Matrigel coated

Matrigel invasion assays, and transwell migration assays were performed using standard protocols. Cells were trypsinized and seeded at a density of 50,000 cells per well on either Matrigel-coated (BD Biosciences) or uncoated (Corning) Transwell filters in a 24-well plate and allowed to invade for 12 hours toward 10% FBS in the lower chamber. Cells invading and migrating through the Matrigel-coated and uncoated filters, respectively, were stained with Three Step stain (Richard-Allan Scientific). Each filter was counted in its entirety with four 10× fields, and invasion or migration was quantified as fold increase relative to control.

Cells were trypsinized and seeded at a density of 50,000 cells/well on both Matrigel-coated (BD Biosciences, San Jose, CA, USA) and uncoated (Corning, Tewksbury, MA, USA) transwell filters in a 24-well plate. Both groups of cells were allowed to invade for 12 h toward 10% FBS in the lower chambers. Three-Step Stain (Richard-Allan Scientific, San Diego, CA, USA) was applied when the cells invaded or migrated through the Matrigel-coated or uncoated filters, respectively. Each filter was counted in its entirety under the rule of four 10× fields, while invasion or migration was quantified as the fold change relative to the control.