Ecm670

Forty‐eight hours post‐transfection of HTB63 cells with NC siRNA (100 nM), WNT5A siRNA #1 (100 nM) or WNT5A siRNA #2 (100 nM), the cells were detached with Versene and resuspended in cell media supplemented with 10% FBS; the cell concentration was then calculated using an automated cell counter. At the start of each experiment, 15,000 cells were suspended in 0.5 ml cell media supplemented with 10% FBS and added to each well of a 8‐well glass chamber slide (BD Bioscience) coated with gelatin‐FITC according to the manufacturer's instructions (#ECM670, Millipore). The cells were then allowed to degrade the gelatin‐FITC coating for 24 h at 37 °C in a humidified atmosphere of 5% CO₂. Next, the cells were fixed with 4% paraformaldehyde, stained with Phalloidin‐TRITC and DAPI and mounted with cover slip glasses using Fluorescent Mounting Medium (Dako). Four representative fields of each treatment were captured at 20x magnification using a confocal microscope (Zeiss LSM 700). Area of gelatin‐FITC degradation (loss of FITC signal) and total cell area (Phalloidin‐TRITC signal) were calculated using the ImageJ software (National Institute of Health). The number of DAPI‐stained nuclei were manually counted to ascertain that an equal number of cells were analyzed for each treatment.

Gelatin degradation was measured using QCMTM Gelatin Invadopodia Assay (Green) following manufacturer’s instructions( EMD Millipore, # ECM 670)