Fig. 1: Long-term severe hypoxia adaptation induces EMT-like morphological changes.

A The definitions of hypoxia and anoxia commonly used in the literature and their effect on HIF activity. B Confocal microscopy images of B16-HG cells under normal culture conditions (left), or after CoCl₂ treatment (200 µM, 24 h). Scale bars: top = 20 µm; bottom = 10 µm. Green: HIF1α-GFP. Red: mCherry (cytoplasmic). C Definition of the Long-Term Hypoxia (LTHY) time course. D GFP levels of B16-HG cells over the course of the LTHY protocol. Numbers represent geometric Mean Fluorescent Intensity (geoMFI) of GFP signal. E B16-HG cell morphology under normal tissue culture conditions (left). B16-HG cell morphology after reaching the 0.3%O₂ timepoint of the LTHY protocol (right). Scale bars: top = 100 µm; bottom = 50 µm. F Western blot (top) and analysis (bottom) of B16-HG harvested at indicated LTHY time points and probed for EMT-associated E-Cadherin (E-Cad), N-cadherin (N-Cad) and vimentin (Vim) and loading control calnexin (Caln). Band intensities were normalized to loading control. Data expressed as expression fold change relative to the 5%O₂ condition.