Hypoxia induces Src kinase-dependent pY654-β-catenin/HIF1α complexes and EMT in human lung adenocarcinoma cells. (a) pY654-β-catenin immunoprecipitation of H358 cells incubated in normoxia (21% O2) (N) or hypoxia (1% O2) (H) for 2 h or 12 h. (b) Immunoblots for HIF1α, pY416-Src, total Src, and Snail1 of H358 cells under normoxia or hypoxia for 24 h ± Src inhibitor. TGFβ1 stimulation-positive control; β-actin blot-loading control. (c, d) Sequential immunoprecipitation for pY654-β-catenin and total β-catenin (c) or two rounds of HIF1α and one round of pY654-β-catenin (d) of H358 cells under normoxia or hypoxia or hypoxia with Src inhibitor SU6656 (5 µM) (H+SU) for 4 h. (e) Myc immunoprecipitation of H358 cells expressing Myc-tagged wt (W) or Y654F mutant (F) β-catenin under normoxia or hypoxia for 4 h. (f) Fibronectin (orange) and E-cadherin (green) staining of H358 cells under normoxia or hypoxia for 56 h ± Src inhibitor. Scale bar, 100 µm.