Luminal Progenitors Restrict Their Lineage Potential during Mammary Gland Development (original) (raw)
Fig 2
Notch1 expression in the postnatal mammary gland is restricted to ERαneg and PRneg luminal cells.
(A–B) Representative sections of ducts from pubertal (6-wk-old) N1CreERT2R26mTmG females analyzed 24 h upon tamoxifen injection. Immunofluorescence was performed with anti-K5 antibodies (in red in A), anti-K8 (in red in B), anti-GFP (to reveal Notch1-marked cells in green) and DAPI stains DNA in blue; n = 3. (C) FACS plots of 6-wk-old N1CreERT2R26mTmG females analyzed 24 h upon tamoxifen injection. Dissociated mammary cells were gated as Linneg cells (CD45/CD31/Ter119)neg, and then as mammary epithelial cells (MEC) using the CD24 and CD29 markers, allowing us to resolve luminal (CD24+CD29low) and myoepithelial (CD24+CD29high) populations. 98.24 ±0.4% of GFPpos cells gated in MEC were found in the luminal subset by FACS analysis. Note that GFPpos cells also display Tomato fluorescence 24 h after induction, as the Tomato protein is still present at this time point, even if recombination has occurred. Values are shown in average ± s.e.m, n = 6. (D–E) The expression of Notch1 in sorted luminal (CD24+CD29low) (LUM) and myoepithelial cells (CD24+CD29high) (MYO) mammary cells from 10-wk-old B6/N wild-type females was analyzed at the mRNA level by qRT-PCR in A and at the protein level by western blot in B. The relative mRNA expression was normalized to the housekeeping gene 18S in A, while lamin B1 was used as a loading control in E. For western blot analysis, we used anti-K8 and anti-K5 antibodies as controls for sorted luminal and myoepithelial cells, respectively. n = 2. (F–G) Representative sections of mammary ducts from 6-wk-old N1CreERT2R26mTmG females show that GFP-expressing cells (in green) are invariably negative for ERα (in red in F) and PR expression (in red in G). DAPI stains DNA in blue, n = 3. Scale bars correspond to 20 µm in C–D and F–G and 10 µm in the insets.