Figure 2.

Nimbolide induces the generation of ROS and mitochondrial dysfunction in human osteosarcoma cells. (A) MG63 cells were treated with control solution or various concentrations of Nimbolide for 24 h. The concentration of H₂O₂ as a measure of ROS production was examined by using flow cytometry; (B) MG63 cells were pretreated for 30 min with N-acetylcysteine (NAC), diphenyleneiodonium chloride (DPI), and catalase followed by treatment with Nimbolide (10 μM) for 24 h. The percentage of apoptotic cells was then analyzed by flow cytometric analysis of PI-stained cells; (C) MG63 cells were treated as described in (A). The mitochondrial membrane potential was examined by using flow cytometry and JC-1 staining; (D) MG63 cells were treated with control solution or various concentrations of Nimbolide for 24 h. The expression levels of Bcl-2, Bak, Bax, mitochondrial cytochrome c, and cytosolic cytochrome c were examined by using western blot analysis; actin and voltage-dependent anion channels (VDAC) were used as internal controls. The data are expressed as the mean ± SEM. * p < 0.05 compared with controls. ^# p < 0.05 compared with the Nimbolide treated groups.