Fig. 2
B5G9 induces apoptotic cell death in HepG2 cells. a HepG2 cells treated B5G9 (6 μM) were stained with Hoechst 33342 (10 μg/mL) and then observed under a fluorescence microscope. Apoptotic cells with chromatin shrinking were observed (as indicated by the arrow). Original magnifications: × 630; scale bar: 10 μm. b The ultrastructure of HepG2 cells after B5G9 (6 μM) treatment for 24 h was observed by transmission electron microscopy. Original magnifications: × 8900; scale bar: CTL, 5 μm; B5G9 (6 μM), 2 μm. c B5G9 triggered the accumulation of HepG2 cells in the sub-G1 phase of the cell cycle. After B5G9 treatment, HepG2 cells were stained with PI (0.02 mg/mL). Cell cycle distribution was determined by flow cytometry. d Apoptotic cells after B5G9 treatment were quantified by the Annexin V/PI assay. HepG2 cells treated with various concentrations of B5G9 were stained using an Annexin V/PI kit and detected by flow cytometry. e Effects of B5G9 on the apoptosis-related proteins level. Total cell lysate from HepG2 cells treated with B5G9 at indicated concentrations for 24 h was evaluated by western blotting, and β-actin was used as a loading control. f B5G9-induced cell death is necrosis-independent. HepG2 cells were cultured with various concentrations of B5G9 in the presence or absence of necrostatin-1 (50 μM) for 12 h. Cell viability was measured by the MTT assay. The results were presented as the mean ± S.D