siRNA-mediated PPARγ suppression

JEG-3 cells were transfected with PPARγ siRNA or scrambled siRNA using GeneMute transfection reagent (SignaGen laboratories), according to the manufacturer's instructions. Briefly, 50% confluent cells in a 6-well culture plate were treated with 50 pM of four MISSION® predesigned siRNAs (Sigma) targeting different sites of PPARγ mRNA, or scramble siRNA, diluted in 100 μl of transfection buffer. After addition of 4 μl of transfection reagent, the mixture was incubated for 20 min at room temperature. The mixture was added dropwise to 900 μl OPTI-MEM medium (Gibco) per well. Cells were incubated with siRNA transfection mixture for 5 h, followed by replacement with 10% FBS-supplemented DMEM/F12. After 48 h, cells were either harvested or treated for another 18 h prior to harvesting.