Fig. 5: 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-PKCα/β 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.