media containing 10% DMSO, and frozen in liquid nitrogen prior to transfer into recipient mice. To identify the population of Lrp5 high expressing cells that were not localized to the MRU sub- population, virgin BALB/c MECs were stained with anti-VWB in addition to CD49f and CD24. Microarray MECs were isolated from 6 groups of Lrp5+/+ and 3 groups of Lrp52/2 mice, as described above. Each sample group comprised 5 mice each that spanned 12–16 weeks of age. Estrous staging was performed on the mice by vaginal cytological examination and mice belonging to different stages of the cycle were assigned to each group randomly. Total RNA was isolated from MECs using the Qiagen RNeasy kit (Qiagen), as described above. The isolated RNA was submitted to the Gene Expression Center, University of Wisconsin-Madison where a quality control test was performed on the RNA samples. RNA from each group was used for cDNA synthesis followed by labeling of the cDNA with Cy3. The labeled cDNA samples were submitted to NimbleGen and hybridized to Mus musculus 1-Plex arrays (Roche NimbleGen, A4543-00-01) that represent 42,586 mouse genes. The single color NimbleGen arrays were scanned with a GenePix 4000B microarray scanner. The data was extracted from scanned images using NimbleScan software and the Robust Multichip Average (RMA) algorithm used to generate gene expression values. The details of labeling, hybridization, scanning and normalization of the data are described in detail on the NimbleGen website (http://www. nimblegen.com). The normalized data was subsequently analyzed using GeneSifter, an online microarray data analysis system (http://www.genesifter.net). Heat maps were generated from pooled Lrp5 +/+ and 2/2 samples. T-tests were used to determine the statistical significance between the two groups which was established at p,0.05. Quantitative RT-PCR Lrp5 +/+ and 2/2 MECs from 12–14 week old mice were cultured on matrigel-coated plates and treated with Wnt-3a (Wnt) or L Cell (Con) conditioned media for 16 h, 1, 2, or 3 days. Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer instructions. cDNA was generated using a mix of oligo dT and random primers using QuantiTect Reverse Transcription Kit (Qiagen). cDNA (100 ng) was amplified by real time PCR using 5 mL SYBR Green qPCR SuperMix-UDG with Rox (Invitrogen) and 4 mL of forward and reverse primers (0.5 mM). The analysis was performed on each sample in triplicate with an ABI 7900-HT (Applied Biosystems, Foster City, CA). Relative transcript levels were calculated using the comparative Ct method and normalized to the previously characterized house- keeping genes, tata binding protein (TBP) and hypoxanthine- guanine phosphoribosyltransferase (HPRT) [75]. Primer sequences and additional methods are described in Methods S1. Mammary Reconstitution Assay Mammary glands of 3-week old BALB/c virgin mice were cleared of endogenous epithelium as previously described [76]. Populations of mammary epithelial cells isolated by flow cytometry were quickly thawed, washed, counted, and re-suspended in DMEM containing 5 mg/mL Matrigel and loading dye (5% glycerol/0.5% trypan blue/25 mM HEPES pH 7.2). A 1 mL volume of cell suspension containing 50–50,000 cells was injected into each cleared fat pad of the recipient mice. The fat pads were removed 8 weeks following transplants, fixed in 2% PFA overnight at 4uC, stained with carmine alum, dehydrated in ethanol, and de- fatted in xylene. Outgrowths were subsequently identified using a Lrp5 in Mammary Stem Cells light microscope, photographed, and the number of outgrowths quantified for each dose of cells transplanted. Estimation of Stem Cell Frequencies and Statistical Analyses Stem cell frequencies were estimated using the limdil software for limiting dilution analysis (http://bioinf.wehi.edu.au/software/ limdil). All other data were compared by either unpaired Student’s T-test or Wilcoxon Sum Rank test, using Mstat 4.0 statistical software (Norman Drinkwater, McArdle Laboratory). Significance was established at p,0.05. Supporting Information Figure S1 Evaluation of Antibody Specificity. To determine whether there is any cross-reactivity between the Lrp5 and Lrp6 antibodies, 293 cells were transfected with plasmids encoding Lrp5 or Lrp6. A) Western blot of transfected 293 cell lysates for Lrp5 and Lrp6. Actin was used as a loading control. B) Immunofluo- rescence of transfected 293 cells stained for either Lrp5 or Lrp6 (red). Nuclei were stained with ToPro3 (blue). C) FACS analysis of transfected 293 cells. Gates were set based on staining levels seen in the untransfected control samples. Scale bar = 50 mm. Found at: doi:10.1371/journal.pone.0006594.s001 (3.84 MB TIF) Figure S2 Specificity of Lrp5 Antibody Staining of MECs by FACS. A) C57Bl6 Lrp5 +/+ MECs were isolated, reduced to single cell suspensions, and stained with anti-Lrp5, in addition to CD24 and CD49f (and Lin antisera) prior to FACS analysis. B) C57Bl6 Lrp5 2/2 MECs were isolated and stained as described in A. Top Panels: gating strategy for exclusion of cell doublets, apoptotic (PI+), hematopoetic (CD45+), and endothelial (CD31+) cells. Bottom Panels: Gates were drawn for Lrp5 expression on the basis of the staining pattern for the Lrp52/2 MEC population. Lrp5 positive (blue) and Lrp5 negative (red) populations were overlaid on the CD24/CD49f staining profile. Found at: doi:10.1371/journal.pone.0006594.s002 (3.01 MB TIF) Figure S3 Ductal Outgrowth of Lrp5 2/2 MECs is Enhanced by the Presence of GFP Labeled Control Cells. MECs were isolated from C56Bl6 Lrp5 2/2 and Actin-GFP mice (12–14 weeks old). They were counted and mixed in various proportions to total 10,000 cells, and then tested for their relative reconstitution activity after transfer into cleared fat pads of C57Bl6 Lrp5 +/+ mice. Outgrowths were analyzed for the presence of GFP positive cells, and subsequently Carmine stained. They are presented as % colonization of glands, and % of each outgrowth that is GFP+ (black = proportion of unlabeled cells; green = proportion of GFP+ cells). Found at: doi:10.1371/journal.pone.0006594.s003 (1.33 MB TIF) Figure S4 The Ductal Stem Cell Deficiency Observed in Lrp52/2 Glands does Not Affect Ductal Integrity after Multiple Rounds of Parity. Control (A) and Lrp52/2 (B) glands were evaluated by whole mount staining, and show similar structure and fat pad colonization. Scale bar = 1 mm. Found at: doi:10.1371/journal.pone.0006594.s004 (3.89 MB TIF) Figure S5 K5 mRNA Expression is Decreased in Lrp5 2/2 MECs and Embryos. A) Quantification of K5 mRNA expression in uncultured Lrp5 +/+, 2/2, and MMTV-Wnt1 MECs. B) Quantification of K8 mRNA expression in Lrp5 +/+, 2/2, and MMTV-Wnt1 MECs. C) Quantification of K5 mRNA expression in E10-12 Lrp5 +/+, +/2, and 2/2 embryos. D) Quantification of K8 mRNA expression in E10-12 Lrp5 +/+, +/2, and 2/2 embryos. All data were normalized to the housekeeping genes TBP and HPRT. PLoS ONE | www.plosone.org 12 August 2009 | Volume 4 | Issue 8 | e6594