NADPH oxidase 4 regulates anoikis resistance of gastric cancer cells through the generation of reactive oxygen species and the induction of EGFR

a ROS generation by adherent or suspension MKN-45 and AGS cultures was measured by flow cytometry using carboxy-H2DCFDA. The solution that contains GC cells without treatment using ROS-sensitive substrate H2DCFDA was used as the negative control. b ROS generation by MKN-45 and AGS suspension cultures treated with H2O or NAC was measured by flow cytometry using carboxy-H2DCFDA. c Anoikis rates of MKN-45 and AGS cells treated with H2O or NAC in suspension conditions for 24？h were measured by flow cytometry using Annexin V-FITC. ***P？<？0.001. d MKN-45 and AGS cells were treated with exogenous H2O2 (0, 0.1, 0.5, 1, 2, 5, 10, 50, 100？μM), and the rate of anoikis detected by flow cytometry using Annexin V-FITC. e Cellular morphology of MKN-45 and AGS suspension cells treated with H2O or NAC captured by microphotography after 24？h of culture (magnification ×100). f The average number of aggregated MKN-45 and AGS suspension cells treated with H2O or NAC in each field was measured after 24？h of culturing. **P？<？0.01. All experiments were performed three independent time