Cell migration and invasion assay

For the cell invasion assay, 100 μL transfected MDA-MB-453 and MCF7 cells (1×10⁵ cells per well) in serum-free medium were seeded into the Matrigel-coated upper chamber of transwell inserts (Corning), whereas 500 μL medium was added into the lower chamber. After incubation for 12 hours at 37 °C, the non-invasion cells on the upper chamber were removed using cotton swabs, then the cells that invaded through the Matrigel-coated filters were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet. The number of cells on the lower surface of the filter was counted using a microscope in five random fields, and all samples were performed in triplicate. The migration assay was the same as the invasion assay except that the top chambers were not coated with Matrigel (BD Biosciences).