Supplementary Figure 2: Lgr6-expressing cells in postnatal mammary gland development. | Nature Cell Biology

Supplementary Figure 2: Lgr6-expressing cells in postnatal mammary gland development.

From: Lgr6 labels a rare population of mammary gland progenitor cells that are able to originate luminal mammary tumours

Supplementary Figure 2

(a) K8 immunostaining of Lgr6-CreERT2+/− mammary glands at postnatal day 14 (P14) and P30. EGFP+ cells in luminal (arrowheads) and basal cells (arrows). Scale bars, 25 μm. (b) Recombination efficiency 24 h after tamoxifen administration to 2w-old (n = 5 mice) and 4w-old (n = 4 mice) Lgr6-CreERT2+/−:tdTomato+/− (LT) females. Mean ± s.e.m. (c) FACS plot showing the distribution of tdTomato+ cells over MECs 24 h after tamoxifen administration to 2w-old (left) and 4w-old (right) LT mice. (d) FACS analysis to exclude recombination of the Rosa26-tdTomato allele by Lgr6-CreERT2 in the absence of Tamoxifen. Left/middle panel: MECs from 10w old wildtype (WT) or LT mice injected with sunflower seed oil at P12 and analysed 8w later Right: Detection of tdTomato+ cells 8w after administration of 1 mg tamoxifen at P12. (e) Mammary gland from LT mouse injected with sunflower seed oil at P12 and imaged 8w later. Scale bars, 1 mm (f) F-actin staining of LT mammary glands 22w p.i. in pre-puberty (right panel) and 24w p.i. in puberty (left panel). Scale bars, 100 μm, 20 μm (insets). (g, h) Distribution of basal and luminal multi-cellular tdTomato+ clones, and cell patches spanning both compartments after induction in pre-puberty (2w, g). and in puberty (4w, h). Numbers indicate % of multicellular areas comprising adjacent basal and luminal cells. (Analysed clones pooled from n = 3 mice per timepoint: n = 1,157, n = 344, n = 542, n = 645, n = 691 for 2w, and n = 531, n = 1,476, n = 1,733, n = 163, n = 782, n = 597 for 4w). Mean ± s.e.m. (i) K14/K8 immunostained mammary gland 16w p.i. (4w) showing a rare tdTomato+ ‘clone’ spanning the basal (arrow) and luminal (arrowhead) compartments. Scale bar, 25 μm. (j) RNA in situ hybridization (ISH) analysis. Left: Lgr6 (blue arrowheads) and Axin2 (red arrowheads). Lgr6/Axin2 co-expression (arrows) can be observed in some cells. Right: Rare cells in the terminal end buds (TEBs) of pubertal mammary glands co-express Lgr6 and Lgr5 mRNA (arrows; inset). Scale bars, 10 μm (left), 20 μm (right). (k) Overview of transplantation assays of basal EGFP+ and EGFP− cells from adult virgin LT females into emptied fat pads of NOD/SCID mice. See Supplementary Table 2 for source data for g,h.

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