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Fig. 8 | Journal of Translational Medicine

Fig. 8

From: Suppression of p66Shc prevents hyperandrogenism-induced ovarian oxidative stress and fibrosis

Fig. 8

Sirt1 suppresses fibrotic factors activation in a p66Shc-dependent manner. Sirt1 in granulosa cells was silenced using siRNA. After 48 h, the cells were treated with DHT, resveratrol, H2O2, or EX527. a p-p66Shc and Sirt1 were analyzed by double immunofluorescence staining (40×). Images are representative of three independent experiments with similar results. b Quantification of the p-p66Shc and Sirt1 fluorescence is shown. c ROS was measured using a DCF-DA probe, and assessed by confocal microscopy (40×). Images are representative of three independent experiments with similar results. d Quantification of DCF fluorescence is shown. e Granulosa cells were treated with various concentrations of EX527. The expression of p66Shc, p-p66Shc, Sirt1, TGF-β, and AR was analyzed by western blot assay. f The expression of p66Shc, Sirt1 and TGF-β was assessed by western blot assay in granulosa cells after treatment with DHT, resveratrol, or EX527. g mRNA levels of p66Shc, TGF-β, α-SMA, and CTGF were measured by real-time PCR. h The expression of p66Shc, Sirt1, TGF-β, CTGF, β-catenin, and AR in granulosa cells treated with DHT or H2O2 was analyzed by western blot after Sirt1 silencing. Three independent experiments were performed with similar results. Data are shown as the mean ± SD. *p ≤ 0.05. DHT dihydrotestosterone, TGF-β transforming growth factor-beta, p-p66Shc phosphorylated 66-kDa Src homology 2 domain-containing protein, Sirt1 sirtuin 1, AR androgen receptor, α-SMA alpha-smooth muscle actin, CTGF connective tissue growth factor, GCs granulosa cells, DCF dichlorodihydrofluorescein, ROS reactive oxygen species, EX527, a Sirt1 inhibitor

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