Western blot analysis

DPCs were plated in 25-cm² dishes at a density of 1 × 10⁶ cells per dish. The following day, cells were transfected with TCF4- and MAD2B-expressing vectors and siMAD2B, individually or in combination. After 48 h of incubation, cells were washed twice with cold phosphate-buffered saline, followed by cell lysis using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China). Cell lysates were collected and the protein concentration was measured using bicinchoninic acid protein assay reagent (Thermo Fisher Scientific). Subsequently, proteins (20 μg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were subsequently blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20, and then incubated with a primary antibody against β-actin, hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), or vascular endothelial growth factor (VEGF) (R&D Systems, Minneapolis, MN, USA) for 1–2 h at room temperature. This was followed by incubation with horseradish peroxidase–conjugated secondary antibody for 1 h. After that, signals were detected using enhanced chemiluminescent substrates (Thermo Fisher Scientific). Images were acquired and quantified using a Bio2 Rad imaging system (Bio-Rad).