Figure 5: Resveratrol-induced autophagy in db/db mice and podocytes. | Scientific Reports

Figure 5: Resveratrol-induced autophagy in db/db mice and podocytes.

From: Resveratrol protects podocytes against apoptosis via stimulation of autophagy in a mouse model of diabetic nephropathy

Figure 5

(a) Representative photomicrographs of immunofluorescence for LC3-II and synaptopodin in mouse renal glomeruli. LC3-II exhibits green fluorescence and synaptopodin exhibits red fluorescence. Magnification: 400×. (b–g) Immunoblot and densitometric analysis of LC3, Atg5, p62 and β-actin in cells treated with indicated treatments for 48 h. (b) Cells were treated with HG (30 mM) for the indicated time points (0, 6, 12, 24, 36, and 48 h). (c) Quantitative densitometry for LC3-II, Atg5, and p62. Protein levels were normalized to that of β-actin. Data are the mean ± SEM of three experiments. *P < 0.05 vs. control (0 h), #P < 0.05 vs. control (0 h), △P < 0.05 vs. control (0 h). (d) Human podocytes (HPC) were treated with different concentrations of resveratrol (0, 5, 10, and 15 μM) followed by HG treatment for 48 h. (e) Relative abundances of LC3-II, Atg5, and p62 normalized to that of β-actin. Data are the mean ± SEM of three experiments. *P < 0.05 vs. NG, #P < 0.05 vs. NG, △P < 0.05 vs. NG, **P < 0.05 vs. HG, ##P < 0.05 vs. HG, △△P < 0.05 vs. HG. (f) Human podocytes (HPC) were treated with NG (5.6 mM), HG, HG plus resveratrol (15 μM), HG plus resveratrol (15 μM) and bafilomycin A (10 nM) for 48 h. (g) Relative abundances of LC3-II, Atg5, and p62 normalized to that of β-actin. Data are the mean ± SEM of three experiments. *P < 0.05 vs. NG, #P < 0.05 vs. HG, △P < 0.05 vs. HG + RES. (h) Representative immunofluorescence staining for LC3-II. Podocytes were treated with NG (5.6 mM), HG, HG plus resveratrol (15 μM), HG plus resveratrol (15 μM) and bafilomycin A (10 nM) for 48 h. Magnification: 100×. (j) Electron microscopic evaluation of autophagy in podocytes. Equal numbers of human podocytes were incubated in medium containing NG, HG, HG plus resveratrol, HG plus resveratrol and bafilomycin A for the indicated periods. Magnification: 20,000×; scale bar, 1 μm. The numbers of autophagosomes represent autophagic activity. (i) Representative microphotographs of GFP-LC3-transfected podocytes incubated in medium containing NG, HG, HG plus resveratrol, HG plus resveratrol and bafilomycin A for the indicated periods. Magnification: 400×.

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