Immunoblotting analysis of stemness markers

Cells were lysed in Radioimmunoprecipitation assay buffer (Solarbio) containing phenylmethylsulphonyl fluoride protease inhibitor. Protein samples were subjected to 10% SDS polyacrylamide gel electrophoresis. The separated proteins were subsequently transferred to a polyvinylidene fluoride membrane. The membrane was blocked using 10% non-fat milk for 1 h at room temperature and then incubated with primary antibodies overnight at 4 °C. Primary antibodies recognizing the following proteins were used: CD24 (1:1000, Proteintech, Wuhan, China, Cat#18330-1-AP), SOX2 (1:1000, Cell Signaling Technology, Cat#14962), ALDH1 (1:1000, Cell Signaling Technology, Cat#54135), OCT4 (1:1000, Wanlei, Shenyang, China, WL02020), BMI1 (1:1000, Cell Signaling Technology, Cat#6964), YAP1 (1:1000, Cell Signaling Technology, Cat#14074), GAPDH (encoding glyceraldehyde-3-phosphate dehydrogenase) (1:20000, Proteintech, Cat#60004-1). anti-phospho Histone H2A.X (Ser139) (1:1000, Cell Signaling Technology, Cat#9718). Thereafter, the membranes were incubated with secondary antibodies corresponding to the primary antibodies for 1 h at room temperature. Finally, the immunoreactive protein bands were visualized using ECL (NCM Biotech, Suzhou, China) Western blot detection reagent.