Lrp5 in Mammary Stem Cells Figure 6. In Lrp5 Null Glands, the Basal Cell Population is Depleted. A) FACS analysis of Lrp5 +/+, 2/2, and MMTV-Wnt1 MECs stained with CD49f and CD24. Gates were drawn to indicate the luminal and basal lineages. Right panel, quantification of the ratio of basal to luminal cells from three independent FACS analyses of CD24/CD49f profiles, shown in left panel. B) Western analysis of lysates prepared from uncultured Lrp5 +/+, 2/2, and MMTV-Wnt1 MECs. Blots were probed with the basal cell marker, K5, and re-probed with tubulin, as a loading control. C) Representative heatmap and statistical analysis of basal markers from microarray of Lrp5 +/+ and 2/2 MECs. Samples were analyzed by GeneSifter software and compared by t-test, *p,0.05. D) Immunofluorescent staining of cultured Lrp5 +/+ and 2/2 MECs. Cells were cultured for 3 days in normal culture media and stained for K8 (red), SMA (green), and DNA (blue), scale bars = 20 mm. Insets depict 56enlargements, scale bars = 4 mm. The number of large, flattened SMA positive cells were quantified from several fields of immunofluorescent staining of Lrp5 +/+ and 2/2 cultured MECs from two independent experiments. E) Quantification of p16Ink4a mRNA levels from Lrp5 +/+ and 2/2 MECs, after 3 days of culture. F) Quantification of p16Ink4a mRNA expression levels of purified luminal and basal Lrp5 +/+ and 2/2 MECs after culture. mRNA levels were normalized to the housekeeping genes, TBP and HPRT. Normalized expression = 22DCt (see Methods S1). G) Quantification of TA-p63 and DN-p63 mRNA expression levels of uncultured Lrp5+/+, 2/2, and MMTV-Wnt1 MECs. Data is expressed as fold change over Lrp5 +/+. Data were compared by Wilcoxon Rank Sum Test. Significance was established at p,0.05. doi:10.1371/journal.pone.0006594.g006 PLoS ONE | www.plosone.org 8 August 2009 | Volume 4 | Issue 8 | e6594