Cell culture and siRNA silencing of HUVECs

Pooled Human Umbilical Vein Endothelial Cells (HUVEC) were obtained from Pelobiotech. HUVECs received at P0 were expanded over 2 passages in 100 µg/ml collagen 1 (from rat tail, BD) coated flasks in complete EGM-2 medium (Lonza) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in a 5% CO₂ − 3% O₂ humidified chamber. HUVECs at passage 3 were transfected twice at 24 h-intervals with 30 nM siRNA and Lipofectamine RNAi max (Life Technologies, ref. 13778-150) according to the manufacturer’s instructions in OptiMEM at 37 °C in a regular 5% CO₂ in a humidified chamber. For double transfections, 30 nM of each siRNA duplexes (Dharmacon smartpool ON-TARGET plus Thermo Scientific) was used: Non-targeting siRNA #1 (ref. D-001810-01), Ccm2 siRNA (ref. L-014728-01), Rock1 siRNA (ref. L- ref. L-003536-00) and Rock2 siRNA (ref. L-004610-00). siRNA transfected HUVECs were transduced with adenoviral LifeAct-GFP2 (Ibidi, Martinsried, Germany) at a multiplication of infection (MOI) of 5 and incubated for 16 h while the wild-type HUVECs used in mosaics were similarly transduced with LifeAct-RFP2.