Wnt/FZD7 signaling is active in basal-like mammary tumors.

(A) Representative immunostaining images detecting the levels of active β-catenin (non-phosphorylated form), K14, K8 in tumor tissues from Trp53/Brca1(p53/BRCA1)-deficient, MMTV-PyMT and C3(1)-Tag breast cancer mouse models. DAPI staining marks the cell nucleus. Scale bar = 50 μm. (B) Quantitation of active β-catenin positive cells in (A). (C) Immunoblot analysis of active β-catenin expression in mammary tumors from models described in (A and B). Actin serves as a loading control. (D) qRT-PCR analysis of expression of Fzd family genes in p53/BRCA1-deficient (n = 3), C3(1)-Tag (n = 3), and MMTV-PyMT (n = 2) tumors; error bars indicate mean ± SEM. (E) Upper plots: FACS analysis using antibodies against CD24 and CD29 to differentiate basal versus luminal normal MECs in female mice; lower plots: FACS analysis using a FZD7-specific antibody identified FZD7+ in Lin- (negative for lineage markers, CD45, CD31, TER119) cells and in luminal and basal MEC populations. (F) The percentages of FZD7+ cells in p53/BRCA1-deficient, C3(1)-Tag and MMTV-PyMT tumors, as well as in normal mouse mammary glands, were analyzed by FACS. The FZD7+ peaks are marked and compared in the upper panel and quantified in the lower panel (the P values for p53/BRCA1-deficient, C3(1)-Tag and MMTV-PyMT models were S5C Fig. Numerical values are in S1 Data. FACS, fluorescence-activated cell sorting; MEC, mammary epithelial cell; qRT-PCR, quantitative real-time PCR.