Fcs endothelial cell growth medium 2

Rap1 siRNA (90 nM, SASI_Hs01_00040403; Sigma-Aldrich), R-Ras siRNAs (90 nM, siRNA mix, EHU0225113; Sigma-Aldrich), or negative control siRNA (90 nM, SIC-001; Sigma-Aldrich) were transfected into cells according to the manufacturer's instructions. Briefly, cells were incubated in cell culture inserts for four days (medium changed every second day). The medium was changed to 10% FCS Endothelial Cell Growth Medium 2 (C-22211; PromoCell) without heparin (10% EGM-2/heparin(−)). Then, a mixture of MISSION siRNA Transfection Reagent (S1452; Sigma-Aldrich) and siRNA was pre-incubated for 10 min, and then transfected. Two days later (6th day after starting cultivation), the medium was changed to 10% EGM-2/heparin(−) and siRNA transfection, and BSA and TAGE treatment were performed.

Ninety nanomolars of Rap1 siRNA (SASI_Hs01_00040403) or negative control siRNA (SIC-001) (SIGMA Aldrich, St. Louis, MO, USA) were transfected into cell with according to the manufacturer's instructions. Briefly, cells were seeded at 2.0 × 10⁵ cells/mL and, after 6 h, medium was changed to 10% FCS Endothelial Cell Growth Medium 2 (PromoCell) without heparin. Then, a mixture of MISSION® siRNA Transfection Reagent (SIGMA Aldrich) and siRNA was preincubated for 10 min and transfection was performed.

Rap1 siRNA (90 nM; SASI_Hs01_00040403), R-Ras siRNAs (90 nM; EHU022511, siRNA mix), RasGRP2 siRNA (180 nM; SASI_Hs02_00319692), TC21 siRNAs (90 nM; EHU108181, siRNA mix), or negative control siRNA (90 or 180 nM; SIC-001) (SIGMA Aldrich) were transfected into cells, according to the manufacturer’s instructions. Briefly, the cells were seeded, and after 6 h, medium was changed to 10% FCS Endothelial Cell Growth Medium 2 (PromoCell) without heparin. Then, a mixture of MISSION® siRNA Transfection Reagent (SIGMA Aldrich) and siRNA were pre-incubated for 10 min and transfection was performed.