Figure 3

Effects of autophagic inhibition on GANT-61 treated NB cells. (A) 3-MA pre-treatment enhanced GANT-61 toxicity on NBL-W-S cells. Cell viability was measured by MTT assay. (B) The effect of 3-MA on the expression of autophagic proteins in NBL-W-S cells. Western blot analysis was performed with anti-LC3, anti-BECLIN-1 and anti-ATG5 antibodies. The densitometry ratios of LC3 II/β-ACTIN, BECLIN1/β-ACTIN and ATG5/β-ACTIN were plotted as histogram. (C) 3-MA pre-treatment did not affect the level of AKT phosphorylation in NBL-W-S cells after GANT-61 treatment. Values of P-AKT/AKT ratio were listed under p-AKT blots. (D) 3-MA increased cell apoptosis in GANT-61 treated NBL-W-S cells. Apoptotic cells were analyzed using Annexin V/PI double staining. (E) The effect of 3-MA on apoptotic protein expression. Western blot analysis was performed with anti-BCL-2 and anti-cleaved CASPASE3 antibodies. The BCL2/β-ACTIN and Cleaved-CASPASE3/β-ACTIN ratios were listed under blots. (F) Knockdown of essential autophagic component ATG5 or ATG7 by shRNA in NBL-W-S cells was verified by Western blot analysis with anti-ATG5 or anti-ATG7 antibodies. The ATG-5/β-ACTIN and ATG-7/β-ACTIN ratios were listed under blots. (G) Knockdown of ATG5 or ATG7 completely abolished GANT-61 induced LC3 conversion, even in the presence of lysosomal inhibitor BafA1. Knockdown 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. (H) A higher level of cleaved CASPASE3 and a lower level of BCL2 were detected in ATG5 or ATG7 knockdown NBL-W-S cells. The BCL2/β-ACTIN and cleaved-CASPASE3/β-ACTIN ratios were plotted as histogram. (I) Representative flow cytometry analysis of apoptosis in GANT-61 treated cells. siCON: scramble shRNA knockdown control, siATG5: ATG5 shRNA knockdown, siATG7: ATG7 shRNA knockdown cells. CON, control. Data are expressed as the mean ± SD, *P < 0.05, **P < 0.01, n.s., no statistical significance.