Additional systemic overexpression of TM4SF5 in ApcMin/+ mice increased β-catenin stabilization and transcriptional activity. (A) We analyzed the intestines of ApcMin/+ (n = 4) or ApcMin/+:TgTM4SF5 (n = 6) mice at 26 weeks old using immunohistochemistry (IHC) with mouse IgG or anti-β-catenin antibody. β-catenin immunostaining intensities were categorized as explained in the Materials and Methods section or quantitative comparison between conditions. (B) We analyzed HT29 cells via luciferase reporter assay following transfection with the β-catenin-responsive LEF/TCF-1 reporter pTOP-FLASH vector with shRNA plasmids (shControl or shTM4SF5) for 24 h; cells were treated with (+) or without (−) Wnt-3a for 12 h before the analysis. *, **, *** depict P < 0.05, 0.01, and 0.005, respectively. (C-E) HT29 (C, D) or HT116 (E) cells were independently transfected with control or Apc-full (C), shControl (shCon) or shTM4SF5 (D), or Mock-Flag or TM4SF5-Flag (E) plasmids for 24 h, and the cells were treated with recombinant Wnt-3a for 12 h, as explained in (B), prior to whole-cell extract preparation for standard Western blots for the indicated molecules. The data represent three independent experiments.
