Supplementary Figure 6: Lgr6+ cells in MMTV-PyMT-expressing mammary glands. | Nature Cell Biology

Supplementary Figure 6: Lgr6+ cells in MMTV-PyMT-expressing mammary glands.

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

Supplementary Figure 6

(a) Mammary glands of Lgr6-CreERT2+/− (left) and Lgr6-CreERT2+/−:MMTV-PyMT+/− females (right) at P14. PyMT-induced lesions are found at P14 (inset). Scale bars, 50 μm, 20 μm (inset). (b) Mammary glands of Lgr6-CreERT2+/−(left) and Lgr6-CreERT2+/−:MMTV-PyMT+/− females (right) at P28. Middle T antigen-induced hyperplasia in puberty (right, inset). Scale bars, 100 μm, 20 μm (inset). (c) Middle T expression in early lesions of Lgr6-CreERT2+/−:MMTV-PyMT+/− mammary gland at P14. Lesions stained positive for PyMT (arrows) whereas normally appearing ducts were negative (arrowhead). Arrowheads in inset: PyMT− cells embedded within PyMT+ region. Scale bars, 100 μm, 20 μm (inset). (d) Middle T and K14 expression in pre-malignant mammary gland tissue of 2w-old (left panel) and mammary tumour of 15w-old (right) Lgr6-CreERT2+/−:MMTV-PyMT+/− females. Only rare Middle T oncogene expressing cells are positive for EGFP (arrows) and EGFP+ cells negative for PyMT oncogene are found (arrowheads). Scale bars, 50 μm (left), 200 μm (right panel), 50 μm (inset). (e) Adjacent EGFP+/tdTomato+ (yellow arrows/arrowheads) and tdTomato+ (red arrows/arrowheads) clones 2d post P28-induction. Green arrows/arrowheads depict EGFP+ cells. Scale bar, 50 μm. (f) GFP/EdU co-stained Lgr6-CreERT2+/−:MMTV-PyMT+/−mammary glands at 2w (left) and 15w of age (right). Left: EdU+ cells expressing Lgr6 (arrows). Scale bars, 50 μm. Right: Tumour regions with EdU+/Lgr6+ cells (arrows and inset). Scale bar, 200 μm, 25 μm (inset). (g) GFP immunostaining in mammary tumour of 24w-old PLT female induced at P29. Scale bars, 100 μm, 10 μm (inset). (h) Tumours from PLT females injected with sunflower-seed oil at 2w (left) or 4w (right) did not present with tdTomato+ cells. Scale bars, 1 mm. (i) Tamoxifen administration and time-points to analyse the contribution of Lgr6+ cells to tumour maintenance. (j) PLT mammary tumours 1d (inset) and 1w p.i. Scale bars, 100 μm (inset), 100 μm (inset). (k) Quantification of tdTomato+ area in PLT tumours after induction at 14w. (n = 6 tumours pooled from 4 mice) Line indicates mean. (l) Lgr6 in situ hybridization combined with immunostaining 1w after MPA (left) or MPA/DMBA (right) treatment demonstrating basal (arrows) and luminal (arrowheads) Lgr6+ cells in mammary glands undergoing chemical carcinogenesis. Scale bars, 25 μm.

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