siRNA knockdown

Three individual siRNAs (Silencer Select; Ambion, Life Technologies) were pooled and transfected into cells using the Lipofectamine RNAimax transfection reagent (ThermoFisher Scientific) following the manufacturer's instructions. Each siRNA sequence was cross-referenced with siRNA-Check to confirm specificity [64]. Scrambled non-targeting siRNA was used as the negative control for normalization in all knockdown experiments (specific design details proprietary to Ambion). We selected pooled siRNAs over individual siRNAs since it has been reported that siRNA pools show greater phenotypic penetrance than the individual duplexes [65]. siRNAs were used at a final concentration of 25 nM.