Fig. 7

p66Shc silencing inhibits fibrotic factors activation and improves mitochondrial dysfunction. p66Shc in granulosa cells was silenced by lentivirus or overexpressed by a plasmid vector. After 72 h, the cells were then treated with DHT or TGF-β1. a The expression of p66Shc, p-p66Shc, Sirt1, TGF-β and AR was analyzed by western blot assay. mRNA levels of p66Shc (b), Sirt1 (c), and α-SMA (d) was analyzed by real-time PCR. e The expression of p66Shc, p-p66Shc, Sirt1, and TGF-β was assessed by western blot assay. f The expression of p-p66Shc (Alexa Fluor 488) and α-SMA (Cy3) were analyzed using immunofluorescence staining (60×). Images are representative of three independent experiments with similar results. g p-p66Shc (Alexa Fluor 488) and mitochondria were revealed by immunofluorescence and MitoTracker Red staining, respectively using confocal microscopy. h Mitochondrial membrane potential was analyzed by JC-1 monomers/polymers (60×). Images are representative of three independent experiments with similar results. i Quantitative analysis of the ratios of the ratios red to green fluorescence is shown. Three independent experiments were performed with similar results. Data are shown as the mean ± SD. *p ≤ 0.05. DHT, dihydrotestosterone; TGF-β1, transforming growth factor-beta 1; p-p66Shc, phosphorylated 66-kDa Src homology 2 domain-containing protein; Sirt1, sirtuin 1; AR, androgen receptor; α-SMA, alpha-smooth muscle actin; GCs, granulosa cells