Inhibition of Wnt signaling in WS-15 breast cancer cells selected for endogenous Rad6B overexpression downregulates Rad6B gene expression and β-catenin transcriptional activity. (A) Fluorescence imaging of vector control R6B-Zshigh (a) or R6B-Zshigh/LRP6D173 (b) WS-15 cells. (B) Relative Rad6B promoter activities in WS-15 subpopulations. (C) Relative pTOP/Flash or pFOP/Flash activities in WS-15 subpopulations. The data shown in C and D are averages (± S.E.M) of three separate experiments done in duplicate. (D) Steady-state levels of indicated proteins in the cytosols of vector control, R6B-Zshigh or PLKO-Rad6BshRNA WS-15 cells. (E) Tumor morphologies of vector control (a), R6B-Zshigh (b), and Rad6BshRNA (c) WS-15 xenografts by H&E staining. Immunohistochemical analysis of Rad6 (a', b' and c') and β-catenin (a", b" and c") in vector control, Rad6B-Zshigh and Rad6BshRNA WS-15 tumors. Short arrow in panel a shows hyperplastic region and long arrow shows invasive carcinoma. Long arrow in b and b' show blood vessels in tumors, and the short arrow in b' shows nuclear Rad6 staining. Note the loss of cell membrane staining of β-catenin in R6B-Zshigh tumors (compare a" and b"). Insets in c' and c" show magnified images of Rad6 and β-catenin staining, respectively, in hyperplastic ducts of Rad6BshRNA tumors. Original magnification ×4 (a and b); ×10 (c); ×20 (c' and c"); ×40 (a', a", b', b"). (F) Comparison of tumor masses of vector control, R6B-Zshigh and Rad6BshRNA WS-15 derivatives.