Additional file 10 of Changes in correlation between promoter methylation and gene expression in cancer

Hierarchical clustering of breast cancer patients based on the average methylation level of CGI + SS associated with cluster 3down. (PDF 379 kb

Concordance of the prediction with molecular signatures combination.

<p><b>Combination of 2 to 7 molecular signatures.</b> (A)Overall population (B) NPI risk groups.</p

Performance of the eight molecular signatures

<p>(A)Breast Cancer Specific Survival (Kaplan Meier curves) (B) Discrimination performances (ROC curves)</p

Clinical, histological and molecular signatures of 769 ER-positive HER2-negative patients presenting breast carcinoma^*.

<p>Clinical, histological and molecular signatures of 769 ER-positive HER2-negative patients presenting breast carcinoma<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148957#t001fn001" target="_blank">^*</a>.</p

Concordance of the 8 molecular signature, according to the modified-NPI risk groups*.

<p>*We considered concordance for 0 or 1 signature and 7 or 8 signatures.</p

The transcriptional signature of ERα^neg mammary luminal progenitors is conserved in their derived lineages.

<p><b>(A)</b> Genome-wide heat map showing the pattern of GFP^neg and GFP^pos luminal cells (Lin^-CD24⁺CD29^low) from 10-wk-old N1Cre^ERT2R26^mTmG females induced for 24 h. Biological triplicates for each group were performed by pooling two females per experiment and are represented by different columns in the unsupervised clustering. <b>(B)</b> GSEA analysis showing the enrichment plots for GFP^neg and GFP^pos transcriptome profiles in four published gene signatures (Luminal_Mature_Up, Luminal_Mature_Down, Luminal_Progenitor_Up, Luminal_ Progenitor_Down). Sorted GFP^neg cells correlate with luminal mature cells, while sorted GFP^pos cells correspond to luminal progenitors. ES: Enrichment Score. <b>(C)</b> qRT-PCR analysis, in adult N1Cre^ERT2R26^mTmG females induced for 24 h, of the top-ten ranked genes in the transcriptomic analysis (first row: up-regulated in GFP^pos and second row: down-regulated in GFP^pos) confirms the differential expression of each of these genes detected in the microarray experiments. The expression levels of each gene in sorted myoepithelial cells are also shown (Myo). Relative mRNA expression was normalized to the housekeeping gene 18S. (*) <i>p</i> < 0.05, (**) <i>p</i> < 0.01, (***) <i>p</i> < 0.001 with <i>t</i> test. <i>n</i> = 2. <b>(D)</b> FACS analysis of sorted cells from N1Cre^ERT2R26^mTmG females induced at 4 wk of age and analyzed 10 wk later (<i>n</i> = 8) shows that GFP^pos cells (Notch1-derived progeny, in green) overlap with ER^neg luminal progenitor markers (CD49b^pos, Sca1^neg and CD133^neg in blue) gated as Lin^-CD24⁺CD29^low luminal cells, showing an identical profile as Notch1-expressing cells 24 h post-induction (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002069#pbio.1002069.g004" target="_blank">Fig. 4D</a>).</p

Repartition of patients according to number of poor signature.

<p>Repartition of patients according to number of poor signature.</p

Breast Cancer Specific Survival according to the Nottingham Prognosis Index.

<p>Breast Cancer Specific Survival according to the Nottingham Prognosis Index.</p

Proposed model for luminal cell hierarchy during mammary gland development.

<p><b>(A)</b> Lineage tracing from embryos reveals that Notch1-expressing (N1^pos) multipotent stem cells exist only during embryonic mammary development, when they co-express myoepithelial cytokeratin (K5) and luminal cytokeratin (K8). These multipotent stem cells can generate all mammary lineages (labeled in green postnatally). <b>(B)</b> After birth, luminal stem cells resolve in two distinct luminal progenitors: ERα^pos and ERα^neg cells, which maintain exclusively their own lineage throughout adulthood. Unipotent progenitors lacking ERα expression (ERα^neg) marked by Notch1 are responsible for generating milk-producing alveoli at pregnancy and define self-renewing luminal cells able to survive mammary gland involution.</p

Notch1 is expressed in multipotent embryonic stem cells.

<p><b>(A)</b> Schematic representation of the Notch1-CreERT2^SAT knock-in mice (referred to as N1Cre^ERT2) and Rosa26^mTomato/mGFP reporter mice (called R26^mTmG) used in this study. Pregnant females were induced with tamoxifen to label their embryos at embryonic day E15.5 and double transgenic N1Cre^ERT2R26^mTmG littermates were analyzed 24 h later <b>(B–D)</b> or 5 wk after birth (<b>E–G</b>). <b>(B–D)</b> Representative embryonic mammary bud sections show that Notch1^pos cells (marked by GFP in green) express both myoepithelial (K5, in red in B) and luminal markers (K8, in red in C) and they are negative for ERα (in red in D), <i>n</i> = 2. (<b>E–G</b>) Representative pubertal mammary gland sections show that Notch1-derived clones (in green) contain myoepithelial (K5^pos in red in E) and luminal cells (K8^pos in red in F) as well as ERα^pos and ERα^neg cells (ERα in red in G), <i>n</i> = 3. 4',6-diamidino-2-phenylindole (DAPI) stains DNA in blue. Scale bars correspond to 20 µm in B–G, and 10 µm in the magnifications of the insets.</p