Transwell migration and invasion assays

Transwell chambers were used for transfected cancer cell migration and invasion assays. The 24-well culture inserts (BD Biosciences) with 8-µm pores coated with Matrigel (1 mg/ml; BD Biosciences) were used. The membranes of upper chamber were coated with Matrigel (BD) and then incubated for 6 h at 37°C. For the migration assay, 2×10⁵ transfected cells suspended in 200 µl of serum-free medium were seeded in the upper chambers. Medium containing 20% FBS was added to the lower chamber. For the invasion assay, a Chemicon Cell Invasion Assay kit (Chemicon International; Thermo Fisher Scientific, Inc.) was used. The non-migrating/invading cells were removed from the upper surface of the membrane with cotton swabs following an incubation for 24 h at 37°C, and the migrating/invading cells were stained with 0.1% crystal violet for 30 min at RT and images captured with an inverted fluorescence microscope (magnification: ×20; Nikon Corporation).