Endothelial cell growth supplement (ecgs)

Human umbilical vein endothelial cells (HUVECs), obtained from American Type Culture Collection (ATCC, VA, USA), were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), endothelial cell growth supplement (ECGS, ScienCell) and 100 U/ml penicillin-streptomycin at 37 °C in a humidified incubator containing 5% CO₂. Human aortic endothelial cells (HAECs) were purchased from ATCC and cultured in endothelial culture medium (ECM, ScienCell, CA, USA) supplemented with 5% FBS, ECGS and 100 U/ml penicillin-streptomycin at 37 °C with 5% CO₂.
HUVECs and HAECs were stimulated with various concentrations of ox-LDL (0, 50 and 100 μg/ml) for different durations (0, 4, 8 and 12 h), or treated with a combination of ox-LDL (100 μg/ml) and CQ (50 uM) for 12 h. In control groups, cells were treated with phosphate buffered saline (PBS). For serum starvation-induced autophagy, HUVECs were cultured in serum-free DMEM for various durations (0, 2, 4 and 6 h).

Human umbilical vein endothelial cells (HUVECs; ScienCell, USA) were cultured in endothelial cell medium (ECM; ScienCell, USA) with 5% fetal bovine serum (FBS; ScienCell, USA), 1% endothelial cell growth supplement (ECGS; ScienCell, USA), and 1% penicillin/streptomycin solution (P/S; ScienCell, USA) in 37 °C incubator with 5% CO² and 95% air. HUVECs were passaged when the cells reached 80–90% confluency. Cells were used for subsequent experiments at passages 4–7. The HUVECs were preconditioned by LV-FGF-2⁺-hGMSC-CM, hGMSC-CM, or LV-vector⁺-hGMSC-CM for 3 days, and ECM (ScienCell, USA) without CM was chosen as the negative control. Then, the cells were cultured by ECM with 5% fetal bovine serum (FBS; ScienCell, USA), 1% endothelial cell growth supplement (ECGS; ScienCell, USA), and 1% penicillin/streptomycin solution (P/S, ScienCell) for 7 to 10 days.

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.