Silencer select small

Silencer Select small interfering RNAs (siRNAs) against VEGFR2 (s7823; Thermo Fisher Scientific) and Silencer Select negative-control siRNA number 2 (Thermo Fisher Scientific) were used for RNAi assays. Briefly, 5,000 cells of HUVEC in EGM-2 were plated in each well of a 96-well plate and incubated at 37°C for 6 h. The medium was replaced with 0.1 mL of M199–10% FBS, and the cells were transfected with siRNA (0.5 pmol/well) using Lipofectamine RNAiMAX (Thermo Fisher Scientific) in accordance with the manufacturer's instructions. After 24 h, the cells were washed and replaced with fresh M199–10% FBS and then used for cell proliferation assays against VEGF-A₁₆₅ and BafA_Bba. VEGFR2 knockdown at 24 h after transfection was confirmed by quantitative reverse transcription-PCR (qRT-PCR).