Figure 2.
HP induces apoptosis in HepG2 and Huh7 cells in vitro. (A) Apoptosis was analyzed by flow cytometry, and the dot plots of cells treated with 100 µg/ml HP for 0, 12, 24 and 48 h were obtained. (B) Comparison of apoptosis rates at different times. The data are presented as the mean ± SEM from three independent experiments. *P<0.05 indicates a significant increase in the apoptosis rate in a time-dependent manner. **P<0.01 and ***P<0.001. (C) The cells were treated with HP for 0, 12, 24 and 48 h, stained with Hoechst 33258, and the cell morphology was analyzed by fluorescence microscopy (magnification, ×200). (D) Detection of the activated forms of caspases-3, −8 and −9 and cleaved PARP. The protein levels of glyceraldehyde 3-phosphate dehydrogenase served as a loading control. The proteins were analyzed at the indicated time points, and all immunoblotting experiments were performed twice. (E) Measurements by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of cell viability upon treatment with 100 µg/ml HP, with or without Z-VAD-FMK for 24 h. Data are presented as the mean ± SEM from three independent experiments. *P<0.05 indicates a significant increase in the cell viability upon treatment with Z-VAD-FMK. (F) HepG2 and Huh7 cells were treated with HP (100 µg/ml), with or without 50 µM Z-VAD-FMK for 24 h. The cell lysates were prepared for immunoblotting to examine the expression of caspase-3 and PARP. UL, upper left; UR, upper right; LL, lower left; LR, lower right; PI, propidium iodide; PARP, poly (ADP-ribose) polymerase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HP, Huaier polysaccharide; SEM, standard error of the mean.