Ampk sirna

For stably knockdown of CD44 with lentivirus shRNA, 2 × 10⁵ cells were plated onto 6-well plates. After 24 h, the liquid containing shRNA was added to the cultural medium according to protocol. AMPK siRNA, CD44 siRNA, and control siRNA (Riobio, China) were transfected into cells using lipofectin 2000 according to the manufacturer’s instructions. Followed by siRNA or mimic treatment for 48–72 h, total protein and RNA were extracted for western blot and Q-PCR analysis.

CRA was purchased from Jianfeng Natural Product R&D Co., Ltd (Tianjin, China). The autophagy inhibitor Chloroquine diphosphate (CQ) was obtained from ABCAM, U.S.A., and was applied to cardiomyocytes at a concentration of 10 μM. The autophagy inhibitor 3-methyladenine (3-MA) was obtained from Selleck, U.S.A., and was applied to cardiomyocytes at a concentration of 10 mM. AMPK siRNA was purchased from Guangzhou RiboBio Co., LTD (Guangzhou, China).

For stably knockdown of HMGB1 with lenti-virus shRNA, 2 × 105 cells were planted onto 6-well plates. After 24 h, the liquid containing shRNA was added to the cultural medium according to protocol. To select stable transfectants, cells were cultured in complete DMEM with 10 μg/ml puromycin (Sigma-Aldrich, USA) for some weeks. HIPK2 siRNA, AMPK siRNA, Siah2 siRNA, p53 siRNA, HMGB1 siRNA, and control siRNA (Riobio, China) were transfected into cells using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions. At the end of the siRNA treatment (48-72 h), the cells were collected for immunoblot analysis and Q-PCR. Expression plasmids for human HMGB1-cDNA (pEnter) and ZEB1-cDNA (pEnter) were purchased from Vigene Biosciences, lnc. For the overexpressing or rescue experiments, HMGB1-cDNA, and ZEB1-cDNA were transfected into a standard or stable shRNA cell line by Lipofectamine 3000 according to the manufacturer’s instructions.