Ebm 2 single quots kit

Cells, 5 × 10⁵ cells, were collected, resuspended in endothelial basal medium EBM2 media (EBM-2 Single Quots Kit, Lonza, Switzerland) and seeded in 12-well plates coated with growth factor-reduced Matrigel (Corning Inc., Corning, USA) for 24 h in the presence or absence of angiogenic factors (human epidermal growth factor (5 ng/mL), vascular endothelial growth factor (VEGF; 0.5 ng/mL), R3-insulin-like growth factor-1 (20 ng/mL), ascorbic acid (1 µg/mL), hydrocortisone (0.2 µg/mL), human basic fibroblast growth factor (FGF; 10 ng/mL), heparin (22.5 µg/mL) and FBS (0.02 mL/mL)). Experiments were performed in triplicate. Images of formed tubes were captured by Olympus IX81 microscope (Olympus, Japan).

Human umbilical vein ECs (HUVECs) and ECs from healthy donor and patients were cultured in EGM‐2 (EBM‐2 + SingleQuots™ Kit) and 2% foetal bovine serum (FBS) (Lonza). HUVECs and ECs were used between P2 and P6 passages. HEK293T (ATCC) were grown in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/l glucose, 2 mM l‐glutamine, without Na pyruvate (Lonza), 10% FBS and 1% penicillin/streptomycin (Pen/Strep). Reagents and doses were as follows: LysoTracker (Thermo Fisher), bafilomycin A1 (Sigma, 200 nM), torin‐1 (CST, 10 μM), BAY11‐7082 (Abcam, 300 nM), TYRPHOSTIN AG1288 (Abcam, 300 nM), SB202190 (Tocris, 300 nM), cycloheximide (Sigma, 2 μM) and MG132 (Sigma, 2 μM).