Fig. 6

B5G9 treatment induces mitochondrial dysfunction. a B5G9 induced mitochondrial morphology changes. HepG2 cells treated with B5G9 (6 μM) for indicated times were stained with MitoTracker Red CMXRos (100 nM) for 20 min, the cells were observed by a fluorescence microscope. Original magnifications: 630 ×; scale bar: 10 μm, (b) B5G9 induced mitochondrial membrane potential collapse. HepG2 cells were treated with B5G9 (6 μM) for indicated times, and then stained with JC-1 (5 μM) fluorescence dye, and changes of ΔΨm were determined by flow cytometry. c B5G9 increased mitochondrial membrane permeabilization. HepG2 cells were treated with B5G9 (6 μM) for indicated times, then cells were incubated with calcein-AM for 30 min, the fluorescence was detected by a microplate reader, ^* P ≤ 0.05, ^** P ≤ 0.01 vs control. d Cyto c released from mitochondria to cytoplasm after B5G9 treatment. After being treated with various concentrations of B5G9 for 24 h, HepG2 cells lysate was divided into mitochondrial fraction and cytoplasmic fraction and detected by western bolt, β-actin and VDAC were used as loading controls for cytoplasm and mitochondria respectively. e O₂ consumption rate was inhibited by B5G9. HepG2 cells were treated with B5G9 (6 μM) for 3, 6, 9 h and antimycin (6 μM) for 3 h, the O₂ consumption rate was measured by a MitoXpress-Xtra-based assay. ^** P ≤ 0.01, ^*** P ≤ 0.001 vs control. f B5G9 impaired mitochondrial ATP production. HepG2 cells were treated with B5G9 (6 μM) for 3, 6 and 9 h in complete medium or no-glucose medium, the ATP level was detected by a CellTiter-Glo Luminescent Assay, ^** P ≤ 0.01, ^*** P ≤ 0.001