KYSE-30 and KYSE-410 cells in the logarithmic growth phase were seeded into a 6-well plate, and the cell density was adjusted to 1×105 cells/ml, with 3 ml per well and three replicates in each group. Until the cells were attached, transient transfection was performed with 500 µl serum-free RPMI-1640 medium diluted shRNA (2.5 µg) and Lipofectamine® 2000 (7.5 µl). After 48 h of transfection, scratch wounds were created with the tip of a 200-µl pipette to scratch the cells when the cell density reached >90%. The cells were then washed twice with 0.01% PBS and serum-free medium was added. Then, cells were maintained at 37°C in an incubator containing 5% CO2. Random images were taken at 0, 24 and 48 h using a microscope (×50 magnification; Leica Microsystems GmbH) to assess the ability of cells to migrate. The independent experiments were repeated three times.

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