Figure 4

Celastrol induces apoptosis through ROS accumulation and subsequent JNK activation. A. ROS mediate the depolarization of mitochondria induced by celastrol. H1299 cells were treated with the indicated concentration of celastrol in the absence or presence of 5 mM NAC for 24 h. Cells were stained with JC-1 and analyzed by flow cytometry. The ratio of JC-1 red/green fluorescence intensity was normalized by comparing it to the control group and is represented as loss of MMP. Representative measurements of at least three independent experiments are shown. The values reported represent the mean ± SD of three separate experiments. *P < 0.01 compared with the control group. B. Celastrol induces caspase-dependent apoptosis. H1299 cells were treated with the indicated concentration of celastrol in the absence or presence of 5 mM NAC for 24 h. Actin and the cleavage of PARP, caspase 9 and caspase 3 were analyzed by western blotting. Representative images of at least three independent experiments are shown. C. ROS mediate JNK activation induced by celastrol. H1299 cells were treated with the indicated concentration of celastrol in the absence or presence of 5 mM NAC for 12 h. The alteration of phospho-JNK (p-JNK), total JNK (t-JNK) and actin were analyzed by western blotting. Representative images of at least three independent experiments are shown. D. JNK mediates celastrol-induced cell death. H1299 cells were treated with the indicated concentration of celastrol in the absence or presence of 40 μM SP for 24 h. Cells were stained with PI and annexin V-FITC and analyzed by flow cytometry. Representative measurements of at least three independent experiments are shown. The values reported represent the mean ± SD. *P < 0.01.