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Fig. 6 | BMC Cancer

Fig. 6

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. 6

LY294002 and PD98059 enhanced the anti-proliferative effect of bicyclol in HepG2 cells. a Cell proliferation after co-treatment with LY294002 and bicyclol or PD98059 and bicyclol. The cells were treated with both 10 μM LY294002 and 200 μM bicyclol for 24 or 48 h, or cells were pre-treated with 20 μM PD98059 for 30 min and then 200 μM bicyclol was added to the media for 24 or 48 h. The A570 was then measured after the MTT incubation. b The percentage of cells in G1 phase after co-treatment with LY294002 and bicyclol or PD98059 and bicyclol. The cells were treated as in (A) for 24 h, and the percent of cells in G1 phase was determined by flow cytometry. c The bicyclol-mediated protein levels after co-treatment with LY294002 and bicyclol. Cells were pre-treated with both 10 μM LY294002 and 200 μM bicyclol for 6 h. Then, the cells were disrupted, and cellular β-actin, p-AKT (Ser 473), total AKT, p-ERK1/2 (Thr202 and Tyr 204), total ERK, p-Rb (Ser 807) and LC3 I and II were analyzed by western blotting. d The bicyclol-mediated protein levels after co-treatment with PD98059 and bicyclol. The cells were pre-treated with 20 μM PD98059 for 30 min and then 200 μM bicyclol was added to the media for 6 h. Then, the cells were disrupted, and cellular β-actin, p-AKT (Ser 473), total AKT, p-ERK1/2 (Thr202 and Tyr 204), total ERK, Ras, p-Rb (Ser 807) and LC3 I and II were analyzed by western blotting. e The autophagosomes and autolysosomes were increased by LY294002 and PD98059. The cells were transiently transfected with GFP-RFP-LC3 vectors using Lipofectamine 2000 and incubated for 48 h. Then the cells were treated with both 10 μM LY294002 and 200 μM bicyclol for 24 h, or cells were pre-treated with 20 μM PD98059 for 30 min and then 200 μM bicyclol was added to the media for 24 h. 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. Bar graphs represent the means ± SD from three independent experiments. (*p < 0.05 versus bicyclol treatment)

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