Cell migration and invasion assays were performed using Transwell chambers (8-mm pore size; Corning, Inc.). Briefly, for the migration assay, HSC-3 (3x104) or SCC-15 (8x104) cells were plated in serum-free medium into the upper chambers of the Transwell plates. For the invasion assay, HSC-3 (6x104) or SCC-15 (2x105) cells were seeded into the upper chambers with Transwell plates precoated with Matrigel for 6 h at 37°C. The lower chambers were filled with medium supplemented with 10% FBS. Following incubation for 24 h at 37°C, the medium was removed and the cells were fixed with 4% formaldehyde for 20 min at room temperature. Cells that had not migrated or invaded through the membrane were gently removed using a cotton swab. The fixed cells were subsequently stained with 0.4% crystal violet for 15 min at room temperature and observed under a light microscope (Zeiss GmbH). A total of five representative fields at x100 magnification were randomly imaged. ImageJ v1.8.0 (National Institutes of Health) was used for quantification, and the average counts of the five images represented the migrated cells.

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