Pparα sirna

Vigene Biosciences (Shanghai, China) constructed AAV9-shPPARα and negative shRNA (AAV9-shRNA). We purchased PPARα siRNA from Santa Cruz (SC-422360). Two weeks prior to AB surgery, randomly selected mice received an AAV9-shPPARα or AAV9-shRNA myocardial injection of 1 × 10¹¹ vp (virion particles) per animal. Briefly, after the mice were anesthetized with 3% sodium pentobarbital (80 mg/kg, ip), the hearts of the mice were exposed and the pericardium was removed. We injected the following areas with a #29 syringe: the anterior wall, the side walls, and the apex of the left ventricle. A single needle (10 μl) was inserted at the apex of the heart, and the two needles were inserted at the anterior wall and the side wall. The total amount of each adenoviral vector injection (1 × 10¹¹ vp) was 50 μl, and the injection interval was approximately 5 mm.

For peroxisome proliferator-activated receptor (PPAR) α knockdown experiments, cells were transfected with 10 nM PPARα siRNA (Santa Cruz Technology, Santa Cruz, CA) or silencer select control siRNA (Thermo Fisher Scientific, Waltham, MA) using RNAiMax lipofectamine reagent (Thermo Fisher Scientific). Cells were assayed for knockdown 6 days following transfection. For PPARα reporter assays, cells were transfected with Cignal PPAR Reporters (Qiagen, Germantown, MD) using Lipofectamine 2000 (ThermoFisher Scientific) and cells were assayed 48 h later.

PPARα silencing was achieved using Pparα siRNA (Santa Cruz Biotechnology), with DsiRNA NC1 (Integrated DNA Technologies, Coralville, IA) as a negative control. RNA oligonucleotides were delivered to MLO-A5 cells using the X-treme Gene siRNA Transfection Reagent (Roche; Cat#04476093001) according to a protocol provided by the manufacturer. αA5^KD cells and their scrambled (Scrl) control were used for assays 36 h after transfection.