Celastrol triggers caspase activation in Kasumi-1 cells. (A) Caspase activity assays using Kasumi-1 cells treated with celastrol (2.5 μM) for different periods of time; (B) Celastrol induced PARP cleavage and activated caspase-3, -8 and-9. Kasumi-1 cells were treated with 2.5 μM celastrol for various time periods, and the cell lysates were subjected to Western blot using the indicated antibodies; (C) Celastrol induced cell apoptosis in a caspase-dependent manner. Kasumi-1 cells were preincubated with 40 μM Z-VAD-fmk for 2 h prior to treatment with 2.5 μM celastrol for another 24 h. Cell apoptosis was analyzed using Annexin V-FITC/PI staining; (D) Celastrol decreased cell viability in Kasumi-1 cells in a caspase-dependent manner. Kasumi-1 cells were preincubated with 40 μM Z-VAD-fmk for 2 h prior to treatment with 2.5 μM celastrol for another 24 h, and cell viabilities were analyzed by CCK-8 assay; (E,F) Kasumi-1 cells were incubated with 20 μM caspase-8 inhibitor (Z-IETD-fmk) (E) or 40 μM caspase-9 inhibitor (Z-LEHD-fmk), then treated with 2.5 μM celastrol for 24 h. Cell viabilities were analyzed using CCK-8 assays. Data are from three independent experiments. * p < 0.05, and ** p < 0.01.