Immunoblotting Analysis of EMT Markers

Immunoblotting were executed as described previously.^{47 (link)} Simply, cells were washed with PBS and lysed in RIPA buffer (Invitrogen) added protease inhibitor (Sigma). Protein concentration of each sample was calculate by BCA protein assay kit (Thermo Fisher Scientific) to equal protein loading. Equivalent protein were underwent to SDS-PAGE, shifted to polyvinylidene fluoride membrane, interdicted in 5% skim milk for about 2 h at about 25 °C, then checked with relative primary antibodies. The following antibodies were used for analysis: anti-E-cadherin, anti-N-cadherin, anti-Vimentin and anti-SOX2 (Abcam plc, Cambridge, UK), anti-Nanog (Cell Signaling, Beverly, MA, USA), anti-β-actin (Sigma) and anti-β-tubulin (BD Biosciences, CA, USA). β-actin and β-tubulin was served as loading controls. Horseradish Peroxidase (HRP) Secondary Antibodies (Abcam) and an ECL Chemiluminescence Detection Kit (Thermo Fisher Scientific) were used to test combined antibody.