NVP-BEZ235 up-regulates the MEK/ERK pathway, and ERK inihibition increases NVP-BEZ235–induced cytotoxicity. (A) BCWM.1 cells were cultured with NVP-BEZ235 (5-100nM) for 6 hours. Whole-cell lysates were subjected to Western blotting using anti–p-ERK, anti-ERK1/2, anti–p-c-Raf, anti-Rheb, and α-tubulin antibodies. (B) BCWM.1 cells were cultured for 48 hours with NVP-BEZ235 (5nM, 10nM) in the absence or presence of MEK1/2 inhibitor U0126 (5μM, 10μM). Cytotoxicity was assessed by the MTT assay. Data represent mean (± SD) of triplicate experiments. (C) BCWM.1 cells were cultured with NVP-BEZ235 (5nM, 10nM) in the absence or presence of MEK1/2 inhibitor U0126 (5μM, 10μM) for 6 hours. Whole-cell lysates were subjected to Western blotting using anti–p-ERK, anti-ERK1/2, and α-tubulin antibodies. (D) BCWM.1 cells were cultured with NVP-BEZ235 (5nM, 10nM) in the absence or presence of MEK1/2 inhibitor U0126 (5μM, 10μM) for 16 hours. Whole-cell lysates were subjected to Western blotting using anti–caspase-9, anti–caspase-3, anti–caspase-8, anti-PARP, and anti–α-tubulin antibodies.