Fig. 2: Glaucocalyxin A induced mitochondrial apoptosis in HOS and MG-63 cells.

a The percentage change of ΔΨm was detected by using JC-1 staining. b The generation of intracellular ROS of HOS and MG-63 cells after treatment with Glaucocalyxin A was measured by flow cytometry. c Cells were pre-incubated with/without NAC (5 mM) for 2 h, then exposed to 10 μM Glaucocalyxin A for 24 h, followed by ROS measurement. d The O₂^.− level of HOS and MG-63 cells after treatment with 2.5, 5, and 10 μM Glaucocalyxin A for 24 h and the cells pre-incubated with/without NAC (5 mM) for 2 h, then exposed to 10 μM Glaucocalyxin A for 24 h. e The H₂O₂ level of HOS and MG-63 cells after treatment with 2.5, 5, and 10 μM Glaucocalyxin A for 24 h. f The protein expression of Bax, Bcl-2, pro-caspase-9, cleaved caspase-9, pro-caspase-3, and cleaved caspase-3 was determined by western blot with β-actin as an internal control. g Gray scale analysis was performed to determine the relative ratios of Bax/Bcl-2. The results are shown as means ± SD from three independent experiments. ^**P < 0.01 compared with the control group. h Gray scale analysis was performed to determine the relative ratios of cleaved caspase-9/pro-caspase-9 and cleaved caspase-3/pro-caspase-3. The results are shown as means ± SD from three independent experiments. ^**P < 0.01 compared with the control group. i The mRNA levels of Bax and Bcl-2 were determined by real-time PCR. The results are shown as means ± SD from three independent experiments. ^**P < 0.01 compared with the control group