Transwell invasion assay

A Transwell invasion assay was performed using a 24-well Transwell chamber with a pore size of 8 µm (Costar; Corning Inc., Corning, NY, USA), and the inserts were coated with 20 µl Matrigel (1:3; BD Biosciences, San Jose, CA, USA). After 48 h following transfection, cells were trypsinized (0.25% trypsin) at 37°C for 30 sec and then transferred to the upper Matrigel-coated chamber in 100 µl serum-free medium (1×10⁵ cells/ml). Medium (DMEM for M14 and A375 cells, RPMI-1640 for MV3 cells) supplemented with 10% FBS was added to the lower chamber as the chemoattractant. Cells were incubated for 18 h at 37°C. Non-invading cells on the upper membrane surface were then removed with a cotton tip, and the cells that passed via the filter were fixed in 4% paraformaldehyde at room temperature for 20 min. Cells were stained with hematoxylin at room temperature for 5 min. Cells were observed under a light microscope (magnification, ×200; BX53). The experiments were performed in triplicate.