Cell migration kit

For invasion assays, cells were treated with Hakin-1 or DMSO as vehicle for 48 h using 1% FBS during the last 24 h. Then, 3 × 10⁵ MDCK, Hakai-MDCK or LoVo cells were seeded in a cell invasion chamber (cell invasion assay kit, Chemicon International) containing medium with 2% FBS and after 16 h, invasive cells that reached the lower chamber containing 30% FBS were fixed and stained with crystal violet (Sigma Aldrich, St Louis, MO) following the manufacturer’s specifications. For migration assays, HT-29 cells were cultured with Hakin-1 or DMSO as vehicle for 48 h, using medium without serum for the last 24 h. 3 × 10⁵ HT-29 cells were seeded in the cell migration chamber (Cell migration kit, Millipore, Bedford, MA, USA) containing medium without serum. After 16 h, migrated cells in the lower chamber containing serum with 30% FBS were stained with crystal violet and counted following the manufacturer’s specifications. For both invasion and migration assays, cells were counted in five fields photographed with an Olympus microscope using a 20× objective. Experiments were performed in triplicates for each condition and the assays were repeated at least three times. Results are expressed as mean ± SD.

Migration was evaluated across 8 µm pore size membranes in 96-well tissue culture plates with a cell migration kit (Millipore, Burlington, MA, USA). Cells were cultured in RPMI-1640 B27 for 24 h, trypsinized, and then added with 150 µL of RPMI-1640 B27 to the upper chamber (1 × 10⁵ cells per chamber). The lower chamber contained 500 µL of RPMI-1640 B27 and was coated with (positive control) or without (negative control) Matrigel. The cells were incubated for 12 h at 37 °C, and the cells that had migrated to the lower chamber were detected with CyQuant GR Dye.