RNAi depletion

All siRNA were purchased from Life Technologies (Silencer® Select siRNA). Three different silencers were designed for each target gene and used in three individual reactions. HeLa cells were reverse transfected as follows: 1.5 µl of 20 µM siRNA (10 pM final) and 4 µl Lipofectamine™ RNAiMAX (Life Technologies) were mixed with 500 µl of OPTI-MEM (Gibco) in each well of a 6-well plate. After a 15-min incubation at room temperature, 1.5×105 HeLa cells, resuspended in 2.5 ml of medium without antibiotics, were seeded in each well. Inactivation was carried out for 72 h prior to total RNA extraction. Negative controls were either transfected with a scrambled siRNA (Ref. 4390844) or without siRNA (mock transfected), with similar results. The sequence of the silencers used is available here.