Fig. 7

AKT and ERK siRNAs enhanced the anti-proliferative effect of bicyclol in HepG2 cells. a Cell proliferation after AKT inhibition and bicyclol treatment. The cells were transiently transfected with the AKT siRNA using Lipofectamine RNAiMAX and incubated for 48 h. Then, the transfected cells were treated with 200 μM bicyclol for 24 or 48 h. The A₅₇₀ was then measured after the MTT incubation. b Living cell number after ERK inhibition and bicyclol treatment. The cells were transiently transfected with the ERK siRNA using Lipofectamine RNAiMAX and incubated for 48 h. Then, the transfected cells were treated with 500 μM bicyclol for 24 or 48 h. The A₅₇₀ was then measured after the MTT incubation. c The percent of cells in G1 phase after AKT or ERK inhibition and bicyclol treatment. The cells were treated as in (A) with 200 μM bicyclol for 24 h, and the percentage of cells in G1 phase was determined by flow cytometry. d The transfection efficiency of the AKT and ERK siRNAs. The cells were transiently transfected with AKT or ERK siRNAs using Lipofectamine RNAiMAX and incubated for 48 h. Then, the cells were disrupted, and cellular β-actin, total AKT and total ERK were analyzed by western blotting. e The number of autophagosomes and autolysosomes was increased by AKT or ERK inhibition. The cells were co-transfected with the GFP-RFP-LC3 vector and AKT/ERK siRNAs using Lipofectamine 2000, incubated for 48 h, and then treated with 200 μM bicyclol for another 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 was counted. Bar graphs represent the means ± SD from three independent experiments. (*p < 0.05 versus bicyclol treatment)