Fig 2: 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.

Fig 3: Schematic diagram of the crosstalk among DPP-4, CAV1 and integrin β1 during EMT and fibrosis induced by BSA injection.

Fig 4: Vilda induces FGF21 expression through the Sirt1 pathway in human cardiac fibroblasts (HCF). a Western blot analysis of the effect of vilda treatment on FGF21 protein levels in HCF. Summarized data for FGF21 are shown in b (N = 4 for each experiment). Statistical analysis was performed unpaired two-tailed Student’s t test with Welch’s correction. *P < 0.05 vs 0 nM. c Gene silencing effect of DPP4 induced by siDPP4 transfection on FGF21 expression in HCF. Summarized data for FGF21 are shown in d (N = 3 for each experiment). Statistical analysis was performed unpaired two-tailed Student’s t test with Welch’s correction. *P < 0.05 vs siGFP. e–g Time course after vilda stimulation (10 nM) in HCF. A representative western blot analysis of FGF21, Sirt1, and P-AMPK protein expression in HCF treated with vilda for 8 or 24 h (e). f, g are summarized data for FGF21 (f) and Sirt1 (g) (N = 4 for each experiment). Sirt1 activator (Resveratrol: 50 nM) induced FGF21 protein expression in cell lysates of HCFs (h). Statistical analysis was performed unpaired two-tailed Student’s t test with Welch’s correction. *P < 0.05 vs vehicle. ELISA for FGF21 in medium supernatant of HCF stimulated with Resveratrol (50 nM) for 0.5, 3 or 24 h (i). Open bar, Veh-treated; closed bar, Resveratrol-treated. Statistical analysis was performed unpaired two-tailed Student’s t test with Welch’s correction. N = 4 for each experiment group. *P < 0.05 vs veh. j–l Gene silencing effect for Sirt1 in vilda-induced FGF21 expression. Western blot analysis of the effects of siSirt1 treatment on FGF21 expression in HCFs in the absence and presence of vilda (10 nM) (j). k, l Summarized data for FGF21 (k) and Sirt1 (l). N = 3 for each experiment. *P < 0.01 vs veh. †P < 0.05 for effect from Sirt1 silencing on vilda response, two-way ANOVA

Fig 5: Fibrogenic gene and EMT-related gene expression levels were induced in the BSA-injected diabetic mouse kidney; TENE treatment suppressed these gene expression levels. (a) Microarray analysis of the kidney samples. Heat map analysis of the gene expression. BSA injection (particularly in the diabetic mice) induced genes, such as DPP-4, TGF-β/smad3 signaling, CAV1, integrin β1 and the EMT program; TENE treatment suppressed these alterations (n = 2 mice per group, and the average value is shown in the figure). Red indicates high and green indicates low relative expression levels. (b–p) qPCR analysis of the expression of the indicated genes in the kidney of mice in each group (n = 7 mice per group). Gene expression was normalized to the control mice value. *P < 0.05, **P < 0.01. Data are presented as mean ± s.e.m. (q) Representative western blot analysis. As a densitometric analysis, each protein level was normalized with actin. n = 7 per group were analyzed.