Fig. 6: Over-expression or knockdown of TNFAIP8 inhibits or promotes renal interstitial fibroblast apoptosis in vivo.

a Diagram shows the experimental design. Green arrow indicates the injection of Flag-TNFAIP8 expression vector or shTNFAIP8 knockdown plasmid. b Representative micrographs of immunohistochemical and immunofluorescence staining show an increased tubular TNFAIP8 expression after injection of Flag in the kidney after UUO. Arrows indicate positive staining. Scale bar, 50 µm. c Representative Western blotting show induction of TNFAIP8 expression in UUO kidneys by using different antibodies against TNFAIP8 and Flag, respectively. d Quantitative data of TNFAIP8 levels in different groups as indicated. ^**P < 0.01 (n = 3). e Western blot analyses show the protein levels of TNFAIP8, p53, cleaved caspase-3, FasL, FADD, PARP-1, Fsp-1, fibronectin and cyclinD1 in different groups of primary fibroblasts isolated from kidney at 14 days after UUO. Numbers (1–3) indicate a pool of primary fibroblasts isolated from two animals. f Representative micrographs show collagen deposition, fibronectin and Fsp-1 expression in different groups as indicated. Paraffin sections were subjected to Masson’s trichrome staining (upper), immunostaining for fibronectin (middle) and Fsp-1 (bottom), respectively. Arrows indicate positive staining. Scale bar, 50 µm. Graphic presentations show the quantitative determination of fibronectin (g) and Fsp-1 (h) positive staining. ^**P < 0.01 (n = 6). i Representative micrographs of immunohistochemical staining show tubular TNFAIP8 expression after injection of shTNFAIP8 plasmid after UUO. Arrows indicate positive staining. Scale bar, 50 µm. j Graphic presentations show the relative level of TNFAIP8 mRNA measured by qPCR after various treatments in UUO kidneys. ^**P < 0.01 versus sham controls, ^††P < 0.01 versus UUO injected with shCtrl plasmid (n = 6). k Western blot analyses show the protein levels of TNFAIP8, p53, cleaved caspase-3, FasL, FADD, PARP-1, Fsp-1, fibronectin and cyclin D1 in different groups of primary fibroblasts isolated from kidney at 14 days after UUO. Numbers (1–3) indicate a pool of primary fibroblasts isolated from two animals. l Representative micrographs show collagen deposition and Fsp-1 expression in different groups as indicated. Paraffin sections were subjected to Masson’s trichrome staining (upper), immunostaining for Fsp-1 (bottom), respectively. Arrows indicate positive staining. Scale bar, 50 µm. Graphic presentations show the quantitative determination of collagen deposition (m) and Fsp-1 (n) positive staining. ^**P < 0.01 versus sham controls, ^††P < 0.01 versus UUO injected with shCtrl plasmid (n = 6). Data presented as mean ± S.E.M. of six independent experiments. p values were calculated using Student-Newman-Kuels test for multiple groups comparison.