Endothelial Cell Culture Conditions

Human Coronary Artery Endothelial Cells (HCAECs) from ATCC (Manassas, VA, USA), Human Aortic Endothelial Cells (HAECs) from Thermo Fisher Scientific (Waltham, MA, USA), and Human Umbilical Vein Endothelial Cells (HUVECs) from Thermo Fisher Scientific were grown on 1.5% gelatin-coated tissue culture dishes and maintained in medium M200 (Thermo Fisher Scientific, Waltham, MA, USA) containing 2% fetal bovine serum (FBS) and growth factors (EGM-2, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C with 5% CO₂. Experiments were performed in actively proliferating ECs at passages from 3 to 5. KRIT1^−/− and KRIT1^+/+ mouse embryonic fibroblast (MEF) cell lines [21 (link)] were cultured at 37 °C and 5% CO₂ in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, and 100 U/mL penicillin/streptomycin. Cell treatments were performed with either Tumor Necrosis Factor α (TNF-α) (10 ng/mL for 24 h) or Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid) (0.5 or 1 mM for 24 h).