Figure 6.

EZH2 binds to β-catenin and induces β-catenin nuclear accumulation. A: Immunohistochemical staining for β-catenin protein in the mammary glands of 4 months virgin mice. Note the accumulation of β-catenin in the nucleus of hyperplastic epithelial cells of EZH2 transgenic mice (arrows) that contrasts with the crisp membrane β-catenin in wild-type glands (×400). B: Immunoblot analysis for β-catenin and cyclin D1 in MMTV-EZH2 and wild-type mammary glands. Nuclear and total protein lysates were prepared from 4-month-old virgin mice. MMTV-EZH2 glands show up-regulation of nuclear β-catenin and total cyclin D1 levels compared with wild-type glands. Ponceau S staining shows equal loading. The number beneath each band indicates the fold change in intensity of the corresponding band relative to the control. C: EZH2 interacts with β-catenin in mammary gland tissues from 4-month-old virgin mice. Immunoprecipitation (IP) of EZH2 or β-catenin followed by reciprocal EZH2 or β-catenin immunoblotting from nuclear extracted lysates of EZH2 transgenic and wild-type mammary glands. Inputs represented 10% of extracts. Immunoprecipitation with IgG was used as the negative control and immunoprecipitation with EZH2 and β-catenin served as the positive controls. D: Immunofluorescence of human breast tissue samples. EZH2 and β-catenin proteins colocalize in the nuclei of several hyperplastic epithelial cells but not in normal acini. Intraductal epithelial hyperplasia exhibits up-regulation of EZH2 in the nuclei as well as colocalization of EZH2 and β-catenin proteins in the nuclei of hyperplastic epithelial cells (arrow). EZH2 is red, β-catenin is green, colocalized proteins are yellow, and 4,6-diamidino-2-phenylindole (DAPI) stains the nuclei blue. Original magnification, ×600.