Non targeting 2 on targetplus smartpool control sirna

Cells were seeded overnight in 6- or 12-well plates and transfected at 40–50% confluence DharmaFECT 4 lipid reagent (GE Dharmacon, Lafayette, CO). The siRNA pools were used at 100 nM while the single siRNAs were used at 25 nM. The following siRNAs were used in this study: Dharmacon human VANGL2 (L-010581) and integrin αv (L-004565) ON-TARGETplus SMARTpool siRNA, Dharmacon human integrin αv single siRNAs (J-004565-08 and J-004565-10), Ambion (Thermo Fisher Scientific, Waltham, MA) human MMP14 Silencer (ID#104074) and Silencer Select (ID#s8877) siRNA, and Dharmacon Non-Targeting #2 ON-TARGETplus SMARTpool Control siRNA (D-001810). For all siRNA experiments, cells were transfected for 4 days and protein knockdown was confirmed using western blot (see below for procedure and antibodies). The MMP14 Silencer siRNA (see Supplementary Fig. 1) and the integrin αv siRNA pool (see Supplementary Fig. 2) were used throughout this study.

HT-1080 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained as previously described [26 (link)]. Cells were seeded overnight in multiwell plates and transfected with siRNA at 40–50% confluence using DharmaFECT 4 lipid reagent (Dharmacon Inc., Lafayette, CO). The following siRNAs were used: Dharmacon SiGenome SMARTpool human PRICKLE1 (M-016677); Thermo Fisher Scientific Silencer Select human VANGL1 (S37813); Dharmacon ON-TARGETplus SMARTpool human VANGL2 (L-010581), integrin αv (L-004565), integrin α5 (L-008003), integrin β1 (L-004506), integrin β3 (L-004124), and integrin β5 (L-004125) siRNAs; Dharmacon human integrin αv single siRNAs (J-004565–08 and J-004565–10); Dharmacon Non-Targeting #2 ON-TARGETplus SMARTpool Control siRNA (D-001810). The siRNA pools were used at 100 nM while the single siRNAs were used at 25 nM. For all siRNA experiments, cells were transfected 4 days and protein knockdown was confirmed using western blot. The integrin αv siRNA pool was used throughout this study except as noted in Fig. 1. VANGL2 expression was visualized using a plasmid containing full-length human VANGL2 fused to GFP (GFP-VANGL2). Plasmid transfection was performed as previously described [26 (link)].