Western blot assay to determine protein expression

Proteins from cells and tumor tissues were extracted using RIPA buffer (Sigma) with protease inhibitors, and the extracted proteins were resolved on a 10% SDS-polyacrylamide gel followed by transferring to the polyvinylidene fluoride membranes (Sigma). The membranes were then blocked with 5% non-fat milk at room temperature for 1 h before further incubating with corresponding primary antibodies against vimentin, E-cadherin, N-cadherin, β-catenin, GSK-3β, c-myc, cyclin D1 and β-actin for 12 h at 4 °C. After incubating with primary antibodies, the membranes were further incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. All these antibodies were obtained from Abcam company (Cambridge, UK). The blotting bands of corresponding proteins were visualized by using ECL kit (Thermo Fisher Scientific).