Fig. 2

DHM suppresses PA-induced oxidative stress by induction of autophagy in hepatocytes. A–D HepG2 cells were treated with DHM of different concentrations (0, 5, 10, 20, 40 and 80 μM) for 16 h. The expressions of ATG4B, P62, and LC3 were analyzed by western blotting. Representative photographs (A) and densitometric quantification of ATG4B (B), P62 (C) and LC3 (D). E–G HepG2 cells were pretreated with CQ (20 μM) or 3-MA (5 mM) for 1 h and then treated with DHM (20 μM) for 2 h followed by exposure to PA (0.2 mM) for 16 h. The expressions of P62 and LC3 were analyzed by western blotting (E). The bar graphs showed the quantification of P62 (F) and LC3 (G), respectively. H, I HepG2 cells were treated as indicated. The intracellular ROS level was analyzed by FCM assay (H), and the bar graph showed the quantification. J, K HepG2 cells were treated as indicated. The mtROS were analyzed by FCM assay (J) and the bar graph showed the quantification (K). L, M The MMP levels were analyzed by FCM assay (L) and the bar graph showed the quantification (M). N HepG2 cells were transfected with the GFP-LC3 expression vector for 4 h, followed by the treatment with DHM (20 μM) for 2 h and 0.2 mM of PA for an additional 16 h. Representative images of cells co-expressing MitoTracker Red and GFP-LC3 were showed by confocal microscopy; scale bars: 10 mm. Graphs showed mean ± SEM; n = 3; data were from one experiment out of three. *p < 0.05, **p < 0.01, ***p < 0.001, compared between the marked groups; ns no significant difference