Fig. 4: BDNF is a SASP factor that ensures viability of senescent cells.

a Heat map of the levels of mRNAs encoding neurotrophins as quantified by RT-qPCR analysis in the senescence models described in Fig. 2c. b ELISA measurement of BDNF levels in conditioned media collected for 48 h in the groups shown in a. c Cytokine array analysis of SASP factors and neurotrophins in proliferating and senescent (ETIS) WI-38 fibroblasts. Immunofluorescence micrographs of BDNF signal (red) and DAPI-stained nuclei (blue) (d), % cells with positive BDNF staining (e), and BDNF fluorescence intensity measurements (f) in senescent (ETIS) WI-38 cells. g RT-qPCR analysis of BDNF mRNA levels in senescent (ETIS) WI-38, BJ, and IMR-90 fibroblasts. h RT-qPCR analysis of the levels of BDNF, p21, IL6, and p16 mRNAs in senescent (ETIS) WI-38 fibroblasts transfected with the indicated siRNAs, relative to proliferating cells transfected with siCtrl. i WI-38 fibroblasts and HUVECs transfected with siCtrl or siBDNF were exposed to senescence-inducing treatments (ETIS, OSIS, IRIS); % cells remaining at each time point. j WI-38 fibroblasts were transfected with siCtrl or siBDNF siRNAs, subjected to ETIS, and supplemented or not with exogenous BDNF (200 pg/ml, refreshed every 48 h); % cells remaining are shown. Proliferating and senescent (ETIS) WI-38 fibroblasts were treated with IgG or BDNF-blocking antibodies (4 µg/ml each); % remaining cells 48 h later were counted (k) and S cells were assessed by microscopy (l) and by caspase 3/7 activity (m). n Western blot analysis of BDNF levels and sizes in conditioned media from P and S (ETIS) WI-38 cells. o WI-38 cells undergoing ETIS were additionally treated with DMSO (−), MMP inhibitor II (MMPi, 10 µM), or Furin inhibitor II (FURINi, 15 µM). Remaining cells at the indicated times are shown. p Western blot analysis as in n, but additionally treated with DMSO (−) or MMPi during senescence induction. Scale bars, 100 µm. Data in g, h were normalized to ACTB mRNA. Graphs in b, e–k, m, and o represent 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.