Cytotoxicity assay kit

Cell viability and LDH were determined using Cell Counting Kit-8 (CCK-8) assay kit (catalogue no. 04-11; Dojindo, Kumamoto, Japan) and cytotoxicity assay kit (catalogue no. A020-2-2; Jiancheng, Nanjing, China), according to the manufacturer's protocols. Cells SOD and 15-F2t-isoprostane releases which reflect oxidative stress level were detected using assay kits according to the manufacturer's protocols.

Embryonic rat cardiomyocyte-derived cell line H9C2 was maintained in DMEM (Dulbecco’s modified Eagle’s medium) containing 10 % FBS in a humidified atmosphere (5 % CO₂) at 37 °C. Commercial Cav-3 siRNA or eNOS siRNA (Santa Cruz Biotechnology) was used for inhibition of Cav-3 expression according to the manufacturer’s protocol. After transfection with control, Cav-3 siRNA or eNOS siRNA, cells were treated with N-acetylcysteine (NAC, 1 mmol/L) in the condition of LG or HG for 36 h, then the cells was exposed to 4 h of hypoxia (1 % O₂, 5 % CO₂, 94 % N₂) followed by 4 h of reoxygenation (H/R). LDH (lactate dehydrogenase) release was measured to evaluate the extent of cellular injury using a cytotoxicity assay kit (Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions.