Hmgb1 sirna

Control siRNA and HMGB1 siRNA were obtained from Guangzhou RiboBio Co., Ltd, Guangzhou, China. Control siRNA (sin05815122147) consists of a scrambled sequence that will not lead to the specific degradation of any known cellular mRNA. HMGB1 siRNA is a pool of three target-specific 19- to 25-nt siRNAs (sib0941383447, sib11129130839 and sib11129130857, respectively) designed to knock down gene expression. siRNA was prepared according to the transfection protocol for cell cultures. Briefly, siRNA transfection reagent Lipofectamine 2000 (11668-019; Invitrogen Life Technologies) mixture of 1 ml was co-incubated with cardiac fibroblasts/myofibroblasts for 6 hrs in a 5% CO₂ incubator at 37°C, and then the same amount of DMEM 20% FBS was added. An additional incubation was performed for 18 hrs, and then the procedure for conditioned media was carried out.

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.