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. Author manuscript; available in PMC: 2018 Feb 14.
Published in final edited form as: Nat Cell Biol. 2016 Oct 31;18(12):1346–1356. doi: 10.1038/ncb3434

Figure 7.

Figure 7

Lgr6+ cells contribute to luminal, but not basal, mammary tumours (a) Description of MMTV-PyMT, Lgr6-CreERT2, and Rosa26-tdTomato alleles (“PLT mice”) for detection and lineage tracing of Lgr6+ cells and induction of mammary tumours. MMTV: murine mammary tumour virus promoter; PyMT: polyoma virus middle T oncogene. (b) Representative confocal z-stack images showing rare EGFP+ mammary gland cells in PL females. Left: Hyperplastic mammary gland of 4w-old female. Scale bars: 100 µm; 25 µm (inset). Right: Mammary carcinoma of 14w-old female. Scale bars: 100 µm; 50 µm (inset). (c) Scheme illustrating tamoxifen administration in pre-puberty (green arrows) and puberty (red arrows) and analysis time-points to assess the contribution of Lgr6+ cells to MMTV-PyMT-induced mammary tumours. (d) Frequency of EdU+ cells with respect to EGFP+ cells in mammary glands of PL females of different ages. Lines indicate means. (n = 4 mammary glands pooled from n = 3 mice/time-point). Mean ± s.e.m.; n.s., not significant; P = 0.542 (One-way ANOVA). (e) Confocal z-stack images demonstrating expansion of tdTomato+ clones over time after induction at P12. Scale bars: 100 µm (left), 500 µm (right). Percentage indicates extent of tdTomato-labelled tumour area. (f) Quantification of tdTomato+ mammary gland area or tumour area 4w p.i. at P12 (n = 3 mice) and at carcinoma stage (21w – 24w, n = 5 mice). Lines indicate means. *P < 0.048. (unpaired two-tailed t-test) (g) Confocal z-stack images demonstrating expansion of tdTomato+ clones over time after induction at P28. Scale bars: 100 µm (left), 500 µm (right). (h) Quantification of tdTomato+ mammary gland area or tumour area 4w p.i. at P28 (n = 4 mice) and at carcinoma stage (21w – 24w, n = 7 mice). Lines indicate mean. n.s., not significant; p = 0.166 (unpaired two-tailed t-test). (i) Scheme summarizing the protocol used to study the contribution of Lgr6+ cells to MPA/DMBA induced carcinogenesis. MPA: Medroxyprogesterone acetate; DMBA: 7,12-Dimethylbenz[a]anthracene. (j) 3D reconstruction of K14/K8 immunostaining of MPA/DMBA mammary tumour from LT female induced at 4w. Only sporadic K14+/tdTomato+ cells are observed (dashed box and inset). Scale bars: 200 µm, 10 µm (inset). See Supplementary Table 2 for source data for d, f, h.