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1.
Fig. 6.

Fig. 6. From: Dystroglycan controls signaling of multiple hormones through modulation of STAT5 activity.

DG modulates STAT5 activity in vivo. (A) Frozen sections of L1 stage mammary glands from control or ΔDGK14-Cre mice immunostained with anti-STAT5 antibodies and counterstained with propidium iodide. ΔDGK14-Cre mammary glands show markedly reduced nuclear STAT5 staining as indicated by the arrows and lack of overlap staining (yellow color) in the merged images at right. (B) Frozen sections of L1 stage mammary glands from control or ΔDGK14-Cre mice immunostained with anti-P-STAT5 antibodies and counterstained with propidium iodide. P-STAT5 staining in ΔDGK14-Cre mammary glands is absent. Scale bar: 20 μm.

Dmitri Leonoudakis, et al. J Cell Sci. 2010 November 1;123(21):3683-3692.
2.
Fig. 2.

Fig. 2. From: Dystroglycan controls signaling of multiple hormones through modulation of STAT5 activity.

K14-Cre activity efficiently ablates DG expression. (A) Frozen sections of L1 stage mammary glands from control or ΔDGK14-Cre mice co-stained with anti-laminin (Ln) and anti-DG (DG) antibodies. The right panels show the merged laminin and DG co-staining. DG staining was absent from the ΔDGK14-Cre epithelium. Arrows indicate the green laminin staining the exterior of the gland, whereas arrowheads indicate red DG staining the basal surface of the epithelium. Areas of yellow reveal overlap of laminin with its receptor, DG. Scale bar: 20 μm. (B) Protein extracts of primary cell cultures from control and ΔDGK14-Cre glands immunoblotted with the antibodies indicated on the right. No β-DG was detected in mammary epithelial cells cultured from ΔDGK14-Cre mice.

Dmitri Leonoudakis, et al. J Cell Sci. 2010 November 1;123(21):3683-3692.
3.
Fig. 8.

Fig. 8. From: Dystroglycan controls signaling of multiple hormones through modulation of STAT5 activity.

Laminin-111 enhances the expression of genes induced by growth hormone. MEpL cells expressing WT DG were overlaid with medium containing 100 μg/ml laminin-111 or 1.5% matrigel in the presence or absence of growth hormone for 72 hours. (A) Cell lysates were analyzed for the presence of the milk protein β-casein by western blot. E-cadherin was used as a loading control. The ratio of β-casein to E-cadherin was normalized to untreated cells and is indicated below the immunoblots. (B) Total RNA was extracted from these cells and RT-qPCR was performed for the expression of Igf1 and normalized to Gapdh expression. The data is expressed as the fold change of mRNA in hormone-treated samples versus control, untreated samples (*P<0.01; error bars indicate s.d.).

Dmitri Leonoudakis, et al. J Cell Sci. 2010 November 1;123(21):3683-3692.
4.
Fig. 3.

Fig. 3. From: Dystroglycan controls signaling of multiple hormones through modulation of STAT5 activity.

Knockout of dystroglycan expression in the mammary gland impedes growth of the epithelial compartment. Mammary gland development in control and ΔDGK14-Cre was assessed by whole-mount staining of the fourth (inguinal) gland isolated from nulliparous females at 8 weeks of age. (A) Control glands stained with Carmine Alum. (B) Glands from heterozygous (DGfl/wt;K14-Cre) and (C) ΔDGK14-Cre mice stained with X-gal when the Rosa26R transgene was present, revealing Cre activity. The lymph node appears as dark circles at the center of the gland. Lower panels are enlarged portions (white boxes) of the images above. Scale bars: 5 mm (top) and 500 μm (bottom). (D) Outgrowth of the mammary gland beyond the central lymph node measured for glands of 8-week-old control (n=6) and ΔDGK14-Cre (n=4) mice. *P<0.005.

Dmitri Leonoudakis, et al. J Cell Sci. 2010 November 1;123(21):3683-3692.
5.
Fig. 1.

Fig. 1. From: Dystroglycan controls signaling of multiple hormones through modulation of STAT5 activity.

K14-Cre expression is initiated early in mammary gland development. Histochemical staining for lacZ expression was performed in bi-transgenic K14-Cre+; R26R+ mice to reveal K14-Cre-mediated DNA recombination. (A) In a transverse view of dissected ventral skin from an E14 embryo, lacZ reporter activity (blue) is detected in the epidermal layer (arrow), and in the invaginating mammary bud (box and inset). (B) Staining is evident throughout the mammary epithelium at the P1 stage. (C) Staining of mammary tissue sections at mid-pregnancy shows widespread lacZ expression in the epithelium. (D) Enlargement of boxed area in C shows lacZ expression in luminal and myoepithelial cell populations (white arrows). Scale bars: 0.2 mm (A), 1 mm (B), 0.5 mm (C) and 50 μm (D).

Dmitri Leonoudakis, et al. J Cell Sci. 2010 November 1;123(21):3683-3692.
6.
Fig. 4.

Fig. 4. From: Dystroglycan controls signaling of multiple hormones through modulation of STAT5 activity.

Mammary gland outgrowth is attenuated after mid-pregnancy in ΔDGK14-Cre mice. Mammary gland development in control and knockout mice was assessed by whole mount staining of fourth (inguinal) gland and by hematoxylin and eosin staining of tissue sections. Control glands are stained with Carmine Alum whereas heterozygous and ΔDGK14-Cre glands are stained with X-gal, revealing Cre activity on the Rosa26R transgene. (A) ΔDGK14-Cre glands at mid-pregnancy (day 14.5) show normal tissue architecture and outgrowth compared with control glands. Scale bars: 5 mm (top) and 500 μm (bottom). (B) At lactation day 1 (L1), heterozygous DG glands show normal tissue architecture, whereas ΔDGK14-Cre glands display an apparent reduction in outgrowth. Scale bars: 5 mm (top), 500 μm (middle) and 100 μm (bottom). (C) Digital images of whole-mount L1 mammary glands traced and thresholded to determine percentage epithelial coverage. Compiled data reveal a 20% reduction in ΔDGK14-Cre epithelial outgrowth (n=3; *P<0.05).

Dmitri Leonoudakis, et al. J Cell Sci. 2010 November 1;123(21):3683-3692.
7.
Fig. 5.

Fig. 5. From: Dystroglycan controls signaling of multiple hormones through modulation of STAT5 activity.

Laminin assembly is compromised in mammary epithelial cells lacking DG expression. (A) Primary mammary epithelial cells from control or ΔDGK14-Cre mice were incubated with 10 ng/ml FITC-laminin-111 overnight. DG-expressing cells (WT) showed efficient assembly of laminin, whereas DG-knockout cells (KO) showed little assembled laminin. The phase-contrast images below show the positions of the cells. (B) Cells derived from the immortalized DG-knockout cell line (MEpL), transfected with DG (WT) or a vector control (KO) were overlaid with 10 ng/ml FITC-laminin-111 overnight. WT cells show efficient assembly of laminin, whereas the knockout cells show no assembled laminin. The differential interference contrast images below show the positions of the cells. Scale bar: 10 μm. (C) Fluorescence images as in A and B were thresholded and the resulting pixel intensity was divided by the cell area. The compiled quantified data are normalized to WT. Data were compiled from two experiments and 10 images each. P<0.0001 for both primary mammary epithelial cells and MEpL cells (error bars indicate s.e.m.).

Dmitri Leonoudakis, et al. J Cell Sci. 2010 November 1;123(21):3683-3692.
8.
Fig. 9.

Fig. 9. From: Dystroglycan controls signaling of multiple hormones through modulation of STAT5 activity.

The cytoplasmic domain of dystroglycan is not required for modulation of STAT5 activity or expression of genes induced by prolactin and growth hormone. MEpL cells lacking DG expression (KO), expressing the wild-type DG cDNA (WT) or intracellular C-terminal deletion mutant of DG (ΔC) were grown in 3D rBM cultures for 1 week and treated with prolactin or growth hormone for 24 hours. (A) Protein extracts from MEpL cells grown in 3D cultures and stimulated with hormones were immunoblotted with the antibodies indicated on the right. E-cadherin was used as a loading control. The ratio of β-casein to E-cadherin was normalized to WT DG, untreated cells and is indicated below the immunoblots. (B) Ratio of P-STAT5 to STAT5 quantified from immunoblots as in A (*P<0.05, n=3; error bars indicate s.e.m.). (C) Total RNA was extracted from MEpL cells grown and treated as above. RT-qPCR was performed for the expression of Igf1 and normalized to Gapdh expression. The data is expressed as the fold change of mRNA in hormone-treated samples versus control, untreated sample and normalized to the WT DG untreated sample (*P<0.05, error bars indicate s.d.).

Dmitri Leonoudakis, et al. J Cell Sci. 2010 November 1;123(21):3683-3692.
9.
Fig. 7.

Fig. 7. From: Dystroglycan controls signaling of multiple hormones through modulation of STAT5 activity.

Loss of DG attenuates prolactin and growth hormone signaling through STAT5. (A) Immortalized mammary epithelial cells (MEpL cells) lacking DG expression (KO) or expressing the wild-type DG cDNA (WT) were grown in 3D rBM cultures for 1 week and treated with prolactin or growth hormone or left untreated for 24 hours. Cell colonies were stained with anti-P-STAT5 antibodies and nuclei counterstained with propidium iodide. Confocal images show staining of nuclear P-STAT5. Scale bar: 10 μm. (B) Protein extracts from MEpL cells grown in 3D cultures and stimulated with hormones immunoblotted with the antibodies indicated on the right. (C) Ratio of P-STAT5 to STAT5 quantified from immunoblots as in B (*P<0.05, n=3; error bars indicate s.e.m.). (DF) Total RNA was extracted from MEpL cells grown and treated as above for 24 hours (D,E) or 72 hours (F). Real time-qPCR was performed for the expression of genes encoding β-casein, WAP and IGF-1 and normalized to Gapdh expression. Data are expressed as the fold change of mRNA in hormone-treated samples versus untreated samples (**P<0.001; error bars indicate s.d.).

Dmitri Leonoudakis, et al. J Cell Sci. 2010 November 1;123(21):3683-3692.

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