Helenalin activates caspase cleavage. A2750 cells were treated with increasing concentrations of helenalin for 24 h (A) or with 2uM helenalin for the indicated timepoints (B), following which cells were harvested and cell lysates prepared and subjected to immunoblot analysis for cleaved PARP, caspase 3 and 9. Actin was used as a loading control.A2750 cells were subjected to caspase inhibitor, Z-VAD-fmk treatment two hours before addition of 2uM helenalin for 24 h, following which the cells were lysed and subjected to (C) immunoblot analysis for cleaved caspases and PARP or (D) harvested and analyzed for cell cycle flow distribution using FACS analysis. (E) Percent of cells in Sub-G1 in DMSO, helenalin or Z-VAD-fmk plus helenalin treatment. All figures represent data from three independent experiments.