Additional file 1: of Analysis of tumour-infiltrating lymphocytes reveals two new biologically different subgroups of breast ductal carcinoma in situ

Pathobiological and TIL characteristics of lymphocyte-rich (lyDCIS) and microinvasive carcinomas (miCa) corresponds to the title. (DOCX 15 kb

Regulation of metabolism by BRCA1 protein in SUM1315-LXSN and SUM1315-BRCA1 cell model.

<p>A. Gene expression study: mRNA of SUM1315-LXSN and SUM1315-BRCA1 cells were extracted and analyzed on TaqMan Low Density Arrays as described in materials and methods. The replicates represent independent culture experiments. Low levels of expression are represented in blue and high levels of expression in yellow. B. The major metabolites observed by ¹H-NMR and by LC-MS were analyzed by hierarchical clustering<b>.</b> The replicates represent independent culture experiments. Small quantities of metabolites are represented in blue and high quantities in yellow.</p

Phenotypic characterization of cell lines.

<p>A. Expression of BRCA1 was analysed by Western blotting using anti-BRCA1 antibody. Mouse anti-BRCA1 primary antibody was purchased from Oncogene Research (San Diego, CA). Actin was used as a loading control. B. Proliferation experiments were performed using the Sulforhodamine B technique. Data are means of three independent experiments + SEM. C. Cycle distribution was determined using flow cytometry. Data are means of three independent experiments + SEM. D. Percentage of apoptotic cells was determined at 48 h using the Annexin V test. Data are presented as means of 3 independent replicates + SEM. *: p<0.05.</p

Role of BRCA1 in energetic metabolism.

<p>A. The major enzymes and metabolites involved in energy metabolism were quantified by q-RT-PCR, ¹H-NMR and by LC-MS. Data are presented as means of at least 3 independent replicates + SEM. *: p<0.05; **: p<0.01, +: p = 0.06 for lactate, SUM1315-BRCA1 vs SUM1315-LXSN cell lines. B. Analyses of HK2 and IDH1 enzymes was performed by Western Blot and quantified by ImageJ software. Data are presented as means of 3 independent replicates + SEM. *: p<0.05; ***: p<0.001, SUM1315-BRCA1 vs SUM1315-LXSN cell lines. C. The oxygen partial pressure was measured in the culture medium of SUM1315-LXSN and SUM1315-BRCA1 cell lines subjected to hypoxic atmosphere for the indicated time. *: p<0.05, SUM1315-BRCA1 vs SUM1315-LXSN cell lines. D. Production of lactate and consumption of glucose were measured in cell culture media by enzymatic colorimetric assays. Data are presented as means of 3 independent replicates + SEM.</p

BRCA1-induced increased activity of antioxidation pathways.

<p>A. The major enzymes and metabolites involved in antioxidation metabolism were quantified by q-RT-PCR, ¹H-NMR and by LC-MS. Data are presented as means of at least 3 independent replicates + SEM. *: p<0.05; **: p<0.01, SUM1315-BRCA1 vs SUM1315-LXSN cell lines. B. Schematic representation of glutathione metabolism and its regulation by BRCA1. Genes (<i>italicized</i>) in red: overexpression. Metabolites: grey, not detected; red, increased.</p

Reprogramming of lipid metabolism induced by BRCA1.

<p>A. The major enzymes and metabolites involved in lipid metabolism were quantified by ¹H-NMR, quantitative RT-PCR and HPTLC. Data are presented as means of at least 3 independent replicates + SEM. *: p<0.05; **: p<0.01, SUM1315-BRCA1 vs SUM1315-LXSN cell lines. BOHButyrate: β-hydroxybutyrate. B. Intact whole cell pellets were analyzed by ¹H-NMR spectroscopy in order to reveal differential metabolites the most concentrated in breast tumor cells. Spectra were normalized according to a published procedure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102438#pone.0102438-BayetRobert2" target="_blank">[25]</a>. Representative spectra of intact SUM1315-LXSN (blue) and SUM1315-BRCA1 (red) cells are shown. Arrows: most concentrated differential signals. C. Fatty acid composition was analyzed by GCFS. Results of major groups are given as percentage of total lipids. Data are presented as means of at least 3 independent replicates + SEM. *: p<0.05; **: p<0.02, SUM1315-BRCA1 vs SUM1315-LXSN cell lines.</p

Schematic representation of global metabolism regulation by wild-type BRCA1 transfection.

<p>Enzymes or metabolites up-regulated in SUM1315-BRCA1 cells compared to SUM1315-LXSN cells are represented in red whereas down-regulated ones are presented in green.</p