Phosphonoformic acid

HUVECs were purchased from Lonza (Allendale, NJ), and cultured with endothelial cell growth medium-2 (EGM-2) bullet kit (Lonza) in a humidified atmosphere of 5% CO₂ at 37°C. Pooled complement human serum was purchased from Innovative Research, Inc (Novi, MI) and used as normal human serum (NHS) in all experiments. Heat-inactivation was performed with this serum at 56°C for 30 min. Factor B-depleted human serum, C3-depleted human serum, C6-depleted human serum, purified properdin, purified C3 and purified factor B were purchased from Quidel Corporation (San Diego, CA). Factor P (properdin)-depleted human serum was purchased from Complement Technology, Inc. (Tyler, Tx). EDTA, EGTA, heparin, GW4869, methyl-β–cyclodextrin, chlorpromazine, and phosphonoformic acid were obtained from Sigma-Aldrich (St. Louis, MO).

VSMCs were used from same passage (8 or 9), considering 1:3 splitting during trypsinization. Cells were grown to confluence and used after a quiescent intermediate step (overnight in culture media containing 0.1% fetal bovine serum). Calcification assays were performed on cells incubated for 7 days in MEM supplemented with 1 mM l‐glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, 0.1% fetal bovine serum, and 2 mM phosphate (phosphate‐calcifying medium), as described previously (Villa‐Bellosta, 2018; Villa‐Bellosta et al, 2011, p.; Villa‐Bellosta & Hamczyk, 2015). Phosphate‐calcifying medium was replaced every day. To quantify the calcium content of VSMC, wells were treated with 0.6 M HCl overnight at 4°C and analyzed using a colorimetric QuantiChrom Calcium Assay Kit (BioAssay System, Hayward, CA). Phosphate, magnesium chloride, phosphonoformic acid, and pyrophosphate were obtained from Sigma‐Aldrich. Cells were fixed as described previously (Villa‐Bellosta & Sorribas, 2009).