Neuroblastoma cell growth assay and Western blot assay in vitro

MYCN amplified cell lines, IMR5 and Kelly, and non-MYCN amplified cell lines, SY5Y and SKNAS, were used in this study. IMR5 and SY5Y have normal TP53 gene, whereas Kelly and SKNAS cells harbor mutations in TP53 gene. IMR5 and Kelly cells express high levels of MYCN protein, whereas SY5Y and SKNAS cells express high levels of MYC protein. IMR5, a clone of IMR32, was a gift of Roger H. Kennett (University of Pennsylvania, PA. USA) who derived this clone. Kelly and SY5Y cell lines were obtained from Sigma (St. Louis, MO, USA). SKNAS cell line was a gift of Susan L. Cohn (University of Chicago, IL, USA). Growth of these neuroblastoma cell lines in response to CX-5461 and Halofuginone was assessed by MTS assay as described in our previous study [46]. Neuroblastoma cells were treated with the small molecule inhibitors at the concentrations and durations indicated in Figure ​Figure4.4. Cells were then subjected to Western blot assay using anti-pan-MYC antibody, NCM II 143 [43] and anti-β-Actin antibody (Santa Cruz) as described in our previous studies [47, 48].