Figure 5.

Caspase-3, rather than RhoA, activated ROCK1 during triptolide-induced apoptosis. Caspase inhibitors (z-VAD-fmk or z-DEVD-fmk) and a Rho inhibitor (C3 exoenzyme) were used to explore the mechanism behind ROCK1 activation. The percentage of apoptotic cells was determined by FACS analysis using Annexin V/PI staining. Error bars represent the mean±S.D. (n=5). Total protein lysates were analyzed by immunoblotting using the indicated antibodies. Results are representative of three independent experiments. (a–c) U937 cells and blasts from one AML patient were pretreated with 20 μM z-VAD-fmk or z-DEVD-fmk for 2 h followed by treatment with or without 40 nM triptolide for 24 h. (d–f) U937 cells and blasts from one AML patient were transfected with 5 μg C3 (premixed with FuGENE 6 Reagent in RPMI-1640 medium). After 5 h of incubation, cells were treated with or without 40 nM triptolide for 24 h