Transwell Migration and Invasion Assay

Transwell chambers with 8-μm pore-size polycarbonate membrane (Corning Life Sciences, Tewksbury, MA, USA) were used. For migration assay, FTC-133 cells (0.8 × 10⁴ cells/well) or 8505C cells (1.5 × 10⁴ cells/well) resuspended in serum-free medium with different concentrations of GB1107 or TD139 were seeded onto the upper chamber, while the complete medium containing 10% fetal bovine serum as the chemoattractant was added to the lower chamber [13 (link)]. After 24 h, the migrated cells on the lower surface of the porous membrane were fixed and stained with Diff-Quik (Sysmex, Kobe, Japan). For invasion assay, FTC-133 cells (1 × 10⁴ cells/well), 8505C cells (1 × 10⁴ cells/well), or Nthy cells (2 × 10⁴ cells/well) were allowed to invade through BioCoat cell culture inserts precoated with Matrigel. The invaded cells were fixed and stained after 24 h. Migrated or invaded cells were photographed under the microscope, and five random fields were counted for each assay.