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. 2016 Jun 8;12(2):1058–1066. doi: 10.3892/ol.2016.4686

Figure 2.

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.