Western blot

Cells were lysed for 30 min using radioimmunoprecipitation (RIPA) buffer (Sigma-Aldrich, St. Louis, MO, USA). Then, the cells were centrifuged at 10,000 ×g for 10 min at 4 °C, and the supernatants were collected. After that, cell lysates (50 µg) were resolved in 8–10% SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 4% non-fat milk and then probed at 4 °C for 12 hours with the following primary antibodies: anti-cleaved Caspase-3 (1:500), Ki-67 (1:500), PCNA (1:500), MMP-9 (1:500), MMP-14 (1:500), E-cadherin (1:500), N-cadherin (1:500), Twist1 (1:500), β-Catenin (1:500), Wnt2 (1:500), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:1,000) (Abcam, Cambridge, UK). The membranes were then incubated with the corresponding secondary antibodies at room temperature for 1 hour. GAPDH was used as the internal control.