Fig 2: ERK5 activation by TrkB-BDNF sustains senescent cell survival through BCL2L2.a Western blot analysis using PhosTag gels to separate phosphorylated (p-TrkB) from unphosphorylated TrkB in WI-38 cells progressing to senescence (left), as well as in cells transfected to express normal (siCtrl) or reduced (siBDNF) BDNF levels (right). Phosphorylation ratios (p-TrkB/TrkB) were calculated (means ± SD) relative to day 0. b WI-38 fibroblasts transfected with siCtrl, siNTRK2, or siBDNF were treated with etoposide (50 µM) and cell viability was measured by direct cell counting at early senescence (day 2) and late senescence (day 8). c Experimental design for RNA-seq analysis to evaluate transcriptomic differences among cell groups; created using BioRender. d Transcriptomic analysis of WI-38 cells in the groups explained in c; GSEA associations (left), and heat map depicting genes related to apoptosis that changed significantly in cells transfected with siNTRK2 or siBDNF (right). e WI-38 cells were processed as in b and the levels of PUMA, BCL2L2, and BCL2L1 mRNAs were quantified by RT-qPCR analysis. f WI-38 cells were treated with IgG or BDNF-blocking antibodies (4 µg/ml each) for 48 h, and the levels of PUMA, BCL2L2, and BCL2L1 mRNAs were quantified by RT-qPCR analysis. g WI-38 cells were processed as explained in b, f and the levels of the proteins shown were assessed by western blot analysis. h WI-38 cells were treated as explained in b and western blot analysis was used to assess the levels of effector proteins downstream of BDNF-TrkB, including p-AKT(S473), p-ERK1/2(T202/Y204), p-p38(T180/Y182), p-JNK(T183/Y185), p-STAT3(Y705), p-PKCa/ß II(T638/641), and p-ERK5(T218/Y220). i RT-qPCR analysis of the levels of BCL2L2, PUMA, and BCL2L1 mRNAs in proliferating (P) or etoposide-induced senescent (S) WI-38 cells treated or not with an ERK5 inhibitor (ERK5i, ERK5-IN-1; 48 h, 1 µM). j Cell viability as measured by direct cell counting of the remaining viable cells in the senescent groups described in i. In a, g, h, ACTB was included as loading control. Data in e, f, i were normalized to ACTB mRNA. Graphs (b, e, f, i, and j), represent the values ± SD from n = 3 experiments; significance (*p < 0.05, **p < 0.01, ***p < 0.001) was determined by using two-tailed Student’s t-test.

Fig 3: SRC-FAK activation influences cell viability by signaling through p38 and p53.(A) Viable IMR-90 cells (direct counts) following 48 hours of treatment with etoposide (50 µM) plus p38i (AL 8697, 10 µM), p53a (Nutlin-3a, 10 µM), p53a + p38i, PP1 (5 µM), or PP2 (5 µM); see also fig. S3B. (B) RT-qPCR analysis of PUMA and BCL2L2 mRNAs (normalized to ACTB mRNA) in cells treated as in (A). (C) Western blot analysis of the proteins shown (and loading control ACTB). (D) Cell viability (direct counting) after 48-hour treatment as indicated; doxycycline-induced SRC(CA) expression was performed as in Fig. 1R simultaneously with the other treatments. (E) RT-qPCR analysis of PUMA and BCL2L2 mRNAs (normalized to ACTB mRNA) in cells treated as in (D). (F) Analysis of proteins preferentially phosphorylated at 50 µM etoposide ranked CAS-SRC-FAK at the top using CORUM database and Enrichr analysis using data from Fig. 1H. (G) Western blot analysis of p-FAK (activating phosphorylation Y576/Y577), FAK, and loading control ACTB. (H and I) Viability (direct counting) of cells transfected with the siRNAs shown and treated with etoposide (H), and Western blot analysis (I) of the proteins shown (p-p38 is phosphorylated at T180/Y182). (J and K) GSEA (J) and heatmap (K) analyses of the levels of mRNAs encoding EMT and integrin proteins (z scores of the log2-normalized expression values, n = 3). (L and M) GSEA of relevant gene sets in the conditions indicated after expressing SRC(DN) (see also fig. S3E) (L) or SRC(CA) (M). (N) Proposed model (created using BioRender). Senescence-causing, low-level DNA damage (lower half) activates SRC, decreasing p53 function and activating p38 (lowering PUMA, elevating BCL2L2). Apoptosis-causing, high-level DNA damage (top half) does not activate SRC and instead activates p53 and represses p38, in turn increasing PUMA and lowering BCL2L2 levels and leading to apoptosis. Values shown in (A), (B), (D), (E), and (H) are means ± SD (n = 3); significance (*P < 0.05, **P < 0.01, ***P < 0.001) was determined by using two-tailed Student’s t test.

Fig 4: BCL-W and BCL-XL maintain the viability of senescent cells.(a) IMR-90 human fibroblasts (DIS, and OIS cells, as well as control G or V cells) were treated for 24 h with the indicated concentrations of Obatoclax or with vehicle (DMSO). Cell survival relative to vehicle-treated cells was determined by quantification of the remaining adherent cells. Histograms indicate the percentages of surviving senescent (DIS and OIS) cells compared with G or V controls. Data are presented as mean±s.e.m of three repeats, performed in triplicates. (b) Percentage survival of the cells described in a following 24 h treatment with the indicated concentrations of the BCL-2 inhibitor ABT-199 and of SH-SY5Y cells, which express high BCL-2 levels and serve as positive control for response to the drug. Data are presented as mean±s.e.m of three repeats, performed in triplicates. (c) Percentage survival of DIS cells transduced with siRNAs targeting BCL-2, BCL-W and BCL-XL or their combinations as indicated. (d) Western blot analysis of BCL-2, BCL-W and BCL-XL following siRNA treatment of DIS cells. (e) Percentage survival of DIS cells transduced with siRNAs targeting BCL-W, BCL-XL or both, with or without treatment with the BCL-2 inhibitor ABT-199 (10 µM). Data are presented as mean±s.e.m of three repeats, performed in triplicates. Data were analysed using Student's t-test. *P<0.05, **P<0.005, ***P<0.0005.

Fig 5: ABT-737 eliminates senescent cells from lung of irradiated mice.(a) Seven days post irradiation, mice were treated with ABT-737 (n=5) or vehicle (n=5) for 2 consecutive days. Lungs were dissected 1 day thereafter, sectioned and the sections were stained for SA-ß-Gal. Scale bar, 10 µm. (b) Quantification of SA-ß-Gal staining in the bronchioles area of vehicle- or ABT-737-treated mice. Data were presented as means±s.e.m. from five mice in each group. (c) One day following vehicle or ABT-737 treatment, lung were dissociated, stained for ?H2AX and analysed by FACS. Percentage of ?H2AX-positive cells was quantified from this analysis. Data were presented as means±s.e.m. from five mice in each group. (d) Western blot analysis of Bcl-w, p53 and p21 in the mice described in a. Two mice represent each condition, each line represents a mouse. (e) Lung sections from unirradiated mice and the mice described in a were stained for Bcl-xL, p53 and cleaved caspase-3, a marker of apoptosis. Scale bar, 10 µm. Data in b,c were analysed using Student's t-test. *P<0.05, ***P<0.0005.