Fig. 2.

ATF3 silencing blocked MLN4924-induced autophagy. a Treatment of MLN4924 induced the conversion of LC3-I to LC3-II in a dose dependent manner in both EC1 and Kyse450 cells. Cells were treated with the indicated concentrations of MLN4924 for 48 h and cells were collected and subjected to IB analysis for the expression of LC3. Actin was used as an equal loading control. b MLN4924 induced the conversion of LC3-I to LC3-II in a time-dependent manner in both EC1 and Kyse450 cells. Cells were treated with the 0.6 μmol/L of MLN4924 for indicated time and then cells were collected and subjected to IB analysis for the expression of LC3. Actin was used as an equal loading control. c-d Autophagic flux analysis. EC1 and Kyse450 cells, treated with DMSO or MLN4924 for 48 h, were incubated with or without CQ (50 μM), BafA1 (50 nM) or 3MA (5 mM) for 8 h. The treated cells were then collected and subjected to IB analysis. Actin was used as an equal loading control. e-f ATF3 is required for MLN4924 induced autophagy in esophageal cancer cells. 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. ATF3 Knockdown efficiency and conversion of LC3-I to LC3-II were assessed by IB analysis. g ATF3 overexpression increase autophagy. EC1 and Kyse450 cells were transfected with pcDNA3-ATF3 (0 μg, 0.5 μg, 1 μg, 2 μg) for 48 h. ATF3 overexpression efficiency and conversion of LC3-I to LC3-II were assessed by IB analysis