KYSE-30 and KYSE-410 cells in the logarithmic growth phase were seeded into a 12-well plate. The cell density was adjusted to 1×104 cells/ml, with 3 ml per well and three replicates in each group. After the cells adhered to the wall, shRNA (1.5 µg) and Lipofectamine® 2000 (4.5 µl) were diluted with 200 µl serum-free RPMI-1640 medium for transient transfection. Migration and invasion assays were performed using a Transwell chamber. For the migration assay, 1×104 cells were seeded into the upper chamber of Transwell (Corning, Inc.). For the invasion assay, 1×104 cells were added to the upper chamber (previously coated with Matrigel) and placed in the incubator for 1 h at 37°C. In both assays, cells were maintained in serum-free medium in the upper chamber and medium containing 10% FBS was added as a chemoattractant to the lower chamber. After 24 h of incubation, cells that did not migrate or invade the membrane were removed. The membrane was then fixed with methanol (15 min at room temperature) and stained with 0.1% crystal violet (15 min at room temperature). Each chamber was counted in three random fields using an inverted microscope (×100 magnification; Carl Zeiss AG) and each experiment was repeated three times.

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