Whole-cell protein lysates were prepared with radioimmunoprecipitation (RIPA) buffer containing a protease inhibitor cocktail (Roche, Basel, Switzerland). Membrane, cytoplasmic, and nuclear proteins were extracted using a subcellular protein fractionation kit (78840; Thermo Fisher Scientific) according to the manufacturer's protocols. Then, 40 µg protein lysate (2ug/ul) per sample was loaded onto a 10% sodium dodecyl-polyacrylamide gel and transferred to 0.45-µm polyvinylidene difluoride (PVDF) membranes using a semi-dry transfer system (Trans-Blot Turbo; Bio-Rad Laboratories, Hercules, CA, USA). The membranes were incubated overnight at 4°C with primary antibodies and then incubated with horseradish peroxidase-conjugated secondary antibody (1:4000) for 1 h at room temperature. Protein expression was detected using an electrochemiluminescence kit (Tanon Science & Technology, Shanghai, China), and blot images captured using a chemiluminescence imaging system (6100; Tanon Science & Technology). The primary antibodies used were antibodies against E-cadherin (1:200; BD Biosciences), KRT5 (1:200; Abcam), KRT15 (1:100; Abcam), KRT19 (1:200; Abcam), phosphorylated (p)-FOXO1 (1:1000; Cell Signaling Technology, Beverly, MA, USA), FOXO1 (1:1000; Cell Signaling Technology), β-catenin (1:1000; Cell Signaling Technology), β-actin (1:1000; Cell Signaling Technology), and histone (1:500; Santa Cruz Biotechnology).

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