Figure 5

GANT-61 induces a lower level of autophagy in MYCN non-amplified NB cells. (A) FISH analysis of MYCN copy number in SK-N-AS and SH-SY5Y cells. White arrows indicate positive signals. Scale bars, 10μm. (B) SK-N-AS and SH-SY5Y cells were treated with GANT-61 at various concentration (0.1-15μM) for indicated times and the cytotoxicity was measured using a MTT assay. The percentage of viable cells was calculated as a ratio of treated to control cells. (C) The effect of lysosomal inhibitor BafA1 on LC3 conversion in SK-N-AS and SH-SY5Y cells treated with GANT-61. Cells were first treated with 200nM BafA1 for 30 min and then treated with 10μM GANT-61 for 12h. The LC3 II/β-ACTIN ratio was listed under blots. (D) Fluorescence microscopy of AO stained SK-N-AS, SH-SY5Y, NBL-W-S and NBL-W-S cells treated with 10μM GANT61 for 48h. Top row, phase contrast; Second row, green fluorescence; Third row, red fluorescence; Bottom row, merged images. Scale bars, 100μm. (E) Flow cytometry histograms of AO stained NB cells. The last plot summarizes four histogram profiles to visualize the difference of fluorescence intensities in MYCN non-amplified and amplified NB cells. (F) MYCN overexpression was verified by Western blot analysis with SK-N-AS and SH-SY5Y cells transfected with MYCN plasmid. The MYCN/β-ACTIN ratio was listed under blots. (G) The effect of lysosomal inhibitor BafA1 on LC3 conversion in SK-N-AS/MYCN and SH-SY5Y/MYCN cells treated with GANT-61. Cells were first treated with 200nM BafA1 for 30 min and then treated with 10μM GANT-61 for 12h. SK-N-AS/MYCN: SK-N-AS cells overexpressing MYCN. SH-SY5Y/MYCN: SH-SY5Y cells overexpressing MYCN. Equal loading and transfer were verified by re-probing membranes with anti-β-actin antibody. The LC3 II/β-ACTIN ratio was listed under blots. Data is expressed as the mean ± SD. *P < 0.05, **P < 0.01.