Figure 2.

Evidence that celastrol induces apoptosis in osteosarcoma cells. (a) Apoptotic morphological changes were evaluated by fluorescent microscopy using Hoechst 33258 staining. Arrows indicate chromatin condensation and DNA fragmentation. Bar: 50 μm. (b) HOS and MG-63 cells treated with celastrol were stained with annexin V-PE/7-AAD and analyzed by flow cytometry. The chart illustrates apoptosis proportion from three separate experiments. (c) The mitochondrial membrane potential was measured with JC-1 fluorescent probe and assessed by flow cytometry. The chart illustrates changes of JC-1 red/green rate from three independent experiments. (d, e) Cells were treated with various concentrations of celastrol for 24 h or incubated with celastrol (3 μM) for different hours. The expressions of cleaved PARP, caspase-3, -8, -9, DR5 and Bid were determined by western blot. (f) Caspase activity assay of cells treated with various concentrations of celastrol for 24 h. (g) HOS cells were incubated with or without celastrol for 24 h after 2 h pre-treatment with caspase inhibitors, z-IETD-fmk (10 μM), z-LEHD-fmk (40 μM) or z-VAD-fmk (20 μM). Then cells were stained with annexin V-PE/7-AAD and analyzed by flow cytometry. Results are expressed as the mean±S.D. from three independent experiments. *P<0.05 versus control, ^#P<0.05 versus celastrol treatment