Hypoxia-induced EMT requires both HIF1α and β-catenin expression. (a) HIF1α and pY654-β-catenin sequential immunoprecipitation of AECTs under normoxia or hypoxia ± Src inhibitor for 4 h. (b) Immortalized AECTs with floxed β-catenin were infected with Ad-Cre or Ad-GFP and cultured in hypoxia for 56 h. Cells were stained for E-cadherin (green) and β-catenin (orange). Scale bar, 50 µm. (c–e) AECTs were infected with Ad-Cre or Ad-GFP and incubated in hypoxia for 56 h ± ALK5 inhibitor SB431542 or Src inhibitor SU6656 (5 µM) before immunoblotting for proteins as indicated in panels (c) and (d). Conditioned medium was concentrated 10× for zymography with recombinant MMP-2 as positive control (e). (f, g) AECTs infected with Ad-Cre or Ad-GFP (f) or transfected with HIF1α or non-targeting control siRNA (g) were incubated in normoxia or hypoxia for 24 h before qRT-PCR analysis. Collagen I, Snail1, β-catenin, HIF1α mRNA levels were normalized to β-actin mRNA level. * indicates p<0.05 by t-test.