Pcs 100 020

Human coronary artery endothelial cells (HCAECs; PCS-100-020™, ATCC, Manassas, VA, USA) were cultured in Vascular Cell Basal Medium (ATCC) with 5 ng/ml VEGF, 5 ng/ml EGF, 5 ng/ml FGF, 15 ng/ml IGF-1, 10 mM L-glutamine, 0.75 U/ml heparin sulfate, 1 µg/ml hydrocortisone, 50 µg/ml ascorbic acid, 1% amphotericin B, 1% penicillin-streptomycin, and 10% FBS. Hypoxia-reoxygenation (H/R) injury was induced on cultured cells as described in our previous study 15 (link). In brief, hypoxic stress was induced in a tri-gas incubator with an N₂ concentration of 95% and a CO₂ concentration of 5% for 45 min. Then, cells were cultured under normal oxygen concentration for 2 h. Dapagliflozin (10 μM) was applied to HCAECs 24 h before H/R injury in vitro. BAPTA (50 μM; #A1076, Sigma-Aldrich, Burlington, MA, USA) was added to the medium for 30 min to attenuate intracellular calcium overload. Ionomycin (100 μM; #200-664-3, Sigma) was alternatively applied for 30 min to induce calcium overload. To inhibit F-actin degradation, HCAECs were treated with jasplakinolide (Jas, 2 μM; #ab141409. Abcam) over 2 h before H/R. To induce an oxidative stress microenvironment, HCAECs were treated with 0.3 mM hydrogen peroxide for 6 h. To prevent CaMKII activation, HCAECs were incubated with KN93 (5 μM; #S678, Selleck Chem. LLC, Houston, TX, USA) during 2 h before H/R.

HCAECs (American Type Culture Collection, PCS-100-020™) were cultured with vascular basal cell medium containing 5 ng/mL vascular endothelial growth factor, 5 ng/mL epidermal growth factor, 5 ng/mL fibroblastic growth factor, 15 ng/mL insulin-like growth factor 1, 10 mM L-glutamine, 0.75 U/mL heparin sulfate, 1 µg/mL hydrocortisone, 50 µg/mL ascorbic acid, 1% amphotericin B, 1% penicillin-streptomycin and 10% fetal bovine serum. Hypoxia/reoxygenation was used to induce sI/R injury in vitro. In brief, the medium was changed to an ischemia-mimicking solution containing 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 mM 2-deoxy-D-glucose, 139 mM NaCl, 12 mM KCl, 0.5 mM MgCl₂, 1.3 mM CaCl₂ and 20 mM lactic acid, pH 6.2, and then the cells were incubated under 100% nitrogen (O₂ < 1%) at 37 °C for 45 min. The cultures were then returned to normal culture conditions (10% fetal bovine serum, 37 °C ambient air, 5% CO₂) for 2 h. Empagliflozin (10 µM) was added to the HCAECs 12 h before the sI/R injury. For the induction of oxidative stress, HCAECs were treated with 0.3 mM hydrogen peroxide for six hours. NAC (10 mM) was added to the medium of HCAECs to reduce oxidative stress-induced DNA-PKcs activation in the presence of sI/R, based on a previous study [42 (link)].