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Additional Supporting Information may be found in the online version of this article.

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stem1454-sup-0001-suppfig1.tiff20346K

Supplemental Figure 1. RANK overexpression under the MMTV promoter impairs alveolar differentiation and promotes proliferation. Related to Figure 1.

(A) Representative image showing RANK expression in a normal duct of MMTV-RANK mice as measured by IHC.

(B) Representative images of mammary whole mounts of virgin and late gestation (G18.5) WT and MMTV-RANK mice.

(C) mRNA expression levels of milk protein, Wap relative to β-actin in WT and MMTV-RANK glands at the indicated time points during gestation. Each bar represents mean values for two or three mice and SD are shown. Measurements for each sample were performed in triplicate and mean was used.

(D) Frequency of BrdU positive cells in mammary glands of WT and MMTV-RANK mice at the indicated points. Each bar represents mean values and SD for two mice.

(E) Representative images of BrdU immunostaining at G14.5 in WT and MMTV-RANK mammary glands.

stem1454-sup-0002-suppfig2.tiff25544K

Supplemental Figure 2. RANK overexpression disrupts mammary populations in WT and MMTV-RANK mammary glands. Related to Figure 2.

(A) Expression of Rank and K14 mRNA relative to β-actin in FACS sorted populations of virgin WT and MMTV-RANK mammary glands. Basal (B): CD24lo CD49fhi Sca1-, Luminal Progenitor (LP): CD24hi CD49flo Sca1-, luminal mature (LM): CD24hi CD49fhi Sca1+. Mammary cell pools from 2 WT and 3 MMTV-RANK mice were analyzed. Quantification was performed in duplicate, mean and SD values are shown.

(B) Frequency of Sca1+ cells within the luminal population of virgin WT and MMTV-RANK glands. Each bar represents one mouse.

(C) Quantification of the percentage of CD24lo CD49fhi Sca1- (B:basal) and CD24hi CD49flo Sca1- (LP: luminal progenitor) in the Lin- population of virgin WT and MMTV-RANK mammary glands.

(D) Quantification of the percentage of CD24+ CD49flo (luminal) in the Lin- population, and of CD61+ and CD49b+ referred to luminal cells of WT and MMTV-RANK virgin and pregnant mammary glands at the indicated points in gestation.

(E) Quantification of the percentage of CD61+ cells in luminal cells of young virgin (12-15 weeks), aged virgin or multiparous (32-82 weeks) WT and MMTV-RANK mice.

stem1454-sup-0003-suppfig3.tiff38667K

Supplemental Figure 3. RANK overexpression results in preneoplasic lesions and tumors that are heterogeneous and contain K14+K8+ cells. Related to Figure 4.

(A) Representative images of H&E sections of aged virgin and multiparous mammary glands of WT and MMTV-RANK mice.

(B) Percentage of K14+/K8+ cells versus total number of cells per duct of aged multiparous (MP) MMTV-RANK mice. Quantification in ducts from young females is included for comparison. 5-7 ducts/structures were analyzed per mouse. Mean, SD and t-test p values are shown.

(C) FACS histograms showing CD24, CD49f, Sca1, CD61 and CD49b staining within the Lin-, in four spontaneous MMTV-RANK tumors. For some markers axis for high/low populations are shown. Overlay with corresponding negative controls is shown.

(D) Summary table of the tumor phenotypes. Most frequent profiles of the tumor cells are shown. n.a. not analyzed. Two populations were found in T5 with different CD49b and Sca1 values as indicated. K14 refers to cells K14+ K8-. K5 refers to cells K5+ K8- and K8 to cells K8+K14-K5-.

stem1454-sup-0004-suppfig4.tiff25889K

Supplemental Figure 4 and 5. Keratin profiles of MMTV-RANK spontaneous tumors reveal heterogeneity. Related to Figure 4.

Representative images of DAPI, K5, K14, K8 and SMA immunostaining in spontaneous MMTV-RANK tumors.

stem1454-sup-0005-suppfig5.tiff16431K

Supplemental Figure 4 and 5. Keratin profiles of MMTV-RANK spontaneous tumors reveal heterogeneity. Related to Figure 4.

Representative images of DAPI, K5, K14, K8 and SMA immunostaining in spontaneous MMTV-RANK tumors.

stem1454-sup-0006-suppfig6.tiff40194K

Supplemental Figure 6. Mammary glands of aged multiparous WT mice resemble those of MMTV-RANK mice. Related to Figure 5.

(A) Representative images of whole mounts of multiparous mammary glands of old WT and MMTV-RANK mice.

(B) Quantification of the percentage of CD24lo CD49fhi (basal) and CD24hi CD49flo (luminal) in the Lin- population, of young virgin (12-15 weeks), aged virgin or multiparous (MP; 32-82 weeks) WT and MMTV-RANK mice.

(C) Sox9 and Slug expression relative to K14, and Elf5 expression relative to Κ8 in mammary glands of young virgin, old virgin and old multiparous (MP) WT mice. Each dot represents one mouse. Mean, SEM and significant t-test p values are shown. For each sample measurements were performed in triplicate and mean was used.

stem1454-sup-0007-suppinfo.xlsx108K

Supplemental Table 1. Gene sets for the original signatures that define the mature luminal, luminal progenitor, and mammary stem cell subsets 28. The signatures are subdivided in over- and under-expressed sets. The results from the GSEA analyses are provided for each subset: the gene rank, rank metric, enrichment score, and core enrichment contribution (yes/no) are indicated.

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