Bicinchoninic acid protein assays kit

The protein was prepared using a total protein extraction kit (Solarbio) and protein concentration was determined by bicinchoninic acid protein assays kit (Beyotime), followed by denaturation at 98°C for 10 minutes. Equal amounts of proteins were subsequently subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and membrane transfer using nitrocellulose membranes (Millipore, Billerica, MA, USA). After the blockage of nonspecific binding sites using the western blocking buffer (Beyotime), the membranes were incubated with anti‐SOX5 (#PA5‐95266, 1:1000 dilution, Thermo Fisher) or anti‐GAPDH (#MA5‐15738, 1:5000 dilution, Thermo Fisher) and corresponding secondary antibody. The protein bands were visualized via enhanced chemiluminescence reagent (Beyotime). The gray values were analyzed with GAPDH as a loading control by QuantityOne software (Bio‐Rad, Hercules, CA, USA).

Cell lysates were prepared with total protein extraction kit (Solarbio) on ice and the concentration of total protein was measured by using bicinchoninic acid protein assays kit (Beyotime). Proteins were mixed with loading buffer and subjected to the boiled water bath for 5 min, followed by separation through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and membrane transfer using nitrocellulose membranes (Millipore, Billerica, MA, USA). Following the treatment of Western Blocking Buffer (Beyotime), the membranes were interacted with anti-B-cell lymphoma-2 (Bcl-2, ab196495, 1 : 2000 dilution, Abcam, Cambridge, MA, USA), anti-cleaved caspase 3 (c-caspase 3, ab2302, 1 : 1000 dilution, Abcam), anti-E-cadherin (ab15148, 1 : 500 dilution, Abcam), anti-matrix metalloprotein-9 (MMP-9, ab38898, 1 : 1000 dilution, Abcam), anti-SOX4 (ab80261, 1 : 1000 dilution, Abcam) or anti-GAPDH (ab9485, 1 : 3000 dilution, Abcam) as a loading control and corresponding secondary antibody. Enhanced chemiluminescence reagent (Yeasen Biotech, Shanghai, China) was used for the development of protein signals.