Culturing HeLa and Mouse Endothelial Cells

HeLa cells (ATCC, CCL-2) were grown in DMEM-High Glucose (Euroclone, Pero, Italy) supplemented with 10% FBS (Euroclone), 4 mM L-glutamine (Lonza, Basel, Switzerland), and 100 U/L penicillin/streptomycin (Euroclone). Mouse endothelial cells (moEC), previously referred as vascular endothelial (VE) cadherin-positive ECs and described in [28 (link),31 (link),35 (link),36 (link)], were cultured in DMEM-High Glucose (Lonza) with 10% FBS, 2 mM L-glutamine (Lonza), 100 U/L penicillin/streptomycin (Euroclone), 1 mM sodium pyruvate (Sigma–Aldrich, Merck, Darmstadt, Germany), 25 mM HEPES (Sigma–Aldrich), 100 µg/mL heparin (from porcine intestinal mucosa, Sigma–Aldrich), and 50 μg/mL EC growth supplement (ECGS from bovine pituitary gland, Sigma–Aldrich). Before seeding, plates were coated with 0.1% porcine gelatin (Difco) and incubated overnight at 37 °C. Cells were maintained in a humidified, 5% CO₂ atmosphere at 37 °C. For VEGF stimulation, moEC were grown in a serum-starved (0.2% FBS) medium, without ECGS supplementation, for 2 h prior to treatment with recombinant murine VEGF-165 (100 ng/mL, PeproTech, EC Ltd., London, UK) for 24 h.