Fig. 3
Inhibition of FOXM1/ β-catenin interaction by DFS. (A) GBM TSs were treated with DFS for 72 h and the protein levels of FOXM1, active-β-catenin and total β-catenin were determined by western blotting. (B) DFS-treated GBM TSs were immunoprecipitated with anti-FOXM1 and β-catenin antibody, and endogenous expression of FOXM1 and β-catenin proteins were determined by western blotting to determine interactions between them. (C) GST pull-down assay to confirm whether FOXM1 and β-catenin interactions are direct or indirect. (D) Immunofluorescence to determine whether DFS affects the nuclear translocation of β-catenin (magnification × 20). (E) Levels of FOXM1 and β-catenin proteins in the cytosolic and nuclear fractions of GBM TSs determined by western blotting. The cytosolic and nuclear fractions are denoted by C and N, respectively. Cytosolic marker: β-tubulin; nuclear marker: PARP. (F) Measurements of β-catenin/TCF signaling activity in GBM TSs transfected with TOPflash or FOPflash luciferase vector. (G) Expression of proteins encoded by β-catenin downstream genes (cyclin D1, cMYC) measured by western blotting. Differences among groups were compared by one-way ANOVA with Tukey’s post hoc test; means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, between indicated groups or compared with controls.
