After 24 h of starving, logarithmic growth cells were digested the cells the next day, centrifugated, and resuspended, with a final concentration of 2×105/ml. Each well of the Transwell upper chamber was added with 0.2 ml suspension, and the lower chamber was added with 700 ml of precooled DMEM/RPMI 1640 that contained 10% FBS. Thereafter, we cultured cells within the cell incubator under 37°C and 5% CO2 conditions. After 24 h, we removed the Transwell chamber, and the cotton swabs were used to scrape cells within the upper and basement membranes. Afterwards, methanol was used to fix cells for 30 min, followed by 20 min of 0.1% crystal violet staining. Five fields (100×) were randomly selected to count the number of transmembrane cells.

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