HUVEC Isolation and Treatment Protocols

HUVECs isolated from the umbilical cords of healthy pregnant women are the standard model for studying endothelial cell function in vitro [25 (link),26 (link)]. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the institutional review board of USTC (2020-ky013). In this study, 3rd- to 7th-generation HUVECs from different donors were used. The cells were cultured in ECM endothelial cell growth medium (ScienCell, Carlsbad, CA, USA) containing 1× endothelial cell growth supplement (ScienCell, Carlsbad, CA, USA), 5% fetal bovine serum, and 1% penicillin/streptomycin antibiotics. The culture conditions were 37 °C and 5% CO₂. HUVECs seeded in 12-well plates were treated with DMSO or 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, and 25 μM of RES (#501-36-0, TargetMol, Shanghai, China) for 12 h or 24 h after cells reached confluence. For experiments in which RES affected PHACTR1 expression and functional experiments, HUVECs were also infected with Ad-PHACTR1 (MOI = 1, #NM_030948, WZ Biosciences, Shandong, China) or control adenovirus for 24 h, with/without 10 ng/mL of TNF-α (#300-01A, PeproTech, Rocky Hill, NJ, USA) treatment for 6 h.