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Fig. 4 | BMC Cancer

Fig. 4

From: Bicyclol induces cell cycle arrest and autophagy in HepG2 human hepatocellular carcinoma cells through the PI3K/AKT and Ras/Raf/MEK/ERK pathways

Fig. 4

Bicyclol induced autophagy in HepG2 cells. a Autophagy flux was induced by bicyclol and inhibited by 3-MA. The cells were transiently transfected with GFP-RFP-LC3 vectors using Lipofectamine 2000 and incubated for 48 h, and then treated with 200 μM bicyclol for another 24 h or pre-treated with 5 mM 3-MA for 30 min. The GFP-RFP-LC3 fluorescence was observed by a confocal microscope, and the number of autophagosomes (yellow dots) and autolysosomes (free red dots) in each cell were counted by ImageJ. 50 cells for each condition were counted. b The number of autophagosomes and autolysosomes were increased by bicyclol, and 3-MA suppressed the effect. c The cellular ultrastructure was analyzed by transmission electron microscopy. The cells were incubated in 6-well plates and treated with 200 μM bicyclol for 24 h. Then, the cells were collected and fixed. Next, ultra-thin sections were viewed on a TEM. The autolysosomes were indicated by arrows. d Cell proliferation after co-treatment with bicyclol and 3-MA. The cells were incubated in 96-well plates and then pre-treated with 5 mM 3-MA for 30 min. Next, the 3-MA was removed and the cells were treated with 200 μM bicyclol for 24 h. The A570 was then measured after the MTT incubation. e The levels of the LC3 I and II proteins were influenced by bicyclol. The cells were treated with various concentrations of bicyclol for 12 h and then disrupted. The proteins were collected, and cellular LC3 I and II were analyzed by western blotting. β-actin was used as the loading control. Bar graphs represent the means ± SD from three independent experiments. (*p < 0.05 versus bicyclol treatment)

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