Fig. 3

Celastrol induced oxidative stress in human NSCLC cells. A, B Intracellular ROS generation was measured by DCFH-DA. Cells were exposed to 4 µM celastrol for indicated times. Representative flow cytometric graph is shown in A. Quantification of ROS levels in H460 cells is shown in B (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 compared to 0 h control). C, D. Cells were treated with different concentrations of celastrol (0, 1, 2, and 4 μM) for 2 h. NAC was used at a dose of 5 µM, either alone or as a 1 h pretreatment. Representative flow cytometric graph is shown in C. Quantification of ROS levels in H460 cells is shown in D (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 compared to control). E Intracellular ROS as detected by DCFH-DA staining (green). H460 cells were exposed to 4 µM celastrol for indicated times and fluorescence images were captured. F H460 cells were treated as indicated in C and fluorescence images showing DCFH-DA (green) were captured. G Effect of NAC on celastrol-induced apoptosis. Cells were pretreated with 5 mM NAC for 1 h and then exposed to 4 µM celastrol for 24 h. The apoptotic cells were measured by Annexin V/PI staining. H Western blot analysis of BAX/BCL-2 in H460, PC-9, and H520 cells exposed to celastrol at indicated concentrations for 12 h. NAC pretreatment was carried out at 5 mM for 1 h. All experiments were repeated three times. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)