Fig. 3
Identification of multiple subsets in pubertal and adult mammary glands. a Heatmap showing hierarchical clustering of 343 epithelial cells from pubertal (green) and adult (black) mammary glands based on their expression of lineage-specific genes (200 basal and 200 luminal genes; see Supplementary Table 3). Columns correspond to cells colored by stage, indicated by bars at the top. The cells shown are the same 5 and 10-week-old cells as in Fig. 1b, c and Supplementary Fig. 1b, c. Seven major cell clusters are indicated (gene lists shown in Supplementary Table 4). Basal genes = blue; luminal genes = purple; red = high expression; blue = low expression. b Multidimensional scaling plot of scRNA-seq profiles. These are the same as in Fig. 1b but cells expressing Cd55 (RPKM > 40) are highlighted as blue triangles. Cd55 + cells are present at all three stages but pre-pubertal glands harbor only rare cells (circled in the plot). c CD55 expression within epithelial subsets isolated from mammary glands in pre-puberty (2 wks) (left), puberty (5 weeks) (center), and adult (10 weeks) (right). Representative dot plots of CD29 vs. CD24 expression in Lin– cells (see gating strategy in Supplementary Fig. 3) are shown on the left, while CD29 vs. CD55 expression in either the luminal (top) or basal (bottom) compartment is shown on the right (n = 3 independent experiments per time-point: 12 mice per experiment for pre-puberty, 6 mice per experiment for puberty, and 4 mice per experiment for adult). d The CD14/CD55 flow cytometric plots of freshly sorted Lin–CD29loCD24+ (luminal) mammary epithelial cells isolated from 10-week-old FVB/N mice (representative of 5 independent experiments, 4 mice per experiment). e Hierarchical clustering of 76 purified luminal cells based on Cd55 and Cd14 gene expression (Fluidigm C1 platform; see Supplementary Fig. 4c). Red = high expression; blue = low expression. f Clonogenic activity of CD14–CD55–, CD14–CD55+, CD14+CD55–, and CD14+CD55+ luminal (CD29loCD24+) subsets from 10-week-old FVB/N mice, plated in Matrigel (1000 cells per population). Representative of three independent experiments, 4 mice per experiment. P-value (unpaired t-test) <0.0001. Scale bar; 1000 µm
