HUVEC were obtained from Lonza (Breda, The Netherlands) and the Endothelial Cell Facility of University Medical Center Groningen, The Netherlands. Cells were maintained in endothelial cell medium, composed of, RPMI 1640 basal medium (Lonza, Verviers, Belgium), supplemented with 20% heat-inactivated foetal bovine serum (Invitrogen/GIBCO, CA, USA), 50 μg/ml endothelial cell growth factors supplement (bovine brain extract; homemade), 1% penicillin-streptomycin (Sigma-Aldrich, MA, USA), 2 mM L-glutamine (Lonza) and 5 U/ml heparin (Leo Pharma, Ballerup, Denmark). Cells were used for experiments at passage 6 and 7.
Confluent monolayers of HUVEC were stimulated for 48 h with or without 5 or 10 ng/ml citric acid activated-TGF-β1 (Peprotech, NJ, USA; #100–21 C) in RPMI 1640 basal medium, supplemented with 20% heat-inactivated foetal bovine serum, 2 mM L-glutamine, 5 U/ml heparin and 1% penicillin-streptomycin. Cells treated with endothelial cell medium were harvested as unstimulated controls, Cells were treated with pharmacological inhibitors for 48 h to inhibit desired signalling pathways.
Confluent monolayers of HUVEC were stimulated for 48 h with or without 5 or 10 ng/ml citric acid activated-TGF-β1 (Peprotech, NJ, USA; #100–21 C) in RPMI 1640 basal medium, supplemented with 20% heat-inactivated foetal bovine serum, 2 mM L-glutamine, 5 U/ml heparin and 1% penicillin-streptomycin. Cells treated with endothelial cell medium were harvested as unstimulated controls, Cells were treated with pharmacological inhibitors for 48 h to inhibit desired signalling pathways.