Anti wnt5a sirna 1

Transient siRNA transfections were performed with the Lipo-fectamine 2000 reagent (Invitrogen), according to the manufacturer’s instructions. The following siRNA oligonucleotides were used: from Invitrogen, anti-WNT5A siRNA #1 (s14871; 100 nM), anti-WNT5A siRNA #2 (s14872; 100 nM), Negative Control siRNA (#4390843; 100 nM) After 4 h, the transfection complex was replaced with fresh cell media supplemented with 10% FBS, and the cells were subsequently allowed to grow for 48 h prior to analysis.

Transient siRNA transfections were performed with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The following siRNA oligonucleotides were used at the indicated concentrations: anti‐WNT5A siRNA #1 (s14871; 50 nm), anti‐WNT5A siRNA #2 (s14872; 50 nm), and negative control siRNA #1 (#4390843; 50 nm) (Invitrogen). Anti‐RND3 SMARTPool siRNA (L‐007794‐00‐0050; 100 nm) was acquired from Horizon Discovery (Cambridge, UK). After 12 h, the transfection medium was replaced with fresh cell culture medium supplemented with 10% FBS, and the cells were subsequently allowed to grow for 24 or 48 h prior to analysis or further treatment. For BRAF inhibitor treatment, following siRNA treatment for 24 h, HTB63 cells were treated with 1 µm PLX4720 (Vemurafenib) (Selleckchem, Munich, Germany, Cat# S1267) or DMSO for the final 24 h of siRNA transfection. For C59 (Abcam, Cat#: ab142216) treatment, HTB63 and WM852 cells were incubated with 1 μm C59 or vehicle (DMSO) control in either collagen I gels for spheroid invasion (see Spheroid invasion assay) or while growing subconfluently in a 6‐well plate for the RhoA activity assay and western blotting (see Western blotting).