Chromatin immunoprecipitation: Following the different treatments, proteins and chromatin were cross-linked with 1% formaldehyde at room temperature for 10 min. Glycine was added to a final concentration of 0.125 M for 5 min to quench the reaction. Cells were harvested into 400 µL of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1, 1 mM PMSF, 1 μg/mL leupeptin, 1 μg/mL aprotinin) and left on ice for 10 min. Samples were then sonicated at 4 °C eight times for 15 s with a 30 s gap between each sonication at 50% amplitude (Sonics Vibra-Cell # VCX130). Supernatants were recovered by centrifugation (12,000 rpm for 10 min at 4 °C) before 10% of each sample was stored as input. Remaining samples were split into 120 µL aliquots before being diluted 10-fold in dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 8.1). Diluted samples were precleared for 2 h at 4 °C by incubating with 2 µg of sheared salmon sperm DNA and 20 µL of protein G-Sepharose (50% slurry). Immunoprecipitations were performed overnight with 2 µg of specific antibody or IgG control, with the addition of Brij 35 detergent to a final concentration of 0.1%. Immune complexes were captured by incubation with 40 µL of protein G-Sepharose (50% slurry) and 2 µg salmon sperm DNA for 1 h at 4 °C. The immunoprecipitates were washed sequentially for 5 min each at 4 °C in Wash Buffer 1 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), Wash Buffer 2 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), and Wash Buffer 3 (0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1). Beads were washed twice with Tris-EDTA buffer and eluted with 120 µL of Elution Buffer (1% SDS, 0.1 M NaHCO3). Cross-links were reversed by incubation with 0.2 M NaCl at 65 °C overnight and Proteinase K (20 µg each), 40 mM Tris-HCl pH 6.5, and 10 mM EDTA for 1 h at 45 °C was used to remove protein. DNA was purified using a PCR-product purification kit according to manufacturer’s instructions (NBS Biologicals #NBS363). DNA (3 µL) was used for qPCR with the following primers for the putative hypoxia response element (HRE) sites at the KDM2A and KDM2B promoters: