Fig. 6: BDNF is a marker of surviving senescent cells.
From: A BDNF-TrkB autocrine loop enhances senescent cell viability
WI-38 fibroblasts were treated with the doses of etoposide (Etop) shown; 48 h later, cell viability was assessed by direct cell counts and represented relative to initial counts (red line) (a) and RT-qPCR analysis was used to measure the levels of BDNF, p21, and IL6 mRNAs (b). WI-38 cells were treated with H2O2 and viability (c) was measured as in a and BDNF mRNA levels (d) were measured as in b. e RT-qPCR analysis of BDNF mRNA levels in WI-38 cells treated with etoposide (50 µM, 48 h) after silencing p53 (left) by transfection with siTP53 (siCtrl in control transfections) or inducing p53 levels (right) by treatment with Nut3a (N3a, 10 µM). f RT-qPCR analysis of BDNF mRNA levels in WI-38 cells transfected with either siCtrl or siSTAT3 siRNAs and treated for 48 h with 50 µM etoposide. Western blot analysis of p-STAT3(Y705) and p53 protein levels in WI-38 cells treated as in e (g); p-STAT3(Y705), γH2AX(S139), and p53 protein levels in the groups described in a (h), and in H2O2-treated WI-38 cells as described in c (i). j Expression levels of STAT3-regulated mRNAs in GSEA gene set ‘STAT3 TARGETS AZARE’ across the different clusters set in single-cell RNA-seq analysis of senescent (50 μM etoposide, 8 days) relative to proliferating WI-38 cells. Dot size and color represent the percentage of cells expressing a transcript and the average expression value, respectively. k GSEA plots displaying enrichment scores of gene sets ‘STAT3 TARGETS AZARE’ and ‘STAT3 TARGETS DAUER’ for cluster 5 from the analysis in j. l Immunofluorescence analysis of colocalized signals for p-STAT3(Y705) (green) and BDNF (red) in senescent WI-38 cells (50 μM etoposide, 8 days). Blue, nuclei stained with DAPI; orange arrows, cells co-stained for p-STAT3 and BDNF; red arrows, BDNF-only positive cells. Scale bar, 100 µm. m Quantification of signals from l; percentages ±SD of the resulting staining for each group described. n Schematic depicting the proposed model described in this study; created using BioRender. Data in b, d, e, f were normalized to ACTB mRNA. Values in a–f are the means ± SD; significance (*p < 0.05, **p < 0.01, ***p < 0.001) was determined by using two-tailed Student’s t-test.
