Dharmafect 4 reagent

Small interfering RNA (siRNA) duplexes were purchased from IDT (Integrated DNA Technologies, Iowa City, IA, U.S.A.) individually or as sets of three within TriFECTa kits and transfected into MDA-MB-231 and HeLa cells at 5 nM using 1–2 ul DharmaFECT 4 reagent (Thermo Scientific, Pittsburgh, PA, U.S.A.) per ml final volume in HMEC cultures with DharmaFECT 1. RNA was isolated using the RNAeasy Mini Plus Kit (QIAGEN) and RNA concentrations determined on a Nanodrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). SuperScript III First Strand Synthesis SuperMix (Life Technologies) was used to make cDNA from isolated RNA as per manufacturer's directions using oligo(dT) primers. Quantitative real time PCR (qPCR) using validated PrimerPCR SYBR Green Assay human primer sets (Bio-Rad, Hercules, CA, U.S.A.) were performed using a StepOnePlus Real-Time PCR system (Applied Biosystems/Life Technologies) and quantified by the ΔΔCt method. Knockdown efficiency was consistently greater than 80% after 48 hours determined by qPCR normalized to HPRT1 expression. More detailed information regarding siRNAs is given in Table S2.

MST1 expression was inhibited by transfection with a pool of four MST1-specific siRNAs (Thermo Fisher Scientific) into Jurkat T cells using the DharmaFECT4 reagent (Thermo Fisher Scientific). A non-targeting pool of four individual siRNAs was used as a control. MST1 protein levels were determined by immunoblotting 72 h after transfection. Transfected cells were treated and processed as per the standard protocols.