Fig. 4

ATF3 accumulation is mediated by ROS upon MLN4924 treatment. a MLN4924 treatment induced ROS generation. EC1 and Kyse450 cells were treated with different dose of MLN4924 for 24 h and ROS generation was determined by H2-DCFDA staining. b NAC, a classical ROS scavenger, attenuated ATF3 expression. EC1 and Kyse450 cells were treated with 0.6 μmol/L MLN4924 alone or MLN4924 + NAC for 48 h and subjected to IB analysis for the expression of ATF3. Actin was used as an equal loading control. c Reduction of ROS by NAC significantly inhibited MLN4924-induced autophagy. EC1 and Kyse450 Cells were treated with 0.6 μmol/L of MLN4924 alone or MLN4924 + NAC for 48 h and subjected to IB analysis for the expression of LC3. Actin was used as an equal loading control. d MLN4924 decreased the expression of Catalase and did not affect the expression of SOD1. EC1 and Kyse450 Cells were treated with the indicated concentrations of MLN4924 for 48 h and subjected to IB analysis for the expression of Catalase, SOD1. Actin was used as an equal loading control. e MLN4924 decreased Catalase at the transcriptional level. EC1 and Kyse450 cells were treated with different dose of MLN4924 for 24 h and RNA were extracted and analyzed by qPCR (normalized to GAPDH). f Catalase overexpression attenuated MLN4924-induced ROS production. EC1 and Kyse450 cells were transfected with 2 μg p-CMV-Catalase for 24 h and then treated with 0.6 μmol/L MLN4924 for 24 h. ROS generation was determined by H2-DCFDA staining. g Catalase overexpression reversed MLN4924-induced autophagy. EC1 and Kyse450 cells were transfected with 2 μg p-CMV-Catalase for 24 h and then treated with 0.6 μmol/L MLN4924 for 24 h. Catalase overexpression efficiency and conversion of LC3-I to LC3-II were assessed by IB analysis