Figure 6: Inhibition of autophagy by 3-MA and increased apoptosis by Atg5 shRNA during incubation of podocytes with HG plus resveratrol.
(a) Cells were pretreated with the indicated concentrations of 3-MA for 1 h before treatments for 48 h. Immunoblot analysis of LC3, p62, Cleaved caspase-3, and Bax in cells treated with the indicated treatments for 48 h. (b) Quantitative densitometry for LC3-II, p62, Cleaved caspase-3, and Bax. Data are the mean ± SEM of three experiments. *P < 0.05 vs. NG; #P < 0.05 vs. HG; ∆P < 0.05 vs. HG + RES. (c-g) Podocytes were transfected with Atg5 shRNA or scrambled shRNA (control; shNC); 24 h after transfection, the cells were treated with HG plus resveratrol for 48 h. (c) Cell lysates were prepared and subjected to immunoblot analysis with antibodies for Atg5, LC3, Cleaved caspase-3, and Bax, with β-actin serving as a loading control. (d) Quantitative densitometry for Atg5, LC3-II, Cleaved caspase-3, and Bax. Protein levels were normalized to that of β-actin. Data are the mean ± SEM of three experiments. *P < 0.05 vs. shNC. (e) Electron microscopic evaluation of autophagy in podocytes in different treatment groups. (f) The effects of Atg5 shRNA on HG-induced apoptosis with resveratrol treatment were determined by flow cytometry. (g) Quantitative analysis of Annexin V+ PI− and Annexin V+ PI+ podocytes by flow cytometry. Data are the mean ± SEM of three experiments. *P < 0.05 vs. shNC.
