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. 2017 Feb 22;7:42764. doi: 10.1038/srep42764

Figure 3. Role of oxidative stress in palmitic acid-induced autophagy in podocytes.

Figure 3

(a) Representative immunofluorescence images of ROS in podocytes (200×). Podocytes were treated with 150 μmol/L palmitic acid for 24 hours with or without pretreatment of NAC (150 μmol/L), and the intracellular ROS level was then measured using the fluorescent probe 2′,7′-dichlorfluorescein-diacetate (DCFH-DA). (b) Podocytes stably expressing GFP-LC3 were pretreated with 150 μmol/L NAC for 2 hours, treated with 150 μmol/L palmitic acid for 24 hours and then analyzed by fluorescence microscope (400×). (c) GFP-LC3 puncta (mean ± SEM) were quantified for each experiment (n = 3). At least 30 cells were counted in each individual experiment.∗p < 0.05 vs. control group, #p < 0.05 vs. NAC group, Δp < 0.05 vs. PA group. (d) Podocytes were treated with 150 μmol/L palmitic acid in the absence or presence of 150 μmol/L NAC for 24 hours, and the cell lysates were then analyzed by immunoblot using antibodies against Beclin1, LC3 and LAMP-2. (e) Densitometric analysis of Beclin1, LC3-II/LC3-I and LAMP-2 expression in Figure d. ∗p < 0.05 vs. control group, #p < 0.05 vs. NAC group, Δp < 0.05 vs. PA group. Ctr: Control group, podocytes were treated with 1% BSA. NAC: Podocytes were treated with 150 μmol/L N-acetyl-cysteine (NAC) for 24 hours. PA: Palmitic acid group, podocytes were treated with 150 μmol/L palmitic acid for 24 hours. PA + NAC: Podocytes were treated with 150 μmol/L palmitic acid for 24 hours after pretreatment with 150 μmol/L N-acetyl-cysteine (NAC) for 2 hours.