gastric cancer

Prevent

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

EZH2 depletion cells expressed SA-¦Â-gal and flow cytometric analysis demonstrated that the proportion of cells in the G2/M phase increased.We observed a significant inhibition in cellular proliferation in cells infected with lentivirus or EGCG compared with control treated.

p21//p16

--//--

qRT-PCR//CHIP

Depletion of EZH2 elevated the expression of p21 and p16 in SGC-7901 cells. The effect of loss of EZH2 upon induction of p21 and p16 expression was transcriptional, since the transcript of CDKN1A and CDKN2A, which encodes p21 and p16, respectively, was upregulated in SGC-7901 that exhibited increases in p21 and p16 protein levels.

--

Human

L

delay aging

24,588,771

Gene

0

1

0

1

0

1

0

TNF

7124

protein coding

ISO-HAS

--

Aging

Accelerate

RT-PCR//SA--gal activity assay

Treatment with 10 ng/mL TNF-¦Á for 48 h increased the proportion of SA-¦Â-gal-positive cells in the endothelial cell culture.Gene expression of SHC1 and GLB1 increased at TNF-¦Á concentrations above 1 ng/mL,and the significance of this effect was reached for all three genes at 10 ng/mL.

p21

Upregulation

Western blot

We used Western blotting to examine the protein expression levels of p21 in endothelial cells following 10 ng/mL TNF-¦Á treatment for 8 h. TNF-¦Á exposure induced the expression of p21 in ISO-HAS cells.

--

Human

L

delay aging

26,802,937

Gene

0

1

0

1

0

PTEN

5728

protein coding

MEF

--

Prostate cancer

Prevent

SA--gal activity assay//Western blot

Pretreatment of Ptenlx/lx MEFs with aphidicolin,followed by acute inactivation of Pten,resulted in increased ¦Â-gal senescence staining and p53 accumulation after 24 hours.

--

PI3K-mTOR

--

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

Surprisingly,while?this?procedure?resulted?in?efficient?Pten?loss,?senescence?and?p53?levels?were?completely?abolished?by?rapamycin.?Wealso?tested?genetically?whether?mTOR?is?also?essential?for?PICS,? taking?advantage?of?Pten¨C/¨CmTOR¨C/¨C?double-null?MEFs,and?found this to?be?true.

Human

L

delay aging

20,197,621

Gene

0

1

0

1

0

E2F7

144455

protein coding

IMR-90

--

Aging

Prevent

BrdU assay//Cell counting//Colony formation assay

Still, in marked contrast, cosuppression of E2F7 together with RB was sufficient to bypass senescence, as measured by various proliferation assays such as BrdU incorporation, colony formation, and cell counting.

--

p53

Binding

CHIP-Seq

Interestingly, E2F7 has a p53-binding site in its promoter, and indeed, analysis of chromatin immunoprecipitation (ChIP)/next-generation sequencing (ChIP-seq) data from IMR90 cells under different growth conditions revealed a marked and specific binding of p53 to this site in senescence but not in growing conditions .p53 binds to the E2F7 promoter specifically during cellular senescence.

Human

HL

delay aging

22,802,529

Gene

0

1

0

1

0

1

0

TGFB2

7042

protein coding

TM

Eye

Primary open-angle glaucoma

Accelerate

SA--gal activity assay//qRT-PCR

Exposure to TGF-¦Â2 for 24 and 48 hours markedly increased the proportion of SA-¦Â-Gal¨Cpositive TM cells to 31.6% ¡À 7.7% and 33.7% ¡À 5.4% of total cells.Treatment with TGF-¦Â2 for 12, 24, and 48 hours increased three mRNA expressions of Apo J,SPARC,SM22.

--

p16-Rb

Activation

qRT-PCR//Western blot

Treatment with TGF-¦Â2 for 12 and 24 hours markedly increased the p16 mRNA expression by 1.7 ¡À 0.2- and 3.2 ¡À 0.2-fold, whereas exposure of cells to TGF-¦Â2 for 48 hours did not influence p16 mRNA expression.Treatment with TGF-¦Â2 for 24 hours markedly led to a downregulation of pRb to 42.1% ¡À 5.0% of the level of untreated control cells.

Human

L

delay aging

20,554,622

Gene

0

1

0

1

0

LMNA

4000

protein coding

Fibroblast

--

Lipodystrophic syndromes

Accelerate

Cell morphological analysis//SA--gal activity assay

In contrast, SA-¦Â-galactosidase activity, assessed at pH 6, was absent in control cells up to passage 14, but present, even at early passages, in fibroblasts with LMNA mutations or treated with PIs.

p16//p21

--//--

Western blot//Immunostaining

P16INK4aand p21WAF1,two cell cycle checkpoint inhibitors that participate in the setup of the senescence program,were overexpressed by 250¨C400% in fibroblasts bearing LMNA mutations at passages 9¨C15.Dichloro- fluorescein oxidation was increased two- to fivefold in fibroblasts with LMNA mutations at passages 6 8, as compared to control cells.Microscopic examination confirmed the increased production of ROS in fibroblasts with LMNA muta-tions,as compared to control fibroblasts.

--

Human

L

delay aging

17,612,587

Gene

0

1

0

1

0

NPM1

4869

protein coding

MIP101,RKO,HCT116

--

Colorectal cancer

Prevent

Knockdown//SA--gal activity assay

Based on SA-¦Â-gal staining, there were more senescent cells among all CRC cell lines transfected with NPM1 siRNA compared to those transfected with scramble siRNA. By MUG assay, a 20%, 35% and 45% increase in cellular senescence were observed in MIP101, RKO and HCT116 cells following NPM1 siRNA knockdown, respectively.

p53

--

Knockdown//Immunostaining//Western blot

Interestingly, remarkable increases in the expression of p53 were noted after NPM1 gene silencing in all three CRC cell lines by immunofluorescence staining and confirmed in MIP101 cells by immunoblotting.

--

Human

L

delay aging

23,536,448

Gene

0

1

0

1

0

ATRX

546

protein coding

WD/DDLS

--

Aging

Accelerate

Crystal violet assay//SA--gal activity assay//SAHF//qPCR

Reducing ATRX in these cells affect the accumulation of SA-¦Â-gal-positive cells, SAHF-positive cells, the accumulation of three of the four mRNAs (CXCL1, GM-CSF, IL-6, and IL-8) that increase as part of the SASP in LS8817 cells, and the ability of the cells to return to the cell cycle following drug removal and replating.

HRAS

--

qRT-PCR//CHIP-seq

ATRX binding was strongly enriched at the HRAS locus in senescent LS8817 but not quiescent LS8107 cells.Consistent with the importance of ATRX for repression, HRAS expression increased when ATRX was knocked down in cells that were already senescent.

--

Human

HL

delay aging

28,855,512

Gene

0

1

0

1

0

1

0

1

0

SIAH1

6477

protein coding

MRC-5

--

Aging

Accelerate

Knockdown

The Siah1 knockdown cells had increased proliferation rate in the early phase of the experiment and had an extended replicative lifespan, which was approximately 2 or 3 PDL longer than control cells.

p53//TRF2

Upregulation//Downregulation

Western blot//qRT-PCR

In these experiments, the expression of Siah1 protein was inversely correlated with the expression of TRF2 protein; Siah1 was decreased when TRF2 was increased,and Siah1 was increased when TRF2 was decreased.The overexpression of a stabilized form of Siah-1 resulted in the downregulation of TRF2.Decreased SIAH1 mRNA levels were also confirmed in cells with p53 knockdown.The overexpression of wild-type p53 led to increased Siah-1 and decreased TRF2.

--

Human

L

delay aging

21,057,505

Gene

0

1

0

MYC

4609

protein coding

HFF

--

Aging

Accelerate

SA--gal activity assay

Furthermore, while c-Myc overexpression facilitated cellular senescence, which was detected by ¦Âgalactosidase staining, this phenomenon was subdued following the depletion of USP10.

p14//p53//USP10

--//--//Upregulation

Western blot//qRT-PCR

Interestingly, p14ARF protein stability was increased by c-Myc overexpression. Accordingly, c-Myc also increased the protein stability of p53, a downstream target of p14ARF, through an indirect or a direct pathway.Corroborating these findings, cMyc overexpression increased USP10 protein levels as well as its mRNA in HFF and IMR90 cells.

--

Human

HL

delay aging

29,472,714

Gene

0

1

0

USP10

9100

protein coding

IMR-90,HFF

--

Aging

Accelerate

SA--gal activity assay//Western blot

USP10 overexpression stabilized p14ARF protein levels in both cell lines.In line with these observations, these cells exhibited accelerated cellular senescence with decreased cellular growth. These phenomena were prevented by p14ARF ablation under USP10 overexpression. Overall, these results indicated that USP10 could accelerate cellular senescence through p14ARF stabilization.

p14

--

Western blot

USP10 overexpression stabilized p14ARF protein levels in both cell lines.

--

Human

HL

delay aging

29,472,714

Gene

0

1

0

1

0

SUSD2

56241

protein coding

Ishikawa

--

Endometrial cancer

Prevent

Knockdown//SA--gal activity assay

SiRNA-SUSD2 significantly increased blue staining indicative of SA-¦Â-Gal activity and thus senescent cells.

TGF¦Â//SMAD2/3//LGALS1

Downregulation//--//--

Western blot//Knockdown

At 72h TGF¦Â significantly decreased the number of SUSD2+ cells. The significant decrease of SUSD2 at protein level was paralleled by a similar decline of SUSD2 mRNA level.To our surprise, levels of SMAD2/3 were significantly increased upon SUSD2 knockdown. We next examined the expression of MKi67 and LGALS1 gene (the interaction partner for SUSD2).As a result, LGALS1 was significantly reduced upon SUSD2 knockdown.Moreover, MKi67 encoding the proliferation marker Ki-67 antigen, was significantly attenuated upon SUSD2 knockdown.

--

Human

L

delay aging

28,841,682

Gene

0

1

0

1

0

SPI1

6688

protein coding

BJ,WI-38

--

Acute leukemia

Accelerate

SA--gal activity assay//Western blot

The ectopic expression of Spi1 or HRASV12 both led to senescence that was characterized by stable cell cycle arrest, increased senescence-associated betagalactosidase (SA-¦Âgal) activity, as measured by cytochemical staining and cytometric analyses, and increased protein levels of the senescence biomarker Dec1.

--

p38 MAPK

Activation

GFP localization assay

The treatment of Spi1 overexpressing cells with SB203580 also increased, although partially, the number of GFP-positive cells, indicating that p38MAPK14 controls senescence and additional mechanisms modulating cell number.

Human

L

delay aging

28,912,174

Gene

0

1

0

1

0

IL4

3565

protein coding

CAKI-1,A498,786-O

--

Aging

Accelerate

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

Indeed, after exposure to IL-4 for 3¨C7 days, the cells underwent a profound morphological change characteristic of cellular senescence such as flattening and enlargement . Flow cytometric analysis showed that Caki-1 cells exposed to IL-4 for 7 days increased in size and granularity.Up-regulation of SA-¦Â-gal activity increased protein expression of p21WAF1/CIP1 and p16INK4A and formation of senescenceassociated heterochromatin foci were also shown.

STAT6//p38MAPK

Activation//Activation

Western blot

STAT6 phosphorylation was predominant by 10 min after IL-4 treatment.p38 MAPK as well as its upstream kinases, MEK3/6, were phosphorylated following IL-4 treatment.

--

Human

HL

delay aging

23,935,100

Gene

0

1

0

1

0

1

0

1

0

1

0

MAD2L1

4085

protein coding

IMR-90,MCF 10A

--

Aging

Prevent

Immunostaining//SA--gal activity assay//SAHF

Strikingly, several IMR90- siMAD2 cells were (SA)-¦Â gal-positive (pH 6, 58%). The percentage of positive cells forgH2AX was higher (about 60%) in MCF10A-siMAD2 cells than in MCF10A-siGFP control cells .We then looked for SAHFs presence in primary human fibroblasts after 30 days from siMAD2 transfection and we scored that almost all of the cells (about 85%) showed heterochromatin foci when stained with DAPI.

p14

--

Western blot//qRT-PCR

At 72 h 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, suggesting that p14ARF could mediate the senescence response in these cells by sensing aneuploidy.

p53-p21

--

Western blot//qRT-PCR

Real-time RT-PCR revealed a high increase (sevenfold) of p21waf1 mRNA in primary human fibroblasts with MAD2 haploinsufficiency when compared to IMR90-siGFP control cells . As expected Western blotting confirmed that p21waf1 protein was significantly higher in siMAD2 than in IMR90-siGFP transfected cells. However, by Western blot analysis we detected elevated levels of p53 protein suggesting that the observed p21waf1 accumulation could be induced by p53 trans-activation of the p21waf1 gene. When p53 was posttranscriptionally 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

delay aging

22,170,163

Gene

0

1

0

1

0

1

0