Fig. 7

palmitoylation inhibitor 2BP affects sEV secretion in SCs and causes apoptosis of seminiferous tubules in mice. A Double immunofluorescence staining showed that VMP1 was colocalized with ALIX in normal adult mouse testis. B Immunoprecipitation and Acyl-Biotin Exchange assay of VMP1 palmitoylation in control mouse groups (CTR) and 2BP mouse groups (2BP) that were intraperitoneally injected with palmitoylation inhibitor 2BP for 3 months. Right graph: Quantified data of ratios of palmitoylated VMP1 to total VMP1 protein levels in CTR or 2BP groups. C Co-immunoprecipitation of VMP1 interaction with ALIX in CTR (left) and 2BP (right) mouse testes. D Confocal images of double immunofluorescence staining of SC marker Vimentin and CD63 (the marker of exosomes and MVBs) in CTR or 2BP treated mouse testes. CD63 (red) and Vimentin (green) immunoreactivity are merged and magnified. Right panel is the quantification of CD63 fluorescence intensity. E TUNEL staining of seminiferous tubules in CTR or 2BP testes. The positive cells (green) are representative of apoptosis. Right graph: quantification of the number of positive cells per tubule in two groups. F Western blot of apoptosis-related protein Bax and Bcl-2 expression in CTR or 2BP testes. GAPDH served as a loading control. Right panel is quantification of the Bax/Bcl-2 expression ratio in CTR or 2BP. F Immunofluorescence staining PCNA (proliferating cell nuclear antigen), a marker for cell proliferative activity, in CTR or 2BP testes. Quantification of PCNA⁺ cells per seminiferous tubules was carried out in testes of two groups. H Quantitative PCR assay of relative mRNA levels of PCNA expression in CTR or 2BP testes. All scale bars = 50 μm, Data were obtained from three to five independent experiments and were presented as mean ± SEM, *P < 0.05