*4.3. Cell Invasion*

Transwell chambers (6.5 mm diameter, 8 μm pore size; Corning Life Sciences) were coated with 100 μL of diluted Matrigel. Then, 0.6 mL of medium containing 10% FBS was added to the lower chambers, and cells suspended in serum-free medium at a density of 1.5 × 10<sup>5</sup> cells/mL were seeded (0.1 mL) in the upper chambers. Various concentrations of PSS (25, 50, 100 μg/mL) were added to both of the upper and lower chambers. After incubation for 16 h or 8 h (FGF2-mediated invasion of B16-F10), cells were fixed with cold 4% paraformaldehyde and stained with 0.1% crystal violet, and the cells that had not migrated were removed from the upper chambers. The remaining cells were photographed in five random fields per membrane. The dye was dissolved in 80 μL of acetic acid, and the absorbance of the resulting solution was measured at 600 nm using a microplate reader (SpetraMAX i3, Molecular Devices, Sunnyvale, CA, USA).
