Fig. 4.
Necroptosis is repressed by SERCA through the prevention of calcium overload, MCU activation, and mPTP opening. Primary CMECs were cultured in a vascular-cell basal medium supplemented with the endothelial cell growth kit VEGF. Hypoxia/reoxygenation (H/R) injury was induced through 30 min of hypoxia and 2 h of reoxygenation. SERCA AAV9 or control AAV9 vectors were transfected into CMECs, which were termed SERCAAAV9 group or control group respectively. A–C. Intracellular calcium ([Ca2+]i) and mitochondrial calcium ([Ca2+]m) were stained using Furo-2AM (Molecular Probes) and Rhod-2 (Molecular Probes), respectively. D–E. Proteins were isolated from H/R-treated CMECs, and then the levels of MCU were measured through western blots. F. mPTP opening was measured using the calcein AM probe. *p < .05.