NVP-BEZ235 induced G0/G1 cell cycle arrest but not apoptosis in neuroblastoma cells. (A, B) Morphological changes in SH-SY5Y and SK-N-MC cells treated with NVP-BEZ235 (500, 1000 nM) for 24 h followed by AO/EB staining (magnification, 100 ×). Normal cells showed green fluorescence by acridine orange (AO) staining, apoptotic cells showed yellow-orange fluorescence by merge of ethidium bromide (EB) staining and AO staining. (C, D) Western blot analysis for cleaved caspase-3 and cleaved PARP in SH-SY5Y and SK-N-MC cells treated with serial NVP-BEZ235 for 24 h. (E, F) SH-SY5Y and SK-N-MC cells were treated with NVP-BEZ235 (100, 200 nM) for 24 h. Cell cycles were detected by flowcytometry analysis which determining cellular DNA content with hypotonic PI solution. (G, H) SH-SY5Y and SK-N-MC cells were treated with NVP-BEZ235 (200 nM) for the indicated times. Cyclin D1 and cyclin E1 proteins were performed by western blot analysis.