Transwell insert chambers with 8 μm porous membranes

Transwell insert chambers with 8‐μm porous membranes (Corning Inc., Corning, NY, USA) were used for motility assays. Approximately 2 × 10⁵ cells without FBS were added to the upper chamber, and medium with 10% FBS was added to the lower chamber. The cells were cultured for 24 h. The cells on the upper surface of the upper chamber were scraped off using a cotton bud, and the chamber was rinsed with phosphate buffer. Then, the cells on the lower surface of the chamber were soaked in 4% paraformaldehyde. Once the surface was dry, the cells were dyed with 1% crystal violet for 2 min. The migrating cells were those on the lower surface of the chamber. Migrating cells were counted at ×100 or ×200 magnification in eight fields of view.

Transwell insert chambers with 8 μm porous membranes (Corning) were used for the cell invasion assay. Matrigel (354248; Corning) was diluted with FBS-free medium to 200 μg/ml, and 100 μl of the diluted Matrigel was added to the top chamber and placed in an incubator at 37 °C for 1 h. HCT116 cells with indicated treatments in FBS-free medium were placed in the top chamber at a density of 1.5 × 10⁴ cells per well, complete medium was added to the bottom chamber, and the cells were incubated for 24 h. To quantify the invasive cells, noninvasive cells on the upper side of the membranes were removed. Invasive cells on the lower side were fixed with 4% aqueous paraformaldehyde for 15 min and stained with crystal violet dye (C0121; Beyotime) for 20 min. Subsequently, the cells were washed with PBS and photographed under a microscope. A minimum of five images were photographed.

Transwell insert chambers with 8-μm porous membranes (Corning Incorporated, NY, USA) were used for motility assays. Cells were washed three times with PBS and 5 × 10⁴ cells in serum-free media were added to the top chamber. The bottom chamber was filled with RPMI 1640 medium containing 15% FBS. Cells were incubated for 24 h at 37°C in 5% CO₂. To quantify migrating cells, cells in the top chamber were removed using a cotton-tipped swab, and the migrated cells were fixed in methanol and stained with 0.1% crystal violet.