FIGURE 1.

Tetrandrine treatment induced autophagy in HCC cells. A, Huh7, BEL7402, and HepG2 cells were treated with the indicated concentrations of tetrandrine for 24 h. Then, the cell lysates were subjected to Western blotting using antibodies against pro-poly(ADP-ribose) polymerase, pro-CASPASE-8, pro-CASPASE-3, and LC3. A GAPDH antibody was used as a loading control. The molecular weights of bands are shown on the right. B, after exposure to 5 μm tetrandrine for the indicated time intervals in HCC cell lines (Huh7 and BEL7402) and normal liver cell line L02, the cells were lysed and immunoblotted with an anti-LC3 antibody. C, Huh7 and BEL7402 cells were transiently transfected with the GFP-LC3 plasmid for 24 h, subsequently treated with or without 5 μm tetrandrine for 8 or 24 h and observed with a fluorescence microscope. Representative images are shown to indicate the cellular localization patterns of the GFP-LC3 fusion protein (×40 magnification). The percentage of cells with GFP-LC3 puncta was used to quantify the percentage of autophagic cells. The data represent an average ± S.D. of at least three independent experiments. At least 100 GFP-LC3-transfected cells were counted in each experiment. D, GFP-LC3-transfected cells were pretreated with or without 2 mm 3-methyladenine (MA) for 1 h. The cells were then exposed to 5 μm tetrandrine for 24 h, and the localization of GFP-LC3 was observed using a fluorescent microscope (×40 magnification). The percentage of GFP-LC3-transfected cells with punctate fluorescence is shown. At least 100 cells from each treatment group were examined (* = p < 0.05).