Hypoxia-induced pY654-β-catenin formation and EMT are dependent on ROS. (a) H358 cells were cultured in normoxia or hypoxia for 2 h ± ROS scavenger NAC (10 mM). APF signal (green) indicates ROS activity and the fluorescence intensity was quantified by Image J. * indicates p< 0.05 by t-test. Scale bar, 50 µm. (b) H358 cells were cultured in normoxia or hypoxia for 4 h or treated with TGFβ1 (4 ng/ml) for 2 h ± EUK-134 or NAC. The lysates were immunoprecipitated for pY654-β-catenin and the lysates were blotted for β-catenin, pY416-Src, Src, HIF1α, and β-actin. (c) Immumoblots for Snail1 in H358 cells under normoxia or hypoxia for 24 h ± NAC. The numbers indicate relative Snail1 level normalized to β-actin. (d) H358 cells were incubated in normoxia or hypoxia for 24 h ± EUK-134 or NAC. The conditioned media were concentrated for zymography with recombinant MMP-9 as positive control.