Fig. 3

MLN4924-induced autophagy was a survival signal and ATF3 silencing enhanced drug toxicity via inducing apoptosis. a-b The proliferation inhibition by MLN4924 was significantly increased by simultaneously blocking autophagy with siBeclin1(a) or CQ(b). The combination of siBeclin1 or CQ with MLN4924 in EC1 and Kyse450 cells significantly increased proliferation inhibition by ATPLite assay. c-d Apoptosis induced by MLN4924 was significantly increased by simultaneously blocking autophagy with siBeclin1 or CQ. The combination of siBeclin1(c)or CQ (d) with MLN4924 in EC1 and Kyse450 cells significantly increased apoptosis by Annexin V /PI double staining. e Beclin1 knockdown increased cleaved PARP expression induced by MLN4924. EC1 and Kyse450 cells were transfected with control or Beclin1 siRNA for 48 h and then treated with 0.6 μmol/L MLN4924 for 48 h. Knockdown efficiency and cleaved PARP were assessed by IB analysis. f-h Blocking autophagy by siATF3 remarkably suppressed cell proliferation and induced cell apoptosis compared with MLN4924 alone. EC1 and Kyse450 cells were transfected with control or ATF3 siRNA for 48 h and then treated with 0.6 μmol/L MLN4924 for 48 h. Cell proliferation suppressed by MLN4924 was further significantly decreased by simultaneously silencing of ATF3 expression (f). Knockdown efficiency and cleaved PARP were assessed by IB analysis (g). Apoptosis detection by either annexin V and PI double staining (h)