Western Blot Analysis of Stem Cell Markers

The cells from each sample were collected and lysed in radioimmunoprecipitation (RIPA) buffer (Thermo Scientific), followed by centrifugation at 1,200×g for 10 min and subsequent collection of the supernatant. Western blotting was performed as previously described (Xiong et al., 2019 (link)). The primary antibodies were GAPDH (1:20,000, Proteintech, China), Lgr5 (1:1,000, Abcam, United States), TGF-β1 (1:1,000, Santa Cruz Biotechnology, United States), and β-catenin (1:1,000, Cell Signaling Technology, United States), and the secondary antibody was horseradish peroxidase-conjugated goat antimouse (1:10,000, Abcam, United States). Protein expression was observed and visualized by chemiluminescence using an Alpha Imager scanner (Tecan, Thermo Scientific).