GANT-61 induces cell cytotoxicity and apoptosis in NB cells. (A) FISH analysis of MYCN amplification in NBL-W-S cells. Scale bar, 10μm. (B) NBL-W-S cells were treated with GANT-61 at various concentration (0.1-15μM) for indicated times and the cytotoxicity was measured by MTT assay. The percentage of viable cells was calculated as a ratio of treated to control cells. Data is expressed as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01. (C) Representative flow cytometry analysis of apoptosis after Annexin V and PI-double staining. NBL-W-S cells were treated with the indicated concentration of GANT-61 for 48h. (D) Histogram of flow cytometry analyses from 3 independent experiments. *P < 0.05, **P < 0.01. (E) Western blot analysis was carried out to detect the expression of apoptosis-related proteins. NBL-W-S cells were treated with indicated concentrations of GANT-61 for 48h. The BCL2/β-ACTIN and Cleaved-CASPASE3/β-ACTIN ratios were determined by densitometry (mean ± SD), *P < 0.05, **P < 0.01. The expression of anti-β-ACTIN is used as a loading control. CON, control.