Fig. 4

Myoepithelial layer has fewer cells and is thinner in thickness in AREG^−/− mice. a Representative images of 12-week-old AREG^+/+ (left) and AREG^−/− (right) mammary ducts immunostained with K8 (red) and K14 (green) in merged channel. Arrow indicates discontinuous myoepithelial layer. Scale bar shows 50 μm. At both 6 weeks (b, c) and 12 weeks (d, e), AREG^−/− glands have thinner myoepithelial layer (b, d) and smaller percentage of K14⁺ cells (c, e). At least three animals used in each analysis. f Proportion of K14⁺ cells in ducts and mature duct termini of 12-week-old AREG^+/+ mammary ducts compared (N = 7). g PyMT expression in AT-3 cells suppressed by myoepithelial cells when cocultured. AT-3 cells either cultured alone or with primary myoepithelial cells overnight. RNA extracted and PyMT expression assessed by RT-qPCR. h AT-3 cells cultured without addition of growth factors (control), with 100 ng/ml AREG, 10 ng/ml EGF, or 10 ng/ml bFGF, or with both EGF and bFGF. Statistical analysis performed using t test. *p < 0.05, **p < 0.01, ***p < 0.001. AREG amphiregulin, bFGF basic fibroblast growth factor, EGF epidermal growth factor, PYMT polyoma middle-T antigen