Endothelial cell growth supplements

Gαq/11 WT and Gαq/11 KO MEFs were a kind gift from Dr. S. Offermanns (Max-Planck-Institute for Heart and Lung Research, Germany). Atg5 WT and Atg5 KO MEFs were provided by Dr. N. Mizushima (University of Tokyo, Tokyo, Japan) and DREADD-Gq-HEK-293 cells (human female) by Dr. Silvio Gutkind (University of San Diego, California, USA)^{22 (link)}. CHO cells overexpressing the muscarinic M3 acetylcholine receptor (CHO-M3) (mice female) were a kind gift from Dr. A. B. Tobin (University of Glasgow, UK). Cell lines were authenticated by short tandem repeat (STR) or DNA barcoding analysis and tested for mycoplasma contamination. MEFs and DREADD-Gq cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco Life Technologies), CHO-M3 cells in αMEM (Gibco Life Technologies), supplemented with 10% (v/v) fetal bovine serum (Sigma-Aldrich) and 1 mM l-glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin. Human umbilical vein endothelial cells (HUVEC) (human male) were grown in 199 Medium (Life Technologies, without NaHCO₃) supplemented with 2.2 g/L NaHCO₃ (pH 7.4), 1 mM l-glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 15% FBS. After the first passage, cells were supplemented with 50 µg/ml endothelial cell growth supplements (Sigma-Aldrich) and 100 µg/ml heparin (Sigma-Aldrich). All cell lines were maintained at 37 °C in a humidified 5% CO₂ atmosphere.

Human umbilical vein endothelial cells (HUVECs; BCRC number: H‐UV001) were purchased from Food Industry Research and Development Institute (Hsinchu, Taiwan). The cells were cultured on the 100‐mm gelatin‐coated dishes maintained in M199 medium (#31100‐035; Invitrogen) containing 10% FBS (#10099‐141; Invitrogen), 25 unit/ml heparin, 30 μg/mL endothelial cell growth supplements (#E0760; Sigma‐Aldrich), 2 mM L‐glutamine, 100 units/mL penicillin G, and 100 μg/mL streptomycin sulfates, 1.5 g/L sodium bicarbonate. Cells of the 3^rd–5th passage were used. VSMCs obtained from Food Industry Research and Development Institute (Hsinchu, Taiwan) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L‐glutamine 100 mg streptomycin/mL, and 100 units of penicillin. All cells were incubated at 37C and humidified 5% CO₂. Culture media were changed every 2 days.

A total of 20 consecutive patients with CSF were recruited with the approval of the Ethics Committee of The Fifth Affiliated Hospital of Xinjiang Medical University (XYDWFYLS-2019-08). At the same time, 20 contemporary patients with angiographically normal coronary flow were recruited as controls. The exclusion criteria were adopted from those in a previous study [21 (link)]. Written informed consents were obtained from all patients enrolled.
We collected fasting 10 ml peripheral blood samples from all the study participants in the morning. The peripheral blood mononuclear cells were isolated by Ficoll gradient centrifugation and then inoculated on to a culture plate coated with human fibronectin (BD, U.S.A.). The M199 medium supplemented with 20% foetal bovine serum (Thermo Fisher Scientific, Waltham, MA, U.S.A.), 30 μg/ml endothelial cell growth supplements (Sigma–Aldrich, St. Louis, MO, U.S.A.), 90 μg/ml heparin (Selleck Chemicals, Houston, TX, U.S.A.), and 1% antibiotics solution was changed every 3 days. The adherent cells were screened for markers of peripheral blood EPCs, 7 days later. We identified Dil-AcLDL and FITC-UEA-I (FITC-lectin) (Sigma–Aldrich) double-stained positive cells as differentiated EPCs by IF staining. The purity of isolated EPCs was determined by flow cytometry using anti-CD34 and anti-VEGFR antibodies.

Primary human umbilical vein endothelial cells (HUVECs) were seeded in fibronectin coated tissue culture dishes and cultured in MCDB medium (Sigma) supplemented with 15% FBS, endothelial cell growth supplements (Sigma) and heparin. Murine RM1 prostate cancer cells were cultured in DMEM/F12 medium (Hyclone) supplemented with 10% FBS. All these cultures were maintained at 37 °C in a humidified tissue culture incubator with an atmosphere of 5% CO₂. Targefect reagents (Targeting Systems) were used to transfect HUVECs with siRNA duplex or β3-endonexin plasmid for knocking down endogenous β3-endonexin or overexpressing exogenous β3-endonexin in HUVECs.

Human umbilical vein endothelial cells (HUVECs) were obtained from American Type Culture Collection and cultured in DMEM medium (Hyclone, USA) supplemented with 10% foetal bovine serum (Hyclone), 100 U/mL penicillin, 100 μg/mL streptomycin, 0.1 mg/mL heparin and 0.03 mg/mL endothelial cell growth supplements (Sigma, St. Luis, MO, USA) at 37°C under a humidified atmosphere of 95% air with 5% CO₂ to allow the cells to grow and form a monolayer in the flask. After confluence, cells were trypsinized using 0.25% trypsin in Hanks buffer for 2 minutes and resuspended in complete culture medium.