Matrigel uncoated and coated transwell chambers

We transfected the miR-638 mimics and SOX2 siRNA into HCT-116 and SW1116 cells using an Amaxa Nucleofector (Amaxa, Koeln, Germany). Cell migration and invasion were evaluated using Matrigel-uncoated and -coated transwell chambers (cat. 3422, Corning, NY). Briefly, 5 × 10⁴ cells were suspended in 200 μl of DMEM without serum and placed into cell culture inserts (8-μm pore size; BD Falcon, San Jose, CA) of a companion plate (BD Falcon San Jose, CA) in pre-warmed culture medium containing 10% fetal bovine serum in the well. The cells were incubated overnight at 37°C in 5% CO₂ and then fixed in 4% paraformaldehyde in PBS. Cell migration and invasion were determined by staining the cells with 0.1% crystal violet (Sigma, St Louis, MO) and counting the cells under a light microscope (100× magnification) in eight randomly selected areas.

Migration and invasion assays were performed by matrigel-uncoated and -coated transwell chambers (Corning, NY, USA). A density of 8 × 10⁴ cells per well was seeded in the upper chamber with serum-free DMEM. The lower chamber was filled with 500 μl DMEM medium supplemented with 10% fetal bovine serum (Hyclone, USA). Forty-eight hours later, migratory and invasive cells on the bottom surface of the filters were fixed in 4% paraformaldehyde, stained by 0.1% crystal violet solution (MedChem Express, Shanghai, China) and quantified under an inverted microscope (Olympus, Tokyo, Japan).
When cells seeded in 6-well plates were grown to approximately 80% confluence, a 10 μl sterile pipette tip was used to scratch across wound across the cell monolayer. The wounds were observed at 0, 24 and 48 h under an inverted microscope (Olympus, Tokyo, Japan). Three random fields were selected and measured. Migration index was calculated by the ratio of migrating area of treated cells to their counterparts.