(A) Representative flow cytometry dot-plots showing Sca-1 and ICAM-1 expression in the mammary luminal cell population isolated from mice at different stages of development: V-6w, 6-week-old virgin; V-13w, 13-week-old virgin; P-8d, 8-day pregnant; P-16d, 16-day pregnant mice. Combined with Sca-1, ICAM-1 discriminated four cell populations referred to as Lu1, Lu2, Lu3, and Lu4. (B) Colony formation by Lu1, Lu2, Lu3, and Lu4 cell subsets isolated from mammary glands of mature virgin mice. Upper panel: H&E staining of colonies after 8 days in culture. Lower panel: percentages of clonogenic cells. The results from two independent cell samples (each of which with three separate wells) are presented as mean values ±S.E.M. (C) Heat map of qPCR gene expression analysis performed on Lu1, Lu2, Lu3, and Lu4 cells freshly isolated from mammary glands of mature virgin mice. The qPCR values were normalized to Gapdh expression. Mean values from three independent cell preparations were used to establish the map and determine relationships between the luminal subsets by unsupervised hierarchical clustering. (D) q-PCR analysis of Icam1, Ly6a, and Met expression in Lu1, Lu2, Lu3, and Lu4 cell populations freshly isolated from mammary glands of mature virgin mice. The values were normalized to Gapdh expression and represent mean values ±S.E.M from three independent preparations. (E) Representative phase contrast images of spheres derived from Lu2 and Lu4 cell populations grown in the absence or presence of HGF for 10 days. Bar, 150 μm. (F) Comparative expression levels of basal-specific, EMT-associated and luminal-specific genes in spheres derived from Lu2 and Lu4 cells, as determined by q-PCR. Cells were grown as described above in (E). Results are expressed as Log2 ratios of values normalized to Gapdh. The comparator values were those obtained with cell preparations grown in the absence of HGF.