NVP-BEZ235 induces apoptosis and cell-cycle arrest in WM cells. (A) BCWM.1 cells were cultured with NVP-BEZ235 (10-20nM) for 48 hours, and apoptosis was performed using annexin/PI staining and flow cytometric analysis. (B) BCWM.1 cells were cultured with NVP-BEZ235 (5-100nM) for 16 hours. Whole-cell lysates were subjected to Western blotting using anti–caspase-8, anti–caspase-9, anti–caspase-3, anti-PARP, anti-Smac/DIABLO, anti–c-IAP1, anti-Mcl1, and anti–α-tubulin antibodies. (C) IgM-secreting cell lines (MEC.1; RL) were cultured with NVP-BEZ235 (5-100nM) for 16 hours. Whole-cell lysates were subjected to Western blotting using anti–caspase-8, anti–caspase-9, anti–caspase-3, anti-PARP, and anti–α-tubulin antibodies. (D) BCWM.1 cells were cultured with NVP-BEZ235 (5-100nM) for 16 hours. Whole-cell lysates were subjected to Western blotting using anti–p-foxO1/O4, anti–p-foxO3, anti-p27^kip1, anti-p21^Cip1, anti-BIM, anti-p53, anti-PUMA, anti-Bax, and anti–α-tubulin antibodies. (E) BCWM.1 were cultured with NVP-BEZ235 (0-50nM) for 24 hours, and cell-cycle analysis was performed by PI staining.