surprising that DNp63 is the predominant isoform expressed in human breast cancers [38]. Interestingly, DNp63 has been shown to interact with and regulate the Wnt signaling pathway, promoting cell proliferation [68]. Thus, Wnt signaling through Lrp5 may regulate the proliferative potential of the basal mammary stem cell population by inhibiting senescence (as it does in various human cell lines in vitro, [69]). We conclude that profound differences in regenerative potential are not necessarily reflected at the gross level of epithelial organogenesis. Instead, there are changes in the predisposition of the cellular populations to senescence, and perhaps to growth stimuli and transforming events. Materials and Methods Mice and Materials C57Bl6 Lrp5 +/+, 2/2 [29,70], C57Bl6 MMTV-Wnt1 [71], C57BL/6-Tg(CAG-EGFP)1Osb/J (strain 003291, ubiquitous EGFP expression, driven by b-actin promoter) and BALB/c (Jackson Labs, Bar Harbor, ME) mice were bred and maintained in accordance with the guidelines set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals, published by the U.S. Public Health Service. All experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Wisconsin-Madison. Fluorescent conjugated rat monoclonal antibodies against CD49f (GoH3), CD24 (M1/69), CD45 (30-F11), and CD31 (MEC 13.3) were purchased from BD Biosciences (San Jose, CA). Mouse anti-Lrp6 was generated as previously described [46] and Lrp5 (41–130) was purchased from Abnova (Taipei City, Taiwan). Mouse monoclo- nal antibodies against SMA (1A4), tubulin (JDR.3B8), and actin (AC-15) were purchased from Sigma Aldrich (St. Louis, MO). Rabbit anti-K5 was purchased from Covance (Madison, WI), rabbit-anti VWB was purchased from Dako (Glostrup, Denmark), and rat anti-K8 (Troma-I) was purchased from the Developmental Studies Hybridoma Bank (University of Iowa). All fluorescent conjugated secondary antibodies were purchased from Molecular Probes (Eugene, OR). MEC Isolation and Culture MECs were prepared from mouse mammary glands as previously described [34], using reagents and protocol from Stem Cell Technologies (Vancouver, CA). Briefly, mammary glands were isolated from virgin or 14 day pregnant 12–14 week old C57Bl6 Lrp5 +/+, 2/2, MMTV-Wnt1, or BALB/c virgin mice. The mammary glands were cut into small pieces with fine scissors and digested for 6 hours, 37uC in Epicult-B supplemented with 5% fetal bovine serum, 300 U/mL collagenase and 100 U/mL hyaluronidase. The resulting mammary organoids were washed, counted, and frozen in DMEM containing 10% FBS and 10% DMSO until further use. Prior to cell culture, mammary organoids were reduced to single cells by digesting in consecutive one-minute incubations with trypsin and dispase. The cells were passed through a 70 mm mesh filter prior to seeding into Matrigel-coated plates [72]. Cells were maintained in either normal culture media (DMEM supplemented with 10% FBS, 10 mg/ml insulin, and 100 U/m Pennicillin/Streptomycin) or L-Cell (Con) or Wnt-3a (Wnt) conditioned media [73] diluted 50% with normal culture media. Northern Blotting Lrp5 +/+, +/2, and 2/2 embryos were isolated from approximately 18 day pregnant mice, flash frozen in liquid nitrogen, and transferred to RNAlater -ICE (Ambion, Foster City, Lrp5 in Mammary Stem Cells CA) before RNA isolation. A similar strategy was used for isolated MECs and cell lines (RNAlater (Ambion)/RNeasy mini kit; Qiagen). NMuMG cells were cultured in DMEM supplemented with 10% FBS and 10 mg/mL insulin (Sigma). HC11 cells were cultured in RPMI 1640 supplemented with 10% FBS, 5 mg/mL insulin, and 10 ng/mL rEGF (R&D Systems, Minneapolis, MN). The preparation of Northern blots and probes is described in Methods S1. Western Blotting Embryos isolated from 10–12 day pregnant C57Bl6 Lrp5 +/+ or Lrp5 2/2 mice were homogenized for 1 min/4uC in ‘‘Streuli’’ lysis buffer [74] using a Polytron homogenizer. Cultured cells were harvested by scraping into culture medium, centrifuged at 1806g for 5 min, resuspended in lysis buffer, and incubated for 30 minutes on ice. Fresh, purified mammary epithelial cells were quickly thawed, washed with culture media, resuspended in lysis buffer, and incubated for 30 min on ice. All lysates were cleared at 12,0006g for 30 min. Supernatants were removed and protein concentration determined (using Bradford reagent (Pierce, Rock- ford, IL)) and analyzed by SDS-PAGE and immunoblotting (as previously described [72]). Immuofluorescence Mammary tissue sections were prepared from either virgin or 10–12 day pregnant, adult female Lrp5 +/+ and Lrp5 2/2 mice. Virgin mammary glands were fixed overnight in 2% paraformal- dehyde at 4uC, embedded in paraffin, and sectioned (8 mm). Tissues were deparaffinized, re-hydrated, and blocked with 10% non-immune goat serum for 1 h at room temperature. Pregnant mammary glands were frozen in OCT compound (Electron Microscopy Sciences, Hatfield, PA) prior to sectioning. Frozen sections were fixed in acetone for 20 min at room temperature prior to blocking with 10% non-immune goat serum for 1 h and overnight incubation with anti-Lrp5, K5, SMA, or K8 antibodies. Samples were incubated with anti-mouse IgG-Alexa 488 and anti- rabbit or rat IgG-Alexa 546 secondary antibodies for 2 h at room temperature. Nuclei were stained by incubation with ToPro3 (Molecular Probes) for 30 min prior to visualization using confocal microscopy (Radiance 2100, Biorad, Hercules, CA). For ‘‘cytos- plats,’’ single cell suspensions of FACS sorted mammary epithelial cell populations were dried briefly onto microscope slides and fixed in ice-cold methanol and acetone for 4 and 2 minutes, respectively. Cell preparations were blocked in 10% non-immune goat serum and stained as above. The number of K5 and K8 positive cells were quantified by manual counting of at least 100 cells in three random fields per experimental sample. Flow Cytometry Single cell suspensions of BALB/c mammary epithelial cells were prepared from frozen preparations of mammary organoids, as described above. The resulting single cell suspensions were stained with Lrp5 or Lrp6 in addition to fluorescent conjugated rat monoclonal antibodies to CD49f, CD24, CD45, and CD31 for 30 min on ice. The cells were washed and incubated with anti- mouse IgG-Alexa 405 for 30 min on ice, and analyzed on a FACSVantage cell sorter with DiVa software (Becton Dickson, Franklin Lakes, NJ). Live cells were discriminated by propidium iodide exclusion. CD45 and CD31 positive cells were excluded prior to gating of populations based on Lrp5 expression levels. For functional evaluation, cell populations which exhibited high, low, or negative Lrp5 staining, together with a CD24+CD49fhigh (MRU) fraction for comparison, were sorted into pure fetal bovine serum, centrifuged at 7006g for 5 min, re-suspended in culture PLoS ONE | www.plosone.org 11 August 2009 | Volume 4 | Issue 8 | e6594