Figure 3

Inhibition of MRC complex I activity is involved in mediating ROS accumulation induced by celastrol. A. Celastrol induces time-dependent ROS accumulation. H1299 cells were treated with 6 μM celastrol for the designated time. 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 of at least three independent experiments are shown. The values reported represent the mean ± SD of three separate experiments. *P < 0.01 compared with control cells. B. The alteration of antioxidative proteins induced by celastrol is not involved in promoting ROS accumulation. H1299 cells were treated with the indicated concentration of celastrol in the absence or presence of 5 mM NAC for 24 h. The alteration of SOD, catalase, Trx and actin was analyzed by western blotting. Representative images of at least three independent experiments are shown. C. Celastrol significantly inhibits MRC complex I activity. H1299 cells were treated with or without 6 μM celastrol in the absence or presence of 5 mM NAC for 12 h. The activity of MRC complexes was measured with a Mitochondrial Respiratory Chain Complex Enzyme Activity Assay Kit and is displayed as a histogram. All columns display the mean ± SD from three independent experiments. *P < 0.01. D. Inhibition of MRC complex I activity is involved in mediating ROS accumulation induced by celastrol. H1299 cells were treated with 6 μM celastrol for the indicated time. The activities of MRC complexes were measured by Mitochondrial Respiratory Chain Complex Enzyme Activity Assay Kits and are displayed as a histogram. All columns display the mean ± SD from three independent experiments. *P < 0.01 compared with control cells.