Fig. 6: Macrophages regulate MECs through Wnt5a upon Mcam loss.

a qRT-PCR analysis of Axin2, GSK-3β, β-catenin (n = 5 mice in each group), Wnt5a, Wnt5b, and Wnt11 (n = 3 mice in each group) expression in mammary tissues from WT and cKO mice by MMTV-Cre. b IHC staining of Wnt5a in WT and cKO mice. Scale bar, 20 μm. c qRT-PCR results for Wnt5a in indicated cell populations of breast tissues. Wnt5a expressed in adipocyte of WT was taken as a control. The results are from 3 independent mice. d Representative images (left) and reconstitution efficiency (right) of whole-mount-stained mammary outgrowths derived from transplantation of isolating mammary macrophages transducing with shWnt5a together with WT or cKO basal cells. 200 basal cells and 800 isolated macrophages were transplanted into cleared mammary fat pads of recipient mice. n = 5 for WT group and n = 6 for cKO group. Scale bar, 5 mm. e–g Representative images (e), clone numbers (f, n = 6 for anti-IgG group and n = 5 for anti-Wnt5a group), and clone diameter (g, n = 11 for each group) of colonies formed by cKO mammry cells cocultured with isolated macrophages treated with anti-IgG or anti-Wnt5a, respectively. 200 mammary cells and 800 isolated macrophages were seeded in each group. Scale bar, 100 μm. h qRT-PCR analysis of expression levels of Wnt5a in WT and cKO mammary cells co-cultured with isolated macrophages treated with Il4 or IgG antibodies. n = 3 replicates for each group. i IF staining of Wnt5a in mammary tissues of WT and cKO mice after treatment with Il4 antibody in vivo. Scale bar, 50 μm. Data are means ± SEM. Two-sided Student’s t test was used to evaluate statistical significance. Source data are provided as a Source Data file.