24 well boyden chambers with 8 μm pore polycarbonate membranes

Cells transduced with LRIG1 or control pMX vector were plated at 40,000 cells/well in 24-well Boyden chambers with 8 μm-pore polycarbonate membranes (Corning, Lowell, MA, USA) according to the manufacturer’s protocol. Cells were seeded in serum-free media and allowed to migrate for 12 hrs toward the lower chamber containing media with 10% FBS. In some experiments, the c-Met-specific inhibitor 0.5 μM ARQ197 (Active Biochemicals, Selleck, USA) was added in the upper and low compartments. After 12 hrs, the cells were fixed and stained with Diff-Quick staining solution (Dade Behring, Newark, DE, USA). For the cell invasion assay, the chambers were pre-coated with collagen type I (BD Bioscience San Diego, CA). Migrated cells were imaged and counted in 10 microscopic fields on an Olympus IX81 inverted microscope with cellSens Entry software using a 10x objective. The results were averaged among three independent experiments.