P yap

RIPA lysate (Beyotime Biotechnology) and nuclear protein extraction kit (Solarbio, Beijing, China) with PMSF were used to extract the total proteins and nucleus or cytoplasm proteins, according to the manufacturer's instructions. An enhanced bicinchoninic acid Protein Assay Kit (Beyotime Biotechnology) was used to analyse the protein concentrations. The primary antibodies were diluted as follows: BACH2 (1 : 500) (Cell Signaling Technology, Danvers, MA, USA), FUS (1 : 1000) (ProteinTech, Rosemont, IL, USA), WWC3 (1 : 100) (Abcam, Cambridge, UK), Yes‐activated protein (YAP) (1 : 1000) (ProteinTech), p‐YAP (1 : 500) (ABclonal Technology, Wuhan, China), GAPDH (1 : 10 000) (ProteinTech) and Histone H3 (1 : 2000) (ProteinTech). The assays were performed as previously reported [29]. GAPDH or Histone H3 was used as internal controls to calculate the integrated density values.

Total proteins were extracted from cells with RIPA buffer (Solarbio, Beijing, China), proteins were separated by SDS-polyacrylamide gels, then transferred to polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies directed against LC3B (L7543, Sigma-Aldrich, MO, USA), Atg7 (#8558, Cell Signaling Technology, MA, USA), FTH1 (#4393, Cell Signaling Technology), EGFR (A11351, ABclonal, Wuhan, China), N-cadherin (A19083, ABclonal), YAP (A1002, ABclonal), p-YAP (AP0489, ABclonal), mTOR (A2445, ABclonal), p-mTOR (AP0115, ABclonal), TfR (14-0719-82, Invitrogen, CA, USA), β-actin (TA-09, Zsgb Bio, Beijing, China) overnight at 4 °C. Secondary antibodies conjugated with HRP (Proteintech, Wuhan, China) were used and blots were presented with FluorChem Q Imager (ProteinSimple, CA, USA) after incubating with ECL (Beyotime Biotech, Nanjing, China).

Based on the previous study [21 (link)], 200 μL Radioimmunoprecipitation (RIPA) solution (Beyotime, Shanghai, China), which contains 1x protease inhibitor, was added to crack the crypts and shaken at 4 °C for 20 min to fully crack the crypts. The protein concentration was determined by Bicinchoninic Acid (BCA) kit (Beyotime, Shanghai, China). After adding the protein loading buffer, the protein was denatured at 100 °C for 10 min and stored at −80 °C refrigerator. The protein samples with the calculated concentration were subjected to Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel electrophoresis. Then, they were gummed, transferred, sealed, and incubated primary antibody overnight at 4 °C. Protein primary antibody information and dilution ratio: BAX (1:1000, Proteintech, Wuhan, China), Caspase9 (1:1000, CST, Boston, MA, USA), PCNA (1:1000, Abcam, Cambridge, UK), Olfm4 (1:1000, CST, Boston, MA, USA), P-YAP (1:1000, Abclonal, Wuhan, China), YAP (1:1000, Abcam, Cambridge, UK), Cyclin D1 (1:1000, CST, Boston, MA, USA). Finally, we incubated secondary antibodies, and the ECL solution was used to reveal the bands. A total of 30 experimental animals C57BL/6 mice were used.