Uncoated membrane

The cell migration and invasion capabilities were assessed by transwell assay with 8-μm pore, 24-well transwell plates (Corning). For the migration assays, 10 × 10⁵ Caco-2 LV-DKK4, HCT8 LV-DKK4, HCT116-LV-shDKK4, SW480 LV-shDKK4, or corresponding negative control cells were seeded in the top chamber lined with an uncoated membrane (Millipore). In the invasion experiment, 10 × 10⁵ of the aforementioned cells were plated in the upper chamber coated with Matrigel (BD Biosciences). After incubation at 37 °C with 5% CO₂ for an appropriate duration, cells that had migrated or invaded through the membrane were stained with 0.1% crystal violet and counted under a microscope (Olympus) to determine their relative numbers.

The cell migration and invasion capabilities were assessed by Transwell assay with an 8-µm pore, 24-well Transwell plates (Corning, NY, USA). For the migration assays, 2 × 10⁵ HT29 LV-Ran, SW480 LV-Ran, HCT116 LV-shRan, DLD-1 LV-shRan or corresponding negative control cells were seeded in the top chamber lined with an uncoated membrane (Millipore, MA, USA). In the invasion experiment, 2 × 10⁵ of the aforementioned cells were plated in the upper chamber coated with Matrigel (BD Biosciences). After incubation at 37 °C with 5% CO₂ for an appropriate duration, cells that had migrated or invaded through the membrane were stained with 0.1% crystal violet and counted under a microscope (Olympus, Tokyo, Japan) to determine their relative numbers.