Fig. 5: TNFAIP8 from tubule-derived exosomes plays a major role in protecting fibroblasts from apoptosis in vitro.

a Western blot analyses show the presence of TNFAIP8 protein in the exosomes isolated from TGF-β1-treated HK-2 cells. Exosomes prepared from the equal amounts of HK-2 cells after TGF-β1 treatment were lysed and immunoblotted with antibodies against CD63 and TNFAIP8, respectively. Numbers (1 and 2) indicate each individual treatment in a given group. b Double immunofluorescence staining demonstrates colocalization of CD63 (Red) and TNFAIP8 (Green) in HK-2 cells after TGF-β1 treatment. Scale bar, 10 μm. c Experimental design showing knockdown of TNFAIP8 in HK-2 cells prior to exosomes collection of conditioned media. d, e Knockdown of TNFAIP8 abolished its expression after TGF-β1 treatment in HK-2 cells. qPCR (d) and Western blotting (e) show mRNA and protein levels of TNFAIP8 in different groups as indicated. ^**P < 0.01, ^††P < 0.01 (n = 6). Representative FACS analyses (f) and quantitative data (g) show apoptotic cells after various treatments in NRK-49F cells. ^**P < 0.01, ^††P < 0.01 (n = 3). h Western blot analyses demonstrate protein expression of TNFAIP8, p53, cleaved caspase-3, FasL, FADD, Bax in different groups of NRK-49F cells. Numbers (1–3) indicate each individual treatment in a given group. i Representative micrographs show immunofluorescence staining of fibronectin in different groups as indicated. Arrows indicate positive staining. Scale bar, 50 µm. j Graphic presentation shows the quantitative determination of fibronectin-positive area in different groups as indicated. Each point indicates one of three different random fields of view in one micrograph. ^**P < 0.01, ^††P < 0.01 (n = 3). k–m Graphic presentations show the relative level of cyclin D1 (k), c-myc (l) and α-SMA (m) mRNA measured by qPCR after various treatments in NRK-49F cells. ^**P < 0.01, ^††P < 0.01 (n = 6). Data presented as mean ± S.E.M. of three or six independent experiments. p values were calculated using Student-Nwman-Kuels test for multiple groups comparison.