Migration and invasion assay

Migration and invasion were assessed as described in our previous study [18]. Briefly, we used Transwell membranes (8-μm pore size; Corning Inc., Corning, NY, USA) with or without Matrigel coating. PC3 cells were cultured in the upper chamber of a Transwell insert in serum-free medium containing various concentrations of EPA. The lower chamber was filled with DMEM containing 10% FBS. For the migration assay, Matrigel-uncoated upper chambers were used; cells were seeded at a density of 2 × 10⁵ cells/mL, and after 24 h, the number of cells that had migrated into the lower chamber was counted. For the invasion assay, Matrigel-coated upper chambers were used. Before seeding the cells, Matrigel was fixed by incubation for 90 min, and cells were seeded at a density of 1 × 10⁵ cells/mL. After 48 h, cells that had invaded into the lower chamber were counted.