Fig. 4: Depletion of macrophages blocks the elevating clonogenicity and regenerative capacity of MECs upon Mcam loss.

a–c Representative images (a), clone numbers (b, 6 biological replicates in WT and 5 biological replicates in cKO by MMTV-Cre), and clone diameters (c, n = 10 in each group) of colonies formed by basal cells co-cultured with isolated macrophages. Scale bar, 100 μm. d Percentages of macrophages in mammary tissue of WT and cKO mice treated with clodronate liposome (CL) and their control groups (PBS). n = 5 mice in each group of WT. For cKO, n = 4 mice by PBS treatment, and n = 5 mice by CL treatment. e, f IF images of CD206⁺ macrophages and α-SMA⁺ basal cells (e) and quantification of CD206⁺ macrophages in stromal cells (f) in WT and cKO mice with or without CL treatment. n = 6 sections in each group. Scale bar, 50 μm. g, h Representative images (g) and quantification (h) of the filled fat pad by the mammary duct of the whole-mount-stained mammary gland. Scale bar, 5 mm. n = 7 mice in each group. i, j IF images (i) and their quantification (j) of Ki67⁺ proliferation cells and α-SMA⁺ basal cells. n = 10 sections in each group. Scale bar, 20 μm. k Representative images of whole-mount-stained mammary outgrowths derived from transplantation of WT and cKO MECs mixed with PBS or CL. The number of MECs for transplantation was 1*10⁴. The dose of CL (90–125 μl) was determined according to the body weight (~9–12.5 g) of the mice, and PBS had the same volume as CL. Scale bar, 3 mm. l, m FACS analysis (l) and quantification (m) of basal population. n = 5 mice in each group. Mice used in figures d–m were treated with CL (150–170 μl) at 5 weeks of age (body weight ~15–17 g) every other day for a week before harvesting the mammary gland. 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.