Cell migration assay and invasion assay

Cell migration was assayed by using 24-well transwell chambers (Corning Incorporated, Corning, NY, USA). The upper and lower compartments were separated by polycarbonate filters with 8 μm pore size to permit cell migration. After 24-hour serum starvation, 1×10⁵ cells/well were seeded onto the filters in medium (0% FBS) containing 0, 25, and 50 μM cilengitide and medium containing 10% FBS was added to the lower chambers. As control, the same number of cells was seeded into normal 24-well plates with the same cilengitide concentration. After 48 hours of incubation at 37°C with 5% CO₂, cells passing the filters and attaching to the lower sides of filters were harvested using trypsin and the cell number was quantified. The percentage of migrating cells was calculated after correction for controls grown under identical conditions without filters.

Cell invasion was also assayed by using 24-well transwell chambers. Before the assay, the polycarbonate filters were coated with 70 μL Matrigel (Corning Incorporated; 5 mg/mL). The remaining steps were similar to those followed for cell migration assay.