Matrigel invasion assay

To assess the invasive ability of the transfected cells, Matrigel™ (BD Biosciences) and Transwell^® chambers (Costar; Corning, Inc.) with a pore size of 8 µm were used, according to the manufacturers' instructions. Briefly, 100 µl Matrigel™ (1:7 dilution) was added to each insert and the chambers were placed at 37°C, for at least 2 h. Then, 8×10⁴ cells in 100 µl medium supplemented with 2% FBS were added to the upper chamber. Medium supplemented with 20% FBS was added to the lower chamber as the chemoattractant. After 20 h of incubation, the filters were fixed at room temperature for 20 min and stained with hematoxylin at room temperature for 10 min. The non-invading cells on the upper surface were removed with a cotton swab. The number of invasive cells in 10 high-power fields randomly was counted under an inverted microscope (magnification, ×200). The experiments were performed in triplicate.