Figure 6. Postnatal mammary gland branching requires type I collagen remodeling.

(A) Western blot for MT1-MMP in mammary glands harvested from 5 wk-old Col1a1^+/+, Col1a1^r/+ and Col1a1^r/r mice, as compared to GAPDH.

(B) 3-D reconstructions of CF-CHP immunofluorescence in mammary glands harvested from 6 wk-old Col1a1^+/+ and Col1a1^r/r mice with DAPI staining (scale bar, 30 μm). CF-CHP immunofluorescence is reduced from 116.5±6.3 total pixels/μm² around Col1a1^+/+ ducts (n=10) to 24.9±1.1 total pixels/μm² around Col1a1^r/r ducts (n=9) (p<0.0001, mean ± SEM).

(C) Polarized images of Sirius red staining in 5–6 wk-old Col1a1^+/+ and Col1a1^r/r glands. Insets show bright-field images (scale bar, 200 μm). “D” marks the lumen of each mammary duct.

(D) TEM of 5 wk-old Col1a1^+/+ and Col1a1^r/r glands (scale bar, 500 nm). “Ep” marks the mammary epithelial cells and arrows indicate the mammary epithelial basement membrane.

(E) Carmine staining of 6 wk-old Col1a1^+/+ and Col1a1^r/r glands (scale bar, 5.0 mm).

(F) Quantification of ductal penetration (DP) (mm) and branch points per mm duct (BP/mm) in 5–6 wk-old Col1a1^+/+ (n=6), Col1a1^r/+ (n=3), and Col1a1^r/r (n=5) littermates (****p<0.0001, *p<0.05; mean ± SEM).

(G) Ki67 staining in 6 wk-old Col1a1^+/+ and Col1a1^r/r glands (scale bar, 200 μm).

(H) CK8 and CK14 immunofluorescence with DAPI staining in 6 wk-old Col1a1^+/+ and Col1a1^r/r glands (scale bar, 20 μm).

(I) Senescence-associated β-galactosidase (SA-βgal) staining (pH 6.0) with Eosin counter-staining in 6 wk-old Col1a1^+/+ and Col1a1^r/r glands (scale bar, 200 μm).

See also Figure S5.