Fig. 3

B5G9-induced apoptosis is ROS-dependent. a B5G9 elevated ROS level by a time-dependent manner in HepG2 cells. HepG2 cells were stained with H₂DCFDA (10 μM) after being treated with B5G9 (6 μM) at the indicated times. The H₂DCFDA fluorescence was observed by a fluorescence microscope. Original magnifications: 100 ×; scale bar: 200 μm. b The H₂DCFDA fluorescence intensity of HepG2 cells treated with B5G9 at the indicated times was detected by a microplate reader. ^* P ≤ 0.05, ^*** P ≤ 0.001 vs control. c B5G9 induced lipid peroxidation. After being treated with B5G9 (6 μM) for the indicated times, MDA level were detection by a Lipid Peroxidation MDA Assay Kit. ^** P ≤ 0.01, ^*** P ≤ 0.001 vs control. d The elevated ROS level induced by B5G9 was abrogated by NAC or MnTBAP. HepG2 cells were treated with B5G9 (6 μM) in the presence or absence of NAC (20 mM) or MnTBAP (200 μM) for 3 h. The H₂DCFDA fluorescence intensity was detected by a microplate reader. ^* P ≤ 0.05, ^** P ≤ 0.01. e NAC or MnTBAP alleviates B5G9-induced cell death. HepG2 cells were treated with B5G9 (6 μM) in the presence or absence of NAC (20 mM) or MnTBAP (200 μM) for 12 h. Cell viability was measured by MTT assay. ^** P ≤0.01, ^*** P ≤ 0.001