Fig. 3: Increased TrkB levels in senescent cells promotes resistance to apoptosis.

a, b In WI-38 fibroblasts that were proliferating (P) or undergoing senescence (S) (ETIS, OSIS, IRIS, RS as in Fig. 2c), western blot analysis was used to evaluate TrkA, TrkB full-length (FL), and truncated (T), p21, and ACTB (a), and RT-qPCR analysis to measure the levels of p16 and IL6 mRNAs (b). Western blot analysis (c) and RT-qPCR analysis (d) of the same molecules as in a, b but measured in BJ and IMR-90 fibroblasts and in HSAECs, HRECs, and HUVECs rendered senescent by ETIS (S) as described in Fig. 2c or proliferating (P). e WI-38 cells were transfected with siCtrl, siNTRK1 (directed at TrkA), or siNTRK2 (directed at TrkB) and treated with etoposide (ETIS); % live cells relative to day 0 were counted at the indicated times. f RT-qPCR analysis of the levels of NTRK1 and NTRK2 mRNAs at day 10 in the groups analyzed in e. g Western blot analysis of the levels of TrkB, labile protein p53, and ACTB in proliferating and senescent (ETIS) WI-38 cells at the times shown after treating with CHX (50 μg/ml). h Western blot analysis of the levels of TrkB, p21, and ACTB throughout senescence induction with etoposide in WI-38 cells. i RT-qPCR analysis of the levels of TGFB1, p16, and IL6 mRNAs in the same conditions analyzed in h. j After pulldown of biotinylated cell-surface proteins from WI-38 cells [proliferating (P) or subjected to ETIS (S)], TrkB and DPP4 were assessed by western blot analysis. Ponceau staining of the transferred samples served to assess differences in sample loading and transfer. k Immunofluorescence micrographs showing cells positive for TrkB (red), DPP4 (green), and merged signals (orange/yellow) in the groups studied in j in non-permeabilizing conditions. Blue, DAPI staining to identify nuclei. Scale bar, 100 µm. In a, c, g, h, ACTB was included as loading control. Data in b, d, f, i were normalized to ACTB mRNA. Graphs in b, d–f, i display the mean values ± SD of n = 3 experiments; significance (*p < 0.05, **p < 0.01, ***p < 0.001) was determined by using two-tailed Student’s t-test.