Cell Migration and Invasion Assays

Cells were seeded at a density of 1 × 10⁵ (a density of 5 × 10⁴ for transfected SCC9 cells for the migration assay) in serum-free DMEM/F12 medium in the upper wells of Transwell chambers (8 μm pore size, Corning, New York, NY, USA), while the lower wells were filled with complete medium containing 10% FBS. Chambers for the invasion assay were coated with 100 µl of Matrigel (200 μg/µl, Corning) and incubated for 2 h at 37°C. Cells in the upper layer were removed with a swab after 24 h of culture, and cells on the bottom membrane were fixed with 4% paraformaldehyde and stained with crystal violet. The results of each group were photographed at five randomized visual fields, and the experiments were repeated three times.