Culturing and Validating Cell Lines for Hypoxia Studies

All cell lines, with the exception of SUMs, were obtained from the ATCC. SUMs were purchased from Asterand Bioscience. Cells were cultured per company provided protocols. The MCF10A and MCF10A ER-expressing cells were a kind gift from Ben Ho Park and cultured as previously described (27 (link)). CRISPR edited MCF-7 HIF-1α, HIF-2α and control knockout cell lines were previously generated in our laboratory (8 (link)). All cell lines used in the study were authenticated by STR sequencing and confirmed to be mycoplasma free. Cells were maintained in a humidified environment at 37°C and 5% CO₂ during culture and live-cell imaging. Hypoxic cells were maintained at 37°C in an invivo 200 hypoxia workstation equipped with a digitally controlled oxygen regulator and maintained at 1% O₂, 5% CO₂, and 94% N₂. Live-cell microscopy experiments were conducted in a McCoy incubator maintained at 1% O₂, 5% CO₂, and 94% N₂ and imaged with a Lionheart microscope (Biotek).