Sirna hif1α

The AT1R cloned overexpression plasmid and negative control vector were purchased from Sino Biological Inc. (Beijing, China). AT1R transfection was performed using Lipofectamine 2000 according to the manufacturer’s instructions. In brief, a total of 1 μg AT1R plasmid or control vector with lipofectamine 2000 was added to serum- and antibiotic-free 1640 medium. After mixing, the medium was incubated at room temperature for 5 min and added to cultured macrophages. The cells were harvested at 48 h after transfection. The efficiency of AT1R transfection was confirmed by a more than 3-fold increase in AT1R mRNA expression compared with the control vector.
siRNA transfections for AT1R or hypoxia-inducible factor (HIF)1α were performed using Lipofectamine 2000. siRNA AT1R, siRNA HIF1α, and siRNA control (scrabbled siRNA) were purchased from Santa Cruz Biotechnology. The macrophages were resuspended with trypsin and transfected using siRNA AT1R or siRNA HIF1α (25 nM, 50 nM and 100 nM) in serum- and antibiotic-free transfection medium. Then the cells were incubated for 48 h according to the manufacturer’s instructions (Santa Cruz Biotechnology). The efficiency of siRNA transfection was confirmed by more than 70% reduction in the target mRNA by real-time PCR, compared with the siRNA control group.

HPTCs cells (10×10⁵) were seeded in six-well plates and cultured for 18h to 40–50% confluence. 50 nmol/L Small interference RNA (siRNA)-HIF-1α (Santa Cruz, CA, USA) was mixed with 16μL transfection reagent (INTERFERin®) and diluted with 200 μL transfection buffer (OPTi-MEM). While for miRNA Transfection, 50 nmol/L miR-21 mimic or 100 nmol/L antisense miR-21 inhibitor or 50 nmol/L control miRNA (GeneCopoeia, Guangdong, China) was mixed with 8μL transfection reagent (jetPRIME™) and diluted with 200μL transfection buffer (jetPRIME™ buffer). After incubation for 15 min at room temperature, the mixture was added to the 2 mL DMEM/F12 medium, transfection medium replaced by new DMEM/F12 medium 24h after transfection and the cells were cultured for an additional 24 or 48h.