Transwell units with polycarbonate filters

24-well Transwell units with polycarbonate filters (pore size, 8.0 μm; Corning, New York, USA) were used to measure the invasive ability of GC cells. In brief, the upper Transwell inserts were first coated with 100 µl Matrigel basement membrane (BD Biosciences, San Diego, CA, USA), and then 1× 10⁵ cells in 100 µL of serum-free RPMI medium were seeded on it. Medium (600 μl) containing 10% FBS was placed in the lower chamber as a chemoattractant. Cells were allowed to migrate at 37 °C for 24 h; non-invasive cells were removed with a cotton swab. 4% paraformaldehyde was used to fix the filters, and the cells were stained with a 0.05% crystal violet solution, and counted under a microscope with six randomly-selected fields at 100× magnification for each sample. Invasion assays were performed in triplicate.

GC cell invasive and migratory abilities were assessed using 24-well Transwell units with polycarbonate filters (pore size, 8.0 μm; Corning, Corning, NY, USA). First, the upper Transwell inserts were coated with 100 μl of Matrigel basement membrane (BD Biosciences, San Diego, CA, USA) or left uncoated, and then added with 100 μL of serum-free RPMI medium. Then, 1× 10 ⁵ cells were seeded on the upper Transwell inserts. Medium (600 μL) containing 10% FBS as a chemoattractant was added to the lower chamber. For 24 h at 37° C, the cells were allowed to migrate or invade from the upper chamber, after which the non-migratory or non-invasive cells were removed. 4% paraformaldehyde was used to fix the filters, and a 0.05% crystal violet solution was used to stain the cells. In each sample, six fields were selected randomly to count the cells under a microscope (magnification = 100×). All assays were conducted three times independently.