Figure 4. FAT1 is a β-catenin binding partner, inactivation of which causes aberrant Wnt pathway activation, translocation of β-catenin to the nucleus, and enhanced β-catenin-mediated transcription.

(a) Immunoprecipitation assays in indicated FAT1-expressing cell lines demonstrate that endogenous FAT1 binds endogenous β-catenin (top), and vice versa (middle). Antibody or IgG negative control as indicated.

(b) Immunoprecipitation assays demonstrate binding of transfected FAT1_Trunc to endogenous β-catenin.

(c) Immunoprecipitation of FLAG-tagged FAT1 constructs show loss of β-catenin binding in mutated FAT1.

(d) Translocation of β-catenin to the nucleus after FAT1 knockdown in GBM cells and immortalized human astrocytes (IHA). Cells were treated with indicated siRNAs; 48 hours later, cells were stained with DAPI (blue) and antibody against β-catenin (green). Representative photos from 3 independent repeat experiments, at lower and higher magnification. White arrows denote plasma membrane localization; red arrows, nuclear/perinuclear localization. Scale barsrepresent 50 μm.

(e) Quantification of results from (d). Nuclear staining expressed as pixel intensity, demonstrating increased nuclear localization of β-catenin after FAT1 knockdown.

(f) TOPFLASH reporter assay demonstrates reduction in β-catenin-dependent transcription following expression of non-mutated FAT1, but not FAT1 mutant constructs, in 293T cells. Reporter assays were performed as previously described⁵², and luciferase activity reported as relative fluorescence units for TOPFLASH, divided by fluorescence activity for the control (FOPFLASH). Error bars represent 1 standard deviation.

(g) Luciferase assay shows substantially increased β-catenin-mediated transcription after transfection with constitutively active β-catenin S33Y mutant, repressed with co-transfection of FAT1.

*p<.05, **p<.01, ***p<.001, t-test and ANOVA.