Fibroblast growth factor (fgf)

Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Cat. C2519A). Cells below passage 8 were used for experimental manipulations and grown in Endothelial Cell Growth Medium 2 (EGM-2, Cat. CC-22211, PromoCell, Heidelberg, Germany) supplemented with 2% fetal calf serum, growth factors, such as Epidermal Growth Factor (5 ng/mL), Fibroblast Growth Factor (10 ng/mL), Insulin-like Growth Factor (20 ng/mL), Vascular Endothelial Growth Factor 165 (0.5 ng/mL), ascorbic acid (1 µg/mL), and hydrocortisone (0.2 µg/mL) (Cat. C-39211, PromoCell, Heidelberg, Germany). HUVEC were cultured on 0.2% pre-coated gelatin plates in a 37 °C incubator with humidified atmosphere of 5% CO₂. The chemicals Doxorubicin (Cat. D1515), Chloroquine (Cat. C6628), and Lactacystin (Cat. L6785) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Doxorubicin and Lactacystin were dissolved in DMSO. Puromycin (Cat. A1113802) was purchased from Gibco™ (Shanghai, China).

Primary cultures of human umbilical cord-derived venous vascular endothelial cells (HUVECs, purchased from PromoCell, Heidelberg, Germany), were used for all in vitro experiments under sterile conditions. HUVECs were grown in endothelial cells basal growth medium containing 2% fetal calf serum, 0.1 ng/mL epidermal growth factor, 1 ng/mL fibroblast growth factor (PromoCell), and 1% Penicillin/Streptomycin (Invitrogen, Waltham, MA, USA) in a humidified incubator with 5% CO₂ at 37 °C until passage 6 and experiments were performed at passage 7. Gelatin (0.2%) coated flasks/plates were used for cell culture. 5 × 10⁴ cells/cm² were used for all treatments. To create an inflammatory in vitro model, HUVECs were treated with 20 ng/mL of human recombinant TNF-α (Peprotech, Hamburg, Germany). Different treatment durations and concentrations of BRD4 inhibitors were used for optimization. The final concentrations used were 500 nM JQ1 (ApexBio technologies, Houston, TX, USA) or 60 μM RVX208 (ApexBio technologies) for 12 h. Control cells were treated with an equal volume of DMSO. For treatments, cells were first incubated with inhibitors for 12 h. Later, after washing with PBS, a fresh medium with TNF-α was added for the indicated time to create an inflammation model.