Luminal Progenitors Restrict Their Lineage Potential during Mammary Gland Development (original) (raw)
Fig 4
Notch1-expressing cells define highly clonogenic ERαneg luminal progenitors.
(A) N1CreERT2R26mTmG females induced with tamoxifen at puberty (6 wk of age) and analyzed 24 h later present a high proportion of proliferative GFPpos cells (73.45 ±3.72%), as indicated by Ki67 expression (in red); DAPI stains DNA in blue, n = 5. (B) Left, histogram of the cell cycle profile of total luminal cells (Luminal, in blue) compared to Notch1-expressing cells (GFPpos, in green) sorted by FACS and stained with Hoechst-33342 to evaluate their DNA content. Right, quantification of cycling (S/G2/M) and non-cycling (G0/G1) cells in total luminal cells (blue, S/G2/M = 26.9 ±5.5%) and in Notch1-expressing cells (green, S/G2/M = 47.0 ±5.2%). Data are represented as a mean ± s.e.m. of n = 5 animals, (*) p < 0.05 with t test. (C) N1CreERT2R26mTmG females were induced with tamoxifen at 10 wk of age and analyzed 24 h later. GFPneg and GFPpos cells were sorted as Lin-CD24+CD29low luminal cells by FACS, seeded on a feeder layer of irradiated fibroblast and their clonogenic capacity was evaluated after 7 d in culture. Top, quantification of the number of colonies generated by GFPneg and GFPpos cells. n = 3 experiments with two mice each, (***) p < 0.001 with t test. Bottom, representative pictures of counted colonies stained with Hematoxylin are shown below each bar. (D) Flow cytometry analysis indicates that GFPpos cells (in green) are found within progenitor cell populations (CD49bpos, Sca1neg and CD133neg) gated in the luminal population (Lin-CD24+CD29low) (in blue). GFPpos cells represent 82.02 ±3.28% of CD49bpos cells, 96.9 ±0.6% of Sca1neg cells, and 93.7 ±1.0% of CD133neg cells. n = 10. Scale bars correspond to 20 µm in A and 10 µm in the insets in A.