Fig. 4

Celastrol triggered autophagy in glioma cells, and inhibition of autophagy increased celastrol-induced cell death. a U251 cells were transiently transfected with the mRFP-EGFP-LC3 plasmid for 24 h and then treated with or without celastrol (1.5 μM) for 24 h. The images were captured using a confocal microscope. Scale bars = 25 μm. b Cells were treated with various concentrations of celastrol for 24 h or incubated with celastrol (3 μM) for different durations. The autophagy-related proteins LC3, P62 and Beclin-1 were detected by western blotting. c CQ (25 μM) was added to cells 2 h before celastrol treatment. Then, cells were treated with celastrol for 24 h. The autophagy-related proteins LC3, P62 and Beclin-1 were detected by western blotting. d z-VAD (30 μM) or 3-MA (3 mM) was added to cells 2 h before celastrol treatment. After 24 h, cell viability was determined. e CQ was added to cells 2 h before celastrol treatment. Then, cells treated with celastrol for 24 h. Cell viability was determined by CCK8. β-actin was used as an internal control. Data are presented as the Mean ± SD (n = 3). *P < 0.05, ***P < 0.001, significantly different compared with the untreated control group. ^#P < 0.05, ^##P < 0.01, significantly different compared with the celastrol treatment group. ^△P < 0.05, significantly different compared with the CQ treatment group