Competition between FLYWCH1/β-catenin and TCF4/β-catenin complexes for their interaction to Tcf-DNA-binding sites. Competition between FLYWCH1/β-catenin and TCF4/β-catenin complexes for their interaction to Tcf-DNA-binding sites. A, β-Catenin-ChIP assay shows binding of FLYWCH1/β-catenin and TCF4/β-catenin complexes to the human c-jun promoter in SW480 and DLD-1 cells (right), as well as TIG119 fibroblasts (left). SW480 and DLD-1 cells were transfected to express FLYWCH1 and TCF4. B, β-Catenin-ChIP coupled with qPCR was used to quantify binding of FLYWCH1/β-catenin versus TCF4/β-catenin to the c-jun promoter, as outlined in (A), using real-time PCR. Data are mean ± SD (n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001). C, Western-blotting analysis of mock vs. treated Wnt3a+R-spondin 1 in HCT116 cells after 24 hours using the indicated antibodies. D and E, SW480 and DLD-1 cell-lines were treated with Wnt3a+R-spondin1 conditional media or mock after 24 hours. RNA was extracted and FLYWCH1-mRNA expression was assessed by real-time qRT-PCR (D) and standard RT-PCR (N = 3, 100bp marker was used; E). Data are mean ± SD (n = 6; **, P < 0.01). F, Levels of cyclin D1/D2 gene expression associated with the activation of Wnt signaling altered FLYWCH1 gene expression levels in SW480 cells using WB assays. SW480 cells were transfected to express FLYWCH1 (lanes 3 and 4) and control GFP (lanes 1 and 2). Belal A. Muhammad et al. Mol Cancer Res 2018;16:1977-1990 ©2018 by American Association for Cancer Research