Huvecs cc 2517

HUVECs (CC-2517) were purchased from Lonza (Basel, Switzerland) and cultured in complete endothelial basal medium (EBM) containing an endothelial cell growth medium kit (CC-3125 from Lonza). Trypsin-EDTA (0.25%) and trypsin neutralizing solution were used for HUVEC subculture. HAOECs (PCS-100-011) were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and cultured in vascular cell basal medium (PCS-100-030 from ATCC) containing an endothelial cell growth kit BBE (PCS-100-040 from ATCC). Trypsin-EDTA for primary cells (PCS-999-003 from ATCC) and trypsin neutralizing solution (PCS-999-004 from ATCC) were used for HAOEC subculture. All experiments were performed only between passages 3 and 5. All the cells were cultured in a humidified atmosphere of 5% CO₂ and 21% O₂ for normoxia. For hypoxic treatment, the cells were incubated in an InvivO2 400 Hypoxia Workstation chamber (RUSKINN, Bridgend, UK) at 37°C in a humidified atmosphere of 5% CO₂ and 1% O₂. DMOG, CoCl₂, NaHS, P-(4-methoxyphenyl)-P-4-morpholinyl-phosphinodithioic acid (GYY4137), AOAA, DTT, and MG132 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Biotinylated- HIF-1α 19-mer peptide (HIF-1α, 556 to 574) and FLAG-CBS-pcDNA3 (OHu26151) plasmid obtained from GenScript (Piscataway, NJ, USA). HA-PHD2-pcDNA3 was obtained from Addgene plasmid no. 18963.