Whereas mechanisms driving senescence have been extensively studied, the mechanisms allowing their prolonged retention in tissues are much less well characterized. Recently, the anti‐apoptotic BCL‐2 family members BCL‐W, BCL‐XL, and BCL‐2 were shown to facilitate the resistance of senescent cells to apoptosis (Chang et al , 2016 ; Yosef et al , 2016 ). However, the contribution of pathways that regulate the formation of senescent cells to the resistance of these cells to cell death has yet to be determined. On one hand, senescent cells cannot accumulate p53 protein to the levels required for apoptosis (Seluanov et al , 2001 ). On the other hand, the p53 target p21, via its ability to promote cell cycle inhibition, can protect some cells from apoptosis (Abbas & Dutta, 2009 ). This effect might be governed by both p53‐dependent and ‐independent upregulation of the pro‐apoptotic protein BAX, or by activation of members of the tumor necrosis factor (TNF)‐α family of death receptors, or by effects on DNA repair (Abbas & Dutta, 2009 ). We therefore set out to determine how p21 regulates the viability of senescent cells after DNA damage. We found that following p21 knockdown, senescent cells sustain multiple DNA lesions, leading to further activation of DNA damage response and NF‐κB pathways. This activation was regulated by both TNF‐α secretion and JNK activation, and it mediated senescent cell death in a caspase‐dependent and JNK‐dependent manner. Moreover, p21 knockout in mice led to the elimination of senescent cells from fibrotic scars in the liver and alleviated liver fibrosis. These results uncovered new mechanisms that control the fate of senescent cells.

The senescence program is driven by a complex interplay of signaling pathways. To promote and support cell cycle arrest, p16 INK4A (CDKN2A), accompanied by the p53 (TP53) target p21 (CDKN1A), inhibits cyclin‐dependent kinases (CDKs), thereby preventing phosphorylation of the retinoblastoma protein (pRb) and thus in turn suppressing the expression of proliferation‐associated genes (Narita et al , 2003 , 2006 ; Collado et al , 2007 ). In addition, the nuclear factor kappa B protein complex (NF‐κB) acts as a master regulator of SASP and therefore affects both the microenvironment of senescent cells and their immune surveillance (Acosta et al , 2008 ; Krizhanovsky et al , 2008 ; Xue et al , 2011 ; Lasorella et al , 2014 ). Clearance of senescent cells by the immune system is an essential requirement for limiting their prolonged retention in tissues, a trait that might derive from their intrinsic resistance to apoptosis (Yosef et al , 2016 ).

Cellular senescence indeed plays an important role in tissue homeostasis after short‐term damage, facilitating tissue repair after liver damage, in skin fibrosis, and during wound healing (Krizhanovsky et al , 2008 ; Jun & Lau, 2010 ; Demaria et al , 2014 ). Upon liver damage, for example, hepatic stellate cells (HSCs) differentiate into activated myofibroblasts that extensively proliferate and secrete extracellular matrix (ECM). Accumulation of ECM leads to fibrosis, which aims to maintain tissue integrity while the healing process is taking place. These activated HSCs subsequently undergo senescence, accompanied by a decline in ECM production and an increase in SASP factors that facilitate elimination of these cells by natural killer (NK) cells (Krizhanovsky et al , 2008 ). The matricellular protein CCN1, which is secreted from damaged hepatocytes, can trigger senescence in activated HSCs (Kim et al , 2013 ). Inactivation of the senescence specifically in HSCs results in enhanced tissue fibrosis, indicating that senescence limits fibrosis (Krizhanovsky et al , 2008 ). However, inefficient elimination of senescent cells and their excessive accumulation are associated with increased fibrosis (Krizhanovsky et al , 2008 ; Sagiv et al , 2013 , 2016 ).

Cellular senescence, a permanent form of cell cycle arrest, halts cell proliferation in response to various stressors, including telomere shortening, oncogene activation, and DNA damage (Kuilman et al , 2010 ; Campisi, 2013 ; Munoz‐Espin & Serrano, 2014 ; Salama et al , 2014 ). Senescent cells present elevated activity of senescence‐associated beta‐galactosidase (SA‐β‐gal) and a persistent DNA damage response that distinguish them from other non‐proliferating cell populations (Campisi & d'Adda di Fagagna, 2007 ; Collado et al , 2007 ; d'Adda di Fagagna, 2008 ). In addition, senescent cells produce a variety of characteristic secreted factors, collectively termed the senescence‐associated secretory phenotype (SASP) (Coppe et al , 2008 ), which reinforces senescence arrest in an autocrine manner (Acosta et al , 2008 , 2013 ; Kuilman et al , 2008 ; Wajapeyee et al , 2008 ) and mediates immune surveillance of the senescent cells (Sagiv & Krizhanovsky, 2013 ; Sagiv et al , 2016 ). With aging, however, senescent cells accumulate in the organism promoting local inflammation that drives tissue aging, tissue destruction, and potentially also tumorigenesis and metastasis in a cell non‐autonomous manner (Krtolica et al , 2001 ; Campisi, 2013 ; Munoz‐Espin & Serrano, 2014 ; Ovadya & Krizhanovsky, 2014 ; Salama et al , 2014 ). Recent studies have shown that elimination of senescent cells promotes stem cell proliferation and prolongs lifespan (Baker et al , 2011 , 2016 ; Chang et al , 2016 ; Yosef et al , 2016 ). Therefore, mechanisms that regulate the viability of senescent cells in tissues evidently play an important role in tissue homeostasis.

Results

p21 maintains the viability of DNA damage‐induced senescent cells p21 contributes to the induction of cellular senescence owing to its activity as an inhibitor of CDK2 and CDK4 (Campisi & d'Adda di Fagagna, 2007). To examine the contribution of p21 to the viability of senescent cells, we induced senescence in normal human lung fibroblasts (IMR‐90) by treating them with the DNA‐damaging agent etoposide (“DNA damage‐induced senescence”, DIS). We confirmed the induction of the senescence phenotype in these cells by evaluation of cell cycle arrest, expression of senescence markers p16, p21, and p53 and senescence‐associated β‐galactosidase (SA‐β‐gal) staining (Appendix Fig S1A–E). Oncogene‐induced senescence (OIS) was induced by expression of oncogenic H‐RasV12. Proliferating (growing) cells and empty vector‐transduced cells served as controls for DIS and OIS cells, respectively. Senescent and control IMR‐90 cells were treated with siRNA mixes targeting p21 (sip21) or control siRNAs (siCtrl). The efficiency of the knockdown was confirmed by Western blot analysis (Fig 1A). Surprisingly, knockdown of p21 induced death in 30% of the DIS cells only (Fig 1A). The viability of control or of OIS cells was not affected by p21 knockdown. To determine whether this unexpected observation was limited to a specific cell type, we performed p21 knockdown in mouse embryonic fibroblasts (MEFs) and in normal human skin fibroblasts (BJ cells). Growing (G) and DIS MEFs or BJ cells were treated with siRNA mixes targeting p21, and their viabilities were evaluated. Knockdown of p21 reduced the viability of DIS MEFs by 26% relative to siCtrl cells (Fig 1B). p21 knockdown decreased the viability DIS BJ cells even more strongly, leading to a 54% reduction in sip21 relative to siCtrl‐treated DIS cells (Fig 1C). Similarly, p21 knockdown decreases the viability of DIS cells 3 weeks following the induction of DIS phenotype (Appendix Fig S1F). Thus, these results indicate that p21 is necessary to maintain the viability of DIS cells, regardless of the time passed from the induction of senescence. Figure 1.p21 maintains the viability of DNA damage‐induced senescent cells Normal human IMR‐90 fibroblasts were induced to senesce by DNA damage (DNA damage‐induced senescent (DIS)) or by expression of HrasV12 (oncogene‐induced senescent (OIS)). Proliferating IMR‐90 (growing, G) and empty vector‐transfected (V) cells served as respective controls. The cells were transduced with siRNAs targeting p21 (sip21) or control siRNAs (siCtrl). Histograms indicate the percentages of surviving senescent cells compared with G or V controls. Western blots show p21 protein levels in the corresponding samples. Mouse embryonic fibroblasts (MEF; G and DIS) were transduced with siRNAs targeting p21 or control siRNA, and cell survival was evaluated. Western blots show p21 protein levels in the corresponding samples. BJ human fibroblasts (DIS and G cells) were transduced with siRNAs targeting p21 or control siRNA, and cell survival was determined 4 days following the transduction. Western blots show p21 protein levels in the corresponding samples. Mouse embryonic fibroblasts (MEF) from wild‐type (WT) or p21 knockout mice (p21−/−) were treated with etoposide to induce DNA damage‐induced senescence. Survival of DIS cells was determined by quantification of the remaining adherent cells 5 days following etoposide washout. BJ DIS fibroblasts were transduced with siRNAs targeting p21 and cell survival was determined at the indicated time points. Data information: Data are presented as mean ± SEM of three repeats, performed in triplicates. Data were analyzed using Student's t‐test. *P < 0.05, ***P < 0.0005. Data information: Data are presented as mean ± SEM of three repeats, performed in triplicates. Data were analyzed using Student's‐test. *0.05, ***< 0.0005. To find out whether the induction of cell death is dependent on the time of p21 knockdown, we analyzed cells that were already lacking p21 prior to introduction of the DNA‐damaging agent. To this end, we induced senescence in wild‐type (WT) and p21 knockout (p21−/−) MEFs. After exposure to DNA‐damaging agent leading to senescence induction, the viability of p21−/− MEFs was decreased by 60% relative to WT MEFs (Fig 1D). Therefore, p21 supports viability of cells regardless the timing of the knockdown. Cancer cells can acquire senescence‐like phenotypes in response to DNA damage (Appendix Fig S2A and B). To impose this phenotype, we transduced H1299 cells with small hairpin RNA (shRNA) targeting p21 (shp21) or control shRNA targeting Luciferase (shLuci) and then treated the cells with etoposide to induce DNA damage. Treatment with etoposide induced cell cycle arrest in these cells (Appendix Fig S2C). Knockdown of p21 in this setting caused a 75% reduction in the viability of etoposide‐treated cells relative to shLuci cells (Appendix Fig S2D). Thus, the effect of p21 knockdown on the viability of cells after damage to their DNA is not limited to normal fibroblasts. To determine the time at which cell death occurs after p21 knockdown, we monitored cell viability over time course following knockdown. Importantly, p21 knockdown was followed by continuous reduction in DIS BJ cell viability relative to control cells over time (Fig 1E). These results suggest that the effect of p21 knockdown on DIS cell viability is cumulative.

Molecular pathways activated after p21 knockdown in DIS cells To identify the molecular mechanism controlling DIS cell viability, we studied the expression patterns of DIS and control cells with and without p21 knockdown. Growing and DIS BJ cells were transfected with siRNAs against p21 or with control siRNAs. After 3 days, total RNA was extracted and gene expression was determined using Affymetrix microarrays. K‐means clustering (Fig 2A) and principal component analysis (PCA; Fig 2B) were used to visualize the overall response to p21 knockdown. A massive change in gene‐expression profile was detected after p21 knockdown in DIS cells but not in the growing control cells. Overall, the signal intensity of 1,595 unique genes changed significantly in response to p21 knockdown in DIS cells compared to only 82 in growing cells (Fig 2C). Therefore, p21 knockdown in DIS cells induces widespread albeit specific changes in gene expression. Figure 2.Gene‐expression profiles of growing and senescent BJ cells after p21 knockdown Results are presented as K‐means clustering of the microarray data. Probe sets whose abundance was above the mean are shown in red, those below the mean in blue, and those equivalent to the mean in green. Principal component analysis (PCA) scatterplot. Points are colored according to cell type (G, red; DIS, blue). Squares and triangles are drawn for sip21 and siCtrl siRNA groups, respectively. Venn diagram showing the distribution of shared genes among G and DIS cells after p21 knockdown. Enrichment analysis from the WikiPathways database identified pathways affected in 1,545 genes that were uniquely changed in DIS cells after p21 knockdown. K‐means clustering of the 1,545 genes that were uniquely changed in DIS BJ cells after p21 knockdown. Probe sets whose abundance was above the mean are shown in red, and those below the mean in blue. mRNA expression levels relative to controls of p21, COL1A1, CDK‐1, cyclin A2, Smad‐7, andTNF‐α genes after transduction with sip21 or control siRNA in G and DIS BJ cells. Data are presented as means ± SEM of three repeats, each performed in triplicate. Data were analyzed using Student's t‐test. *P < 0.05, **P < 0.005, ***P < 0.0005. BJ human fibroblasts (proliferating, G; and DNA damage‐induced senescent, DIS) were transduced with either siRNA targeting p21 (sip21) or control siRNA (siCtrl). Cells were harvested and analyzed by Affymetrix PrimeView microarrays (3 replicates). Cluster and principal component analyses describe overall changes in gene expression. To further identify the perturbed biological networks and understand the biological processes and signaling networks invoked when p21 is silenced, we analyzed the data by enrichment analysis for pathways in the WikiPathways database (Kutmon et al, 2015) and by Ingenuity Pathway Analysis (IPA®, QIAGEN Redwood City, www.qiagen.com/ingenuity). WikiPathways analysis combined with K‐means clustering indicated that the cell cycle regulators, transforming growth factor‐β (TGF‐β), the DNA damage response, and focal adhesion signaling pathways were altered in response to p21 knockdown in DIS cells (Fig 2D and E). Lists of the genes altered in each pathway, as well as pathway schemes, can be found in the Appendix (Appendix Figs S3–S6; Appendix Tables S1–S4). These findings were further supported by Ingenuity® Pathway Analysis (data not shown), which also predicted that p21 silencing would affect liver fibrosis via modulation of early signaling pathways in HSCs, mainly by modulation of collagen production and TGF‐β signaling (Appendix Fig S7). To verify the microarray results, we performed RT–PCR analysis on specific genes in the pathways identified by the data analyses. Knockdown of p21 led to reduced expression of collagen, type I, alpha 1 (COL1A1) and increased the mRNA levels of the cell cycle regulators CDK‐1 and cyclin A2 (Fig 2F). In addition, p21 knockdown caused a significant increase (30‐fold) in the mRNA levels of TNF‐α accompanied by an increase in Smad‐7 mRNA (Fig 2F), both known to negatively regulate TGF‐β signaling. Overall, it appears that p21‐knockdown DIS cells exhibit alterations in the cell cycle, TGF‐β, DNA damage response, and focal adhesion signaling pathways.

p21 regulates the DNA damage response in senescent cells We then set out to understand the mechanism of cell death induction in senescent cells after p21 knockdown. We hypothesized that an increase in the level of the DNA damage response (DDR) after the knockdown accounts for this effect. We therefore assessed the phosphorylation levels of proteins involved in DNA damage response: ATM, CHK2, gamma H2A histone family member X (γH2AX), and p53. Immunoblotting revealed a substantial increase in phosphorylation of all these proteins in p21‐knockdown DIS cells (Fig 3A). The increase was accompanied by an increase in the number of foci of phosphorylated ATM (p‐ATM) (Fig 3B and C). To further assess the activation of the DDR in p21‐knockdown DIS cells, we evaluated additional DDR markers, namely foci of the ATM target substrates p53‐binding protein 1 (p53BP1) and γH2AX. The amounts of these foci were increased in the p21‐knockdown DIS cells relative to control cells (Fig 3D–F). In order to assess the accumulation of DDR foci by an additional method, we stained growing and DIS cells following p21 knockdown and the control cells with γH2AX antibody and analyzed the cells in flow by ImageStreamX. The distribution of the cells according to the number of γH2AX foci and quantification of cells with more than two foci are presented in Fig 3G. As expected, this analysis revealed an increased in amount of DDR foci in senescent cells comparing to growing cells. p21 knockdown caused an additional significant increase in the amount of cells with more than two foci (Fig 3G, Appendix Fig S8). Next, we performed a comet assay analysis (single‐cell gel electrophoresis) to measure DNA strand breaks at the level of single cells (Fig 3H). Strong increase in DNA damage following p21 silencing in DIS cells was detected, as can be seen from the representative pictures of the DNA tails and from the calculation of Olive tail moment (OTM). Therefore, p21 knockdown leads to an intensified DDR in DIS cells. Figure 3.p21 regulates the DNA damage response in senescent cells BJ human fibroblasts (proliferating, G; and DNA damage‐induced senescent, DIS) were transduced with either siRNA targeting p21 (sip21) or control siRNA (siCtrl). Representative immunoblots for p‐ATM, p‐CHK2, γH2AX, p53, and p‐p53(Ser 15) of at least two independent experiments are shown. Immunofluorescence analysis of p‐ATM in G and DIS cells transduced with sip21 or siCtrl; representative images of at least two independent experiments are shown. Arrows indicate positive staining. Quantification of the total numbers of p‐ATM foci per cell in (B). Immunofluorescence analysis of γH2AX and p53BP1 in G and DIS BJ cells transduced with sip21 or siCtrl; representative images of at least two independent experiments are shown. Quantification of total γH2AX foci per cell. Quantification of total p53BP1 foci per cell. ImageStreamX analysis. Distribution of numbers of γH2AX foci in G and DIS cells transduced with sip21 or siCtrl 3 days after siRNA washout. DIS cells stained with the secondary antibodies only and DAPI served as a negative control. Comet assay. G and DIS BJ cells were transduced with sip21 or siCtrl. 72 h post‐siRNA washout cells were tested for DNA damage using the comet assay. Olive tail moment values were calculated using ImageJ. Each specimen was evaluated in triplicate. At least 50 cells were analyzed per replica and the quantification is shown on the right panel. Representative DNA tails are shown on the left. Immunofluorescence analysis of BrdU incorporation in G and DIS cells transduced with sip21 or siCtrl, 24 h post‐siRNA washout; representative images of at least two independent experiments are shown. Quantification of BrdU‐positive G and DIS cells. DAPI staining of DIS cells transduced with sip21 or siCtrl 4 days after siRNA washout. SubG1 cell cycle analysis of DIS cells transduced with sip21 or siCtrl 3 days after siRNA washout; representative histograms of three independent experiment are shown. Data information: Data were analyzed using Student's t‐test. *P < 0.05. **P < 0.005, ***P < 0.0005. Data represent mean ± SEM (n = 3). Data information: Data were analyzed using Student's‐test. *0.05. **0.005, ***< 0.0005. Data represent mean ± SEM (= 3). Source data are available online for this figure. Source Data for Figure 3 [embj201695553-sup-0002-SDataFig3.pdf] We considered the possibility that the observed induction of DDR is a result of the attempt of the p21‐knockdown cells to re‐enter the cell cycle. To examine this hypothesis, we assessed DNA synthesis in p21‐knockdown DIS cells by using 5‐bromo‐2’‐deoxyuridine (BrdU) incorporation. Positive BrdU staining was detected in p21‐knockdown DIS cells but not in control DIS cells 24 h following the introduction of sip21 or siCtrl (Fig 3I and J). It therefore appears that ablation of p21 induces resumption of DNA synthesis. Four days after p21 knockdown, DIS cells at metaphase indeed displayed extremely fragmented chromosomes and striking micro‐nucleation that was not detected in siCtrl cells (Fig 3K). The cell cycle analysis of DIS cells following p21 knockdown revealed a significant increase in the sub‐G1 fraction in sip21 transduced cells comparing to siCtrl transduced cells (Fig 3L). The cell cycle plot also suggests that some of the cells in the sip21 sample appear in S‐phase, in concordance with the increase in BrdU incorporation we observed in these cells. These findings might reflect entry into mitosis with numerous double‐strand breaks upon cell cycle reactivation. Overall, p21 knockdown enhances the DDR in DIS cells, and this occurs at least partially via resumption of DNA synthesis.

Cell death of DIS cells after p21 knockdown is caspase‐ and JNK‐dependent Accumulation of p53 establishes the growth arrest of senescence at least partially by inducing expression of p21 (Campisi & d'Adda di Fagagna, 2007). p21 in turn suppresses the phosphorylation and hence inactivates pRb, a regulator of the E2F family of transcription factors. p53 and E2F1 may therefore participate in apoptosis induction. To understand the role of these pathways in the observed phenotype, we examined whether the death of DIS cells after p21 silencing is mediated by p53 or by pRb/E2F. To test this hypothesis, we knocked down p53 or pRb individually or together with p21. Surprisingly, knockdown of p53 reduced DIS cell viability, but to a lesser extent than the reduction observed after individual knockdown of p21 (Fig 4A). This may result from the observed reduction in p21 protein following p53 knockdown (Fig 4A). The effect of combined knockdown of p21 and p53 on cell death did not differ from that of p21 knockdown alone, as detected from cell survival and cleavage of the apoptosis effectors caspase‐3 and PARP (Fig 4A). It thus seems that p21 knockdown might induce death of senescent cells in the absence of p53. Similarly, the effect of combined knockdown of pRb and p21 on senescent cell viability did not differ from that of p21 knockdown alone (Fig 4B). Surprisingly, when only pRb was knocked down, it did not significantly affect cell viability, but rather caused an increase in p21, possibly suggesting a feedback loop in the regulation of the protein levels of p21. This finding suggests that an increase in E2F activity as a result of pRb knockdown might not by itself be sufficient to induce death of senescent cells. Figure 4.Death of DIS cells after p21 knockdown ensued by TNF‐α secretion Survival of DIS BJ cells transduced with siRNAs targeting p21, p53 or their combinations, as indicated. Data are presented as means ± SEM of 3 repeats, each performed in triplicate. Western blots of p21, p53, cleaved PARP, and cleaved caspase‐3 after siRNA treatment. Survival of DIS BJ cells transduced with siRNAs targeting p21, pRb or their combinations, as indicated. Data are presented as means ± SEM of 3 repeats, each performed in triplicate. Western blots depict p21 and pRb protein levels following siRNA treatment. Western blot analysis of p21, p65, p‐p65, JNK, p‐JNK, cleaved PARP, and cleaved caspase‐3 proteins 4 days after sip21 or siCtrl treatment of G and DIS cells; representative blots of at least two independent experiments are depicted. mRNA expression levels of TNF‐α and IL‐8 in DIS cells at the indicated times after sip21 or siCtrl treatment. Data are presented as means ± SEM of three repeats. Survival of DIS BJ cells transduced with siRNAs targeting p21, p65 or their combinations, as indicated, 4 days after siRNA washout. Western blots depict levels of p21, p65, JNK, p‐JNK, cleaved PARP, and cleaved caspase‐3 following siRNA treatment. Data are presented as means ± SEM of three repeats, each performed in triplicate. mRNA expression levels of TNF‐α relative to control following treatment with siRNAs targeting p21, p65, or their combinations. Data are presented as means ± SEM of three repeats. Data information: Data were analyzed using Student's t‐test. *P < 0.05, **P < 0.005; ***P < 0.0005. n.s, not significant. Data information: Data were analyzed using Student's‐test. *0.05, **< 0.005; ***< 0.0005. n.s, not significant. Source data are available online for this figure. Source Data for Figure 4 [embj201695553-sup-0003-SDataFig4.pdf] Extensive DNA lesions have been reported to trigger cytokine secretion and programmed cell death independently of p53 (Biton & Ashkenazi, 2011; Lanigan et al, 2011). When DNA damage is excessive, ATM drives the activation of NF‐κB to accelerate TNF‐α production and to mediate phosphorylation of receptor‐interacting kinase 1 (RIP1). RIP1 kinase promotes the JNK‐mediated induction of interleukin (IL)‐8 production and the recruitment of the FADD adapter protein to activate caspase‐8 and trigger programmed cell death (Biton & Ashkenazi, 2011; Condiotti et al, 2014). In view of the extensive DDR that we observed after p21 knockdown in DIS cells, we evaluated activation of the above TNF‐α‐driven pathway in these cells. In DIS cell, phosphorylation of JNK was substantially increased (Fig 4C). Knockdown of p21 in these cells induced an increase in phosphorylation of the p65 subunit of NF‐κB (Fig 4C). We also detected pronounced expression of TNF‐α in response to p21 knockdown, and this was followed by the production of IL‐8 (Fig 4D). It therefore appears that the activity of NF‐κB is increased after p21 knockdown in DIS cells. We then examined whether the death of DIS cells after p21 knockdown is NF‐κB dependent and is driven by the TNF‐α and TNFR1 signaling loop. Combined knockdown of p21 and p65 did not rescue the cells from death (Fig 4E). It did, however, reduce cleavage of the apoptosis effectors caspase‐3 and PARP (Fig 4E) and completely blocked TNF‐α production (Fig 4F). Moreover, the death of DIS cells after the combined knockdown of p21 and p65 was now also accompanied by an increase in JNK phosphorylation (Fig 4E). We concluded that although NF‐κB is activated after p21 knockdown in DIS cells and drives TNF‐α secretion to activate the extrinsic apoptotic pathway, its silencing is not sufficient to block the death of DIS cells, probably because of the increase in JNK activity. To further characterize the cell death that follows p21 knockdown, we treated DIS p21‐knockdown cells and control cells with the pan‐caspase inhibitor Q‐VD‐OPh (QVD) or with DMSO as a vehicle control. Treatment with the inhibitor rescued cell viability only partially (17% increase in cell survival with QVD relative to DMSO; Fig 5A), whereas the activity of caspase‐3 was completely blocked, as indicated by its cleavage. Similar results were obtained when caspases were blocked by the pan‐caspase inhibitor z‐VAD‐fmk (z‐VAD; Appendix Fig S9). Given that the pan‐caspase inhibitors are not able to completely rescue DIS cells after p21 knockdown, we reasoned that the knockdown induces cell death via both caspase‐dependent and caspase‐independent mechanisms. Figure 5.Death of DIS cells after p21 knockdown is caspase‐ and JNK‐dependent Survival of DIS BJ cells transduced with sip21 or siCtrl, with or without incubation with pan‐caspase inhibitor QVD. Western blots depict levels of p21, cleaved PARP and cleaved caspase‐3 after the siRNA treatment. Survival of DIS BJ cells transduced with sip21 or siCtrl, with or without incubation with the JNK inhibitor SP600125 (JNKi). Western blots depict levels of p21, JNK, p‐JNK, cleaved PARP and cleaved caspase‐3 after JNKi treatment. Survival of DIS BJ cells transduced with sip21 or siCtrl, with or without co‐incubation with QVD and JNKi. Treatment with the two inhibitors rescues the cells from sip21‐induced decrease in cell viability. Survival of DIS BJ cells transduced with siRNAs targeting p21, JNK1 or their combinations, as indicated. Western blots depict levels of p21, JNK, p‐JNK, cleaved PARP and cleaved caspase‐3 following siRNA treatment. Survival of DIS BJ cells transduced with sip21 or siCtrl, with or without transduction of siJNK1 and incubation QVD. Western blots depict levels of p21, JNK, p‐JNK, cleaved PARP and cleaved caspase‐3 after treatments. Data information: For all the survival plots, data are presented as means ± SEM of 3 repeats, each performed in triplicate. Data were analyzed using Student's t‐test. *P < 0.05. **P < 0.005. n.s, not significant. Data information: For all the survival plots, data are presented as means ± SEM of 3 repeats, each performed in triplicate. Data were analyzed using Student's‐test. *0.05. **0.005. n.s, not significant. Source data are available online for this figure. Source Data for Figure 5 [embj201695553-sup-0004-SDataFig5.pdf] Activation of the JNK pathway might induce cell death that is caspase independent (Lanigan et al, 2011; Condiotti et al, 2014). We considered the possibility that the observed increase in JNK activity induces caspase‐independent death of DIS cells after blockage of apoptosis with QVD. To test this hypothesis we applied the JNK inhibitor SP600125 to DIS and control cells with and without p21 knockdown. While a reduction in JNK phosphorylation level was detected, JNK inhibition by itself had no effect on the viability of DIS cells regardless of p21 knockdown (Fig 5B). The cell death that followed p21 knockdown observed in this experiment was accompanied by cleavage of the apoptosis effectors caspase‐3 and PARP, indicating that it might happen through apoptosis (Fig 5B). Importantly, combined treatment with QVD and JNK inhibitor resulted in complete rescue of the senescent cells from death following p21 knockdown (Fig 5C). To test the involvement of the JNK pathway in the regulation of cell death following p21 knockdown independent of the pharmacological inhibitor SP600125, we knocked down JNK alone or in combination with p21 in senescent cells. Similarly to the results obtained with the JNK inhibitor, knockdown of JNK alone had no effect on the viability of senescent cells (Fig 5D). It also was not able to rescue the death of senescent cells induced by the knockdown of p21 (Fig 5D). However, when JNK knockdown was performed in combination with QVD treatment, it rescued the death of senescent cells induced by the knockdown of p21 and substantially reduced the levels of cleaved PARP and caspase‐3 in the p21‐knockdown senescent cells (Fig 5E). Overall, it appears that p21 knockdown in DIS cells induces cell death in a caspase‐ and JNK‐dependent manner.