Lrp5 in Mammary Stem Cells Figure 5. Lrp5-High Cells are Enriched for Stem Cell Activity. A) Mammary epithelial cells were isolated from 10-week, virgin BALB/c mice, stained for Lrp5 and FACS sorted, as described above. Lrp5 high (red) and negative (green) MECs were then transplanted into cleared fat pads of 3- week BALB/c recipient mice. Following 8 weeks, mammary glands were harvested, carmine stained, and scored for primary outgrowths, as described above. Transplantations of the MRU sub-population (black), in addition to the FACS sorted, unfractionated population (Whole Population, gray) served as controls. Left panel is a representative CD24/CD49f FACS profile, depicting overlaid Lrp5- (red), Lrp5 low (blue), and Lrp5 high (green) populations. B) Estimation of stem cell frequencies for each transplanted population. Stem cell frequencies, fold enrichment, and p values were calculated using limdil software (http://bioinf.wehi.edu.au/software/limdil). doi:10.1371/journal.pone.0006594.g005 from virgin Lrp5 +/+ mice (Fig. 7A). In order to assess Wnt transactivation, we used endogenous Wnt target gene expression (to circumvent artifacts associated with transfection of highly refractive primary cells). Axin2 and Gpr49 are downstream target genes in the Wnt signaling pathway. In MECs from virgin mice, the amount of Axin-2 mRNA was not affected by the absence of Lrp5 (Fig. 7B). Cultured MECs were treated with Wnt3a- containing conditioned medium (or control), and Axin2 was found to be induced 66even in Lrp52/2 MECs. Similarly, a 2– 36induction of Gpr49 was observed in Lrp52/2 MECS (Fig. 7C). PLoS ONE | www.plosone.org 7 August 2009 | Volume 4 | Issue 8 | e6594