Gene knockdown strategies

Oligonucleotide siRNA duplexes were purchased from GE Healthcare Dharmacon, Inc. Individual ON‐TARGETplus PARD3 (J‐015602‐05 5′‐AAGCAUGGAUUUAGGUAUA‐3′; J‐015602‐06 5′‐AGACUAAACUCAAUACAGU‐3′; J‐015602‐07 5′‐CGAUAAAGACAGACUGGUA‐3′; J‐015602‐08 5′‐GAUGGCGACCUUCGAAAUA‐3′) or scrambled control (D‐001810‐01 ON‐TARGETplus Non‐targeting siRNA#1, 5′‐UGGUUUACAUGUCGACUAA‐3′) was transfected into HUVECs using oligofectamine (12252011, Thermo Fisher) according to the manufacturer's instructions. Briefly, 4 × 100,000 HUVECs were seeded onto 60‐mm dish and cultured with growth medium (EGM‐2, 2% FBS) for 12 h. Thirty minutes before transfection, cells were washed once with PBS and incubated with Opti‐MEM. Scrambled or siPARD3s were diluted with Opti‐MEM, mixed with oligofectamine, and incubated for 30 min at room temperature. siRNA mixture was added onto the dish at final concentration of 100 nM, incubated for 6 h at 37°C, and added twice the volume of the growth medium containing 6% FBS (final concentration: 2% FBS). After 12 h, medium was changed to normal growth medium (2% FBS). Cells were analyzed 48 h post‐transfection. J‐015602‐08 and J‐015602‐06 were used in this study as siPAR‐3#1 and siPAR‐3#2, respectively.