Enzastaurin induced apoptosis in WM cell line BCWM.1. (A) BCWM.1 cells were cultured with enzastaurin for 48 hours at doses that range from 2.5 to 20 μM, and the percentage of cells undergoing apoptosis was studied by annexin V and DAPI staining at 48 hours. Annexin V– and DAPI-positive cells were considered as apoptotic. Error bars represent the result and SD of 3 different experiments. (B) BCWM.1 cells were cultured with enzastaurin (5 to 20 μM) for 6 hours. Whole-cell lysates were subjected to Western blotting using anti–caspase 9, anti–caspase 3, anti-PARP, and α-tubulin antibodies. Enzastaurin induced a cleavage of caspases and PARP in a dose-dependent fashion. (C) BCWM.1 cells were cultured with enzastaurin (7.5 μM) for the indicated periods. Whole-cell lysates were subjected to Western blotting using anti–caspase 8, anti-PARP, and α-tubulin antibodies. Enzastaurin induced cleavage of caspase-8 and PARP in a time-dependent fashion. (D) Cell cycle was studied using DAPI staining by flow cytometry at 24 hours with control media or enzastaurin 2.5 to 10 μM (E2.5, E5, E10). Percentages indicate cells in sub-G₀/G₁ phase (G), G₀/G₁ phase (H), and G₂/M phase (I).