Transfection of siRNAs

Cells were seeded in a 6-well plate at 80%–90% confluence for 24 h without antibiotics. A total of 2,500 ng DNA was diluted with 5.0 μL RNAi. Small interfering RNAs (siRNAs) were from Dharmacon (designated siRNA #1) and Sigma (designated siRNA #2). Cells in all conditions designated as “Control” were transfected with a pool of siRNAs that do not target human genes using Lipofectamine 2000 in 100 μL Opti-MEM Medium without serum, using the manufacturer’s protocol. Cells were harvested 24 h after transfection. Pools of at least three siRNAs were used to dilute potential off-target effects.