NVP-BEZ235 activates apoptosis in osteosarcoma cells. MG-63 cells were treated with indicated concentration of NVP-BEZ235 (NVP) for 42 hrs, cell apoptosis was tested by caspase-3 activity assay (A), Histone-DNA ELISA assay (B) and TUNEL staining assay (C). The expression of caspase-3 (regular and cleaved) and tubulin (equal loading) was tested by Western blotting (A, upper panel). MG-63 cells were pretreated with z-VAD-fmk (zvad, 25 μM), z-DVED-fmk (dved, 25 μM) or z-ITED-fmk (ited, 25 μM) for 1 hr, followed by NVP-BEZ235 (200 nM) stimulation, cells were further cultured, before cell growth (D) and colony formation (E) were tested. Apoptosis in NVP-BEZ235 (200 nM, 42 hrs)-treated U2OS and SaOs-2 cells was analyzed by TUNEL staining assay (F). n = 5 for each assay. *P < 0.05 vs. group “C” (A-C, F). #P < 0.05 vs. NVP-BEZ235 only group (D-E).