Egm bulletkit

BOECs were isolated from healthy donors as described by Ramirez et al.^{24 (link)}. For all experiments, except the multi-omics and secretome experiments, three different pools of three unique BOEC donors (mixed sexes and ages) were used (Supplementary Table 2). For the multi-omics and secretome experiments, BOECs from three different donors (mixed sexes and ages) were pooled. Culture flasks and dishes were coated with collagen type I (50 µg/ml, BD biosciences) for 1 h prior to use. Cells were cultured in EC basal medium (Lonza) supplemented with 18% FCS (Bodinco) and EGM bulletkit (Lonza) unless stated otherwise. For SILAC labeling, BOECs were maintained for 5 passages as described by Beguin et al.^{36 (link)} in custom-made EGM medium (Lonza), containing EBM2 medium (not containing Arganine and Lysine) (Lonza), EGM bulletkit (Lonza) and 18% FCS for passages 1–3 and in 18% 1 kDa dialyzed FCS for passages 4–5. Cells were SILAC labeled by the addition of isotope-labeled amino acids during all passages (light: Arg0 and Lys0, medium: Arg6 and Lys4, heavy: Arg10 and Lys8, Cambridge Isotopes). After five passage incorporation of labeled amino acids reached >95% in the total proteome.

HUVECs (bought: Lonza Switzerland, Lot 220213, 80016, 482213) were cultivated in endothelial growth medium (EGM, EGM BulletKit, Lonza, Switzerland) until 80% confluence. Tube formation was assessed as published^{64 (link)}. In brief, 5 × 10⁴ cells were plated per 24 pretreated/coated well with 50 µl growth factor reduced Matrigel (BD Biosciences. NJ, USA). To assess the effect extracellular succinate has on tube formation, cells were cultivated in EGM with growth factors, penicillin/streptomycin, and 2% FCS, ± succinate: 50 µM and 500 µM (+/+), in EGM without growth factors, supplemented with penicillin/streptomycin and 2% FCS ± succinate: 50 µM and 500 µM (−/+) and in EGM without growth factors, without 2% FCS but supplemented with penicillin/streptomycin ± succinate: 50 µM and 500 µM (−/−). Tube formation was documented after 18 h of culture by bright field microscopy. Total tube formation length per well was measured using ImageJ (version 1.44; http://rsbweb.nih.gov/ij/) and effect of succinate compared to control for each condition.