Transwell migration assay and wound healing assay

Cell migration was assayed in Transwell chambers constituted of polycarbonate cell culture inserts with 8.0 μm pore size (Corning Costar, Cambridge, MA). Wt or transfected cells in concentration of 2 × 10⁵ were suspended in 100 μl of serum-free medium and added to the upper chamber. Inserts were incubated for 24 hours at 37°C with 600 μl of complete medium in the lower chamber. Cells transmigrated on the underside of the insert were detached using 5 mM EDTA and a plastic scraper, collected together with cells eventually fallen in the underlying well and counted. Not migrated cells were collected from the upper chambers and stained with Trypan Blue to verify their viability. To test the cell invasion capability, wt or transfected cells were plated and grown to ~70% of confluence, then the mono-layers cells were scratched vertically down the center of each well with a sterile tip and each well was washed with fresh medium. Cells were cultured for three days and the area covered by the cells after 72 hours was measured and compared with the wound scratched at time 0.