Figure 5

JNK mediates celastrol-induced cell death by initiating the mitochondrial apoptotic pathway. H1299 cells were treated with the indicated concentration of celastrol in the absence or presence of 40 μM SP for 12 h or 24 h. A. SP does not suppress celastrol-induced ROS accumulation. After 12 h of treatment, cells were stained with DCFH-DA and analyzed by flow cytometry. The relative levels of ROS geometry fluorescence are shown as a ratio compared to the control group. Representative measurements are shown. Each experiment was performed in triplicate, and the values reported represent the mean ± SD. B. JNK mediates mitochondrial depolarization induced by celastrol. Cells were stained with JC-1 and analyzed by flow cytometry. The ratio of JC-1 red/green fluorescence intensity was normalized by comparing the data to the control group and is represented as loss of MMP. Representative measurements are shown. Each experiment was performed in triplicate, and the values reported represent the mean ± SD. *P < 0.01. C. JNK mediates celastrol-induced apoptosis by initiating the mitochondrial apoptotic pathway. Actin, p-JNK, t-JNK and the cleavage of PARP, caspase 9 and caspase 3 were analyzed by western blotting. Representative images of three independent experiments are shown. D. JNK mediates mitochondrial translocation of Bad and cytoplasmic release of Cyt c induced by celastrol. Mitochondria and cytoplasm were separated and used to extract protein. The alteration and subcellular localization of Bad and Cyt c were analyzed by western blotting. Representative images of three independent experiments are shown.