Effects of an apoptotic inhibitor on GANT-61 treated NB cells. (A) NBL-W-S cells were pre-treated with Z-VAD-FMK (50 μM) for 1h and then with 10uM GANT-61 for 48 h. Cell viability was measured by MTT assay. Data are expressed as the mean ± SD of three independent experiments. *P < 0.05. (B) The inhibitory effects of Z-VAD-FMK on apoptosis were detected by Western blot analysis with anti-BCL-2 and anti-cleaved CASPASE3 antibodies. The BCL2/β-ACTIN and cleaved-CASPASE3/β-ACTIN ratios were listed under blots (mean ± SD), *P < 0.05. (C) The expression of autophagic proteins was analyzed by Western blot with anti-LC3 and anti-BECLIN-1 antibodies. The densitometry ratios of LC3 II/β-ACTIN and BECLIN1/β-ACTIN were listed under blots, (mean ± SD), *P < 0.05, n.s., no statistical significance. (D) Flow cytometry histogram of AO stained cells treated with the indicated drug. CON, control. Equal loading and transfer were verified by re-probing membranes with anti-β-ACTIN antibody in Western blot analysis.