FIGURE 4.

BMPR1a deficiency resulted in reduced myoepithelial lineages and compromised repopulating capacity of mammary stem cells. (A) FACS analysis of mammary gland epithelial cells from control and cKO mice at pregnancy day 14.5 using the Lin/CD24/CD29/CD61 surface marker set. n = 4 mice per group. (B) Quantification of Lin^– CD24⁺CD29^high (Myo), Lin^– CD24⁺CD29^low (Lu) and Lin^– CD24⁺CD29^lowCD61⁺ cell populations in control and cKO mice at pregnancy day 14.5. n = 4 biological replicates. (C) Immunofluorescence staining for K14 (red) and K8 (green) in control and cKO mammary glands at pregnancy day 14.5. n = 3 mice per group. Scale bar, 25 μm. (D) Western blotting for K14 in control and cKO mammary glands at pregnancy day 14.5. β-Actin was used as a loading control. Statistical analysis the expression of K14/β-Actin. n = 3 mice. (E) Immunofluorescence staining for K14 (red) and K8 (green) in HC11 mammary epithelial cells treated with BMP4 (50 ng/mL) for 24 h. n = 3 biological replicates. Scale bar, 25 μm. (F) Western blotting for K14 in HC11 cells treated with BMP4 (50 ng/mL) for 24 h. β-Actin was used as a loading control. Statistical analysis the expression of K14/β-Actin. n = 4 biological replicates. (G) Illustration of the mammary transplantation assays using K14-rtTA/teto-Cre/Bmpr1a^fl/fl and control mouse mammary stem cells and the induction strategy. (H) Whole-mount staining of mammary glands from the mammary transplantation assays and quantification of the proportion of ductal trees. n = 5 mice per group. Scale bar, 1 mm. (I) Immunohistochemistry staining for Integrin β3 in control and cKO mammary glands at pregnancy day 14.5. Quantification of Integrin β3⁺ cells in alveoli. n = 3 mice per group. Scale bar, 50 μm. Data were presented as means ± SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.