Cell migration and epithelial-to-mesenchymal transition (EMT)

One hundred thousand cancer cells, transfected with control shRNA or miR-106b shRNA were placed in a constrained area around the center of well. After 72 hours of incubation, cells were fixed with methanol for 5 min, followed by staining with 0.03% methylene blue for 10 min. Cell movement was analyzed by comparing cell distribution between control shRNA- and shRNA-miR106-treated cells. EMT status was examined by the expression of the mesenchymal genes, snail2, fibronectin, twist2 and vimentin using real-time PCR. Gene expression was normalized to the housekeeping genes, GAPDH and β-actin. All primer information is listed in Table ​Table22.