GANT-61 induces autophagy in NB cells. (A) MDC staining revealed that autophagy was activated in NBL-W-S cells after 48h treatment with GANT-61. Scale bars, top: 100μm, bottom: 50μm. (B) Fluorescence microscopy of AO stained NBL-W-S cells treated with the indicated drug for 48h. Top row, phase contrast images; Second row, images captured through green fluorescence filter; Third row, images captured through red fluorescence filter; Bottom row, merged images. Scale bars, 100μm. (C) Flow cytometry analysis of AO stained cells in panel B. (D) The expression of autophagic proteins in NBL-W-S cells treated with various concentrations of GANT-61 for 48h. The densitometry ratios of LC3 II/β-ACTIN, ATG5/β-ACTIN and BECLIN1/β-ACTIN were plotted as histogram (mean ± SD), *P < 0.05. (E) Effect of lysosomal inhibitor BafA1 on autophagic flux induced by GANT-61. LC3 immunoblotting was performed to evaluate LC3 conversion. NBL-W-S cells were first treated with 200nM BafA1 for 30 min and then treated with 10μM GANT-61 for 4h, 12h, 24h or 48h. The LC3 II/β-ACTIN ratio at different time points was plotted as histogram (mean ± SD), *P < 0.05, **P < 0.01. (F) Immunofluorescence with LC3 antibody on NBL-W-S cells after 48h GANT-61 treatment. Scale bars, top: 500μm, bottom: 20μm. CON, control. (G) Quantification of cells with a number of LC3 puncta five times higher than basal level in panel F, **P < 0.01. (H) NBL-W-S cells transfected with GFP-LC3 plasmids were treated with GANT-61 for 48h. A puncta pattern of GFP-LC3 was formed after drug treatment. Scale bar,20μm. (I) Quantification of cells with GFP-LC3 puncta in panel H, **P < 0.01. Equal loading and transfer were verified by re-probing membranes with anti-β-ACTIN antibody in Western blot analysis.