Defatted milk

The HCT116 and LoVo control cells without CoCl₂treatment were collected when their confluence reached 80%. Likewise, the PGCCs and their generated daughter cells were collected when they attained a similar confluence. The cells were lysed on ice with 80‐150 μL of glacial radio‐immunoprecipitation assay (RIPA) lysis buffer (Roche, Germany) for 30 minutes, and then centrifuged at 14,000 rpm/min for 30 minutes at 4°C. The concentrations of the proteins were determined, and they were separated by 10% sodium dodecyl sulfate polyacrylamide gel (SDS‐PAGE). The protein bands were then transferred onto polyvinylidene fluoride (PVDF) membranes (GE, USA). The protein‐containing PVDF membranes were blocked with 5% defatted milk (BD, USA) in 1 × Tris‐buffered saline with 1% Tween‐20 (Sigma, USA) for 2 hours at room temperature. Then, the membranes were incubated with primary antibodies (Table S1) at 4°C for 14‐16 hours. The membranes were then incubated with secondary antibodies at room temperature for 2 hours. Protein expression was finally detected using the Chemidoc imaging system (BioRad, USA). The Image‐J software was used to analyze the gray value of each protein band after capturing images from the film processor. β‐actin was employed as a protein‐loading control, and all the western blot experimental results were repeated multiple times.

Equal volume of serum or plasma of mice was diluted 1:10 with PBS (Hyclone, Utah, USA). Equal volumes of diluted samples were separated with 8% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, Massachusetts, USA). The membrane was blocked with 5% defatted milk (BD, California, USA) for 1 h at room temperature and immunoblotted with specific antibody overnight at 4°C followed by incubation with HRP-conjugated secondary antibody (ZSGB-Bio, Beijing, China) for 1 h at room temperature. The membrane was visualized by ECL (Thermo Fisher Scientific, Massachusetts, USA or GE Healthcare, USA) and exposed with chemiluminescence apparatus (Beijing Sage Creation, Beijing, China). Protein bands' gray values were measured by NIH Image J.

Hct116 and LoVo control cells without ATO treatment and PGCCs with daughter cells after ATO treatment were collected when approximately 80% confluence was reached. The cells were lysed on ice with approximately 100 μL of glacial radio-immunoprecipitation assay lysis buffer (Roche, Germany) for 30 min and then centrifuged at 14,000 rpm for 30 min at 4°C. The concentrations of the proteins were determined and separated by 10% sodium dodecyl sulfate-polyacrylamide gel. The protein bands were transferred onto polyvinylidene fluoride membranes (GE, USA). These membranes were blocked with 5% defatted milk (BD, USA) in 1 × Tris-buffered saline with 1% Tween-20 (Sigma, USA) for 2 h at room temperature. The membranes were then incubated with primary antibodies (Supplementary Table 1) at 4°C for 12 h. Subsequently, the membranes were incubated with secondary antibodies at room temperature for approximately 2 h. β-Actin was used as a protein-loading control. Protein expression was detected using a ChemiDoc imaging system (BioRad, USA).

The cells were lysed as described above. For graft tissue, 20 mg of tissue was weighed, 200μl RIPA buffer was added, and the tissues were then lysed on ice. The remaining steps were the same as those used for cell lysis. Protein samples were loaded on 8% or 10% sodium dodecyl sulfate-polyacrylamide gels and then separated by electrophoresis. After that, the separated proteins were transferred onto PVDF membranes (Millipore, USA). The membranes containing proteins were soaked in 5% defatted milk (BD, USA) up to 2 h at room temperature for blocking. The membranes were then incubated with primary antibodies (Supplementary Table 1) at 4 °C last at least overnight. Next day, secondary antibodies were used to react with primary antibodies through incubating for 2 h at room temperature. Protein expression was evaluated using a ChemiDoc imaging system (Bio-Rad, USA).