1. Volume 3, Issue 4, Pages 429-441 (October 2008)
Notch Signaling Regulates Mammary Stem Cell Function and Luminal Cell-Fate Commitment 
Toula Bouras, Bhupinder Pal, François Vaillant, Gwyndolen Harburg, Marie-Liesse Asselin-Labat, Samantha R. Oakes, Geoffrey J. Lindeman, Jane E. Visvader 
Cell Stem Cell 
Volume 3, Issue 4, Pages (October 2008)
DOI: /j.stem
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
Differential Expression of Notch Pathway Genes in Distinct Mammary Epithelial Subpopulations and Preferential Pathway Activation in Luminal Epithelium
(A) Expression analysis of Notch receptor and ligand genes in the MaSC-enriched (CD29hi) and luminal subpopulations (CD61− and CD61+) sorted by flow cytometry from 8-week-old virgin mouse mammary glands was performed by q-RT-PCR relative to 18S rRNA. Expression is relative to that of the CD29hi cells, set at 1. Data represent mean ± SD of three independent experiments. Statistically significant differences of p < 0.05 (t test) were observed between expression in the basal versus luminal populations for all genes except for Notch3, Notch4, Delta3, and Delta4.
(B) Anti-NICD1 and Anti-Hey2 immunostaining of mouse mammary gland sections. Inset, isotype-matched negative control. Scale bar, 80 μm.
(C) Q-RT-PCR of Notch target genes in the MaSC-enriched (CD29hi) and luminal subpopulations (CD61− and CD61+) sorted by flow cytometry from 8-week-old virgin mouse mammary glands relative to 18S rRNA. Expression is relative to that of the CD29hi cells, set at 1. Data represent mean ± SD of three independent experiments. A statistically significant difference of p < 0.05 (t test) was observed between expression in the basal versus luminal populations for all genes.
(D) ChIP analysis of endogenous NICD1 binding to the Hey2 and Gata-3 promoters in CD29loCD24+ luminal cells. Chromatin that had not been immunoprecipitated provided the input control. NICD1 binding was specific for promoter regions containing consensus Cbf-1 binding sites (specific), and no binding was detected on Hey2 and Gata-3 promoter regions that lacked Cbf-1 binding sites (control).
(E) q-RT-PCR of Hey1 and Gata-3 expression in primary MaSC-enriched (CD29hi) and CD61+ luminal cell subsets grown in Matrigel and treated with vehicle DMSO (veh) or 4 μM GSI (GSI) for 14 days. Data represent mean ± SD of three independent experiments. The asterisk (∗) denotes a statistically significant difference between vehicle and control of p < 0.05.
Cell Stem Cell 2008 3, DOI: ( /j.stem )
Copyright © 2008 Elsevier Inc. Terms and Conditions

3. Figure 2
Knockdown of Cbf-1 Expression in MaSCs Increases the Mammary Repopulating Frequency In Vivo
(A) FACS dot plots of CD29 and CD24 expression in the Lin− population of mammary outgrowths resulting from transplantation of CD29hiCD24+ cells transduced with the nonsilencing retrovirus LMS-nons (control) or Cbf-1-shRNA-LMS retrovirus (Cbf-1 shRNA).
(B) Reduced Cbf-1 mRNA in CD29hiCD24+ cells transduced with Cbf-1-shRNA-LMS retrovirus (Cbf-1 shRNA) compared to LMS-nons (control) retrovirus, as demonstrated by q-RT-PCR relative to 18S rRNA. Data represent mean ± SD of three independent experiments. The asterisk (∗) denotes a statistically significant difference of p < 0.05 compared to control.
(C) Whole mounts of GFP+ ductal outgrowths derived from CD29hiCD24+ cells transduced with the LMS-nons (control) or Cbf-1-shRNA-LMS (Cbf-1 shRNA) retrovirus, at 8 weeks posttransplantation in virgin recipients, showing normal and disorganized ductal architecture with increased end bud density, respectively. The arrow points to hyperbranched ducts. Scale bars, 1.5 mm.
(D) Immunostaining of end bud sections from ductal outgrowths derived from CD29hiCD24+ cells transduced with LMS-nons (control) or Cbf-1-shRNA retrovirus for K18, p63, and K14. Arrows point to regions of basal cell expansion (p63+ and K14+ cells). Scale bars, 100 μm.
Cell Stem Cell 2008 3, DOI: ( /j.stem )
Copyright © 2008 Elsevier Inc. Terms and Conditions

4. Figure 3
Inhibition of Notch Signaling in the MaSC-Enriched Population Leads to Expansion of Basal Stem/Progenitor Cells in Matrigel Cultures
(A) Histogram showing the number of colonies formed in Matrigel from CD29hiCD24+ cells transduced with the LMS-nons (control) or the Cbf-1-shRNA retrovirus. Data represent mean ± SD of three independent experiments. The asterisk (∗) denotes a statistically significant difference of p < 0.05 compared to control.
(B) Morphology and immunostaining of Matrigel colonies derived from CD29hiCD24+ cells transduced as in (A). Left-hand panel, images of the 3D colonies in Matrigel (scale bar, 250 μm). Middle and right panels, K18 and p63 immunostaining of the colonies (scale bar, 100 μm).
(C) Histogram showing the number of colonies formed in Matrigel from CD29hiCD24+ cells treated with the vehicle DMSO (veh) or 2 μM GSI for 10 days. Data represent mean ± SD of three independent experiments. The asterisk (∗) denotes a statistically significant difference of p < 0.05 compared to control.
(D) Morphology and immunostaining of Matrigel colonies from CD29hiCD24+ cells treated as in (C). Left-hand panel, images of the 3D colonies in Matrigel (scale bar, 250 μm). Middle and right panels, K18 and p63 immunostaining of the colonies (scale bar, 100 μm).
Cell Stem Cell 2008 3, DOI: ( /j.stem )
Copyright © 2008 Elsevier Inc. Terms and Conditions

5. Figure 4
Constitutive Activation of Notch Signaling in the MaSC-Enriched Population Promotes Luminal Lineage Commitment and Hyperplasia In Vivo
(A) Whole mounts of GFP+ hyperplastic nodules arising from transplantation of CD29hiCD24+ cells transduced with the empty pMIG (control) or pMIG-NICD1 (NICD1) retrovirus, shown in virgin and 0.5 day lactation (0.5 dL) recipients. Scale bar, 1.5 mm.
(B) Western blot analysis of whole-cell lysates from primary CD29hiCD24+ cells transduced with pMIG (control) or NICD1 retrovirus using anti-Notch1 and GFP antibodies. Tubulin provided a loading control.
(C) Semiquantitative RT-PCR analysis of Hey1 and Hey2 mRNA levels in CD29hiCD24+ cells transduced with the pMIG (control) or NICD1 retrovirus. 18S rRNA served as a loading control.
(D) Immunostaining of GFP+ hyperplastic nodules from NICD1-transduced CD29hiCD24+ cells in a virgin recipient for K18, K8, MUC1, SMA, p63, and E-cadherin. Scale bar, 100 μm.
(E) Immunostaining of GFP+ hyperplastic nodules from NICD1-transduced CD29hiCD24+ cells at 0.5 day lactation for Stat5a, P-Stat5a, and mouse milk protein. Scale bar, 100 μm.
(F) Morphology and immunostaining of Matrigel colonies derived by CD29hiCD24+ cells transduced with pMIG (control) or NICD1 retrovirus. Left-hand panel, images of the 3D colonies in Matrigel (scale bar, 250 μm). Middle and right panels, K18 and p63 immunostaining of the colonies. Scale bar, 50 μm.
(G) Colony staining distribution of CD29hiCD24+ cells transduced with the control pMIG or NICD1 retrovirus following immunostaining for K18 and p63 (n = 300 colonies). The asterisk (∗) denotes a statistically significant difference of p < 0.05 compared to control for each antibody.
Cell Stem Cell 2008 3, DOI: ( /j.stem )
Copyright © 2008 Elsevier Inc. Terms and Conditions

6. Figure 5
Constitutive Notch Activation in Committed CD61+ Luminal Progenitor Cells Leads to Hyperplastic Nodules and Tumors that Phenocopy Those Derived from NICD1-Expressing MaSCs
(A) Whole mount of GFP+ hyperplastic nodules following transplantation of NICD1-transduced CD61+ luminal progenitor cells in a virgin recipient. Scale bar, 0.75 mm.
(B) Western blot analysis of whole-cell lysates from primary CD61+ luminal progenitor cells transduced with the pMIG (control) or NICD1 retrovirus, using anti-Notch1 and GFP antibodies. Tubulin provided a loading control.
(C) Semiquantitative RT-PCR analysis of Hey1 and Hey2 mRNA levels in primary CD61+ luminal progenitor cells transduced with the pMIG (control) or NICD1 retrovirus, relative to 18S rRNA.
(D) H&E-stained sections of hyperplastic nodules derived from NICD1-transduced MaSC-enriched (CD29hiCD24+) and luminal progenitor (CD61+) subpopulations 8 weeks posttransplantation. Shown are hyperplastic cell clusters, the majority of which contained a filled lumen, often with a solid filled or papillary type pattern. Scale bar, 100 μm.
(E) H&E-stained sections of hyperplastic nodules derived from NICD1-transduced MaSC-enriched (CD29hiCD24+) and luminal progenitor (CD61+) subpopulations at 3 months posttransplantation, revealing neoplastic progression and tumor formation. The left-hand panels for each population represent large hyperplastic lesions, while the right-hand panels for each represent palpable tumors. Scale bar, 100 μm.
(F) Immunostaining of GFP+ hyperplastic nodules from NICD1-transduced CD61+ luminal progenitors in a virgin recipient for K18, K8, MUC1, SMA, p63, and E-cadherin. Scale bar, 100 μm.
(G) Immunostaining of GFP+ hyperplastic nodules from NICD1-transduced CD61+ luminal progenitors at 0.5 day lactation for Stat5a, P-Stat5a, and mouse milk protein. Scale bar, 100 μm.
Cell Stem Cell 2008 3, DOI: ( /j.stem )
Copyright © 2008 Elsevier Inc. Terms and Conditions

7. Figure 6
Constitutive Notch Activation Promotes CD61+ Luminal Progenitor Expansion and Appears to Confer Self-Renewing Potential
(A) Colony-forming assays in Matrigel of CD61+ luminal progenitor cells transduced with the pMIG (control) or NICD1 retrovirus. Scale bar, 1 mm.
(B) Histogram showing colony-forming capacity as described in (A). Data represent mean ± SD of three independent experiments. The asterisk (∗) denotes a statistically significant difference of p < 0.05 compared to control.
(C) Colony size distribution (μm; n = 100) of CD61+ luminal progenitor cells transduced with the pMIG (control) or NICD1 retrovirus. The asterisk (∗) denotes a statistically significant difference of p < 0.05 compared to control.
(D) Immunostaining of colonies in Matrigel derived from CD61+ luminal cells transduced with the pMIG (control) or NICD1 retrovirus for K18 and p63. Scale bar, 50 μm.
(E) Colony-staining distribution for p63 immunostaining of CD61+ luminal progenitor colonies in Matrigel, transduced with the pMIG (control) or NICD1 retrovirus (n = 300 colonies). The asterisk (∗) denotes a statistically significant difference of p < 0.05 compared to control.
(F) Images of primary (1°) and tertiary (3°) spheres derived from NICD1-transduced CD61+ cells relative to control cells.
(G) CD61 quantitation in tertiary (3°) spheres (histogram, solid line; dashed line shows the isotype).
(H) Schematic model for the role of Notch signaling within the proposed mammary epithelial hierarchy. Notch has distinct functions in different cell types: Notch signaling normally represses the proliferation of stem cells, and downregulation of the pathway leads to MaSC expansion. Activation of Notch1 in MaSCs directs luminal cell commitment at the expense of differentiation along the myoepithelial lineage. In the presence of continual Notch signaling, luminal progenitor cells expand and self-renew, eventually leading to the development of tumors.
Cell Stem Cell 2008 3, DOI: ( /j.stem )
Copyright © 2008 Elsevier Inc. Terms and Conditions