Transwell Assay

Cells were harvested after transient transfection with siRNA for 48 h, and chambers (8-μm pore size, Costar) with or without Matrigel (BD Science, USA) were used for cell invasion assays or migration assays. Approximately 4 × 10⁴ cells (for the migration assay) or 6 × 10⁴ cells (for the invasion assay) were resuspended in 200 μl of serum-free medium and added to the upper chambers. A total of 600 μl of medium containing 10% FBS was placed into the lower chambers. After incubation for 24 h, the cells in the upper chamber were gently removed with cotton swabs, and cells on the lower surface were fixed with paraformaldehyde and stained with 0.1% crystal violet for 20 min at room temperature. Photographs were taken with a microscope (Olympus, Tokyo, Japan), and the cells were counted for analysis.