Research Article ZNF703 in luminal B breast cancer 20 mM, NaCl 150 mM, Glycerol 10%, Triton X-100 0.1%). Beads were then resuspended in 35 ml of Sample Buffer 2X (Tris pH 6, 8; 120 mM, Glycerol 20%, SDS 4%, b-Mercapto-ethanol), boiled at 998C. After centrifugation, immunoprecipitated complexes were resolved by SDS- PAGE. Mass spectrometry Immunocomplexes were separated on NuPage 4–12% Bis–tris acrylamide gels in Mops buffer according to the manufacturer’s instructions (Invitrogen, Groningen, Netherlands). SDS-PAGE gels were stained with colloidal Coomassie blue (Imperial protein stain, Pierce) and proteins of interest were isolated as previously described (Goncalves et al, 2008). Extracted peptides were subjected to mass spectrometric analysis for identification. Proteins were identified by nanoLC-MS/MS using a nano-LC Ultimate system (Dionex, LC Packing) on line with an ion trap analyser (HCT, Ultraflex, Bruker Daltonics Inc. Bremen, Germany). MS/MS Raw data were processed using DataAna- lysis 3.1 and Biotools 3.1 softwares (Bruker Daltonics Inc.). Protein searches were done using a local Mascot search engine (Version 2.2.03) (Matrix Science Ltd., London, UK) against SwissProt database (release 57.12, release date December 17, 2009; 513877 sequences; Homo sapiens: 20401 sequences) using the following parameters: human for taxonomy, 0.5 Da mass tolerance for parent and fragments search, one missed cleavage side for trypsin was allowed. Fixed carbamidomethylated cystein and potential methionine oxidation were selected as modifications. Protein identification was based on at least three unique peptides for each protein. Mascot Ions score was set to >30 and each score was further validated against ions score corresponding to sequence identity. Proteins identified were validated by immunoblotting as described in supplementary methods. Immunofluorescence MCF7 GFP and GFP-ZNF703 cells were grown on labtek chambers. After 48 h, cells were fixed 20 min in 3.7% paraformaldehyde, PBS; permeabilized 5 min with PBS, 0.1% Triton X-100; blocked with protein block serum-free (Dako, X0909); and incubated 1 h with primary antibodies (anti-PHB2; Abcam ab71970, 1:100), anti-SMRT (BD Biosciences 611386, 1:100), anti-HSP60 (Abcam ab46798, 1:50), anti-PML (Santa-Cruz Sc-966, 1:20), anti-GFP (Roche 11814460001, 1:250), anti-ERa (Neomarkers Fremont CA, RB-9016-P1, 1:100) and anti-DCAF7 (Sigma–Aldrich, HPA022962, 1:50), and with correspond- ing secondary antibody Alexa Fluor 594 (Invitrogen) conjugated for 20 min incubation. Images of the nuclei were counterstained with DAPI/antifade (Invitrogen) and coverslipped. Sections were examined with a fluorescent microscope (Leica). For ER-a and ZNF703 expression scoring, around 100 cells were scored for three indepen- dent chambers. Flow cytometry analysis Staining for CD49f was done using a PE-labelled anti-CD49f antibody (diluted 1:100; BD Biosciences). Fresh cells were stained with 1 mg/ml PI (Sigma–Aldrich) for 5 min for viability. Analysis of CD49f expression was done on FACS LSRII (BD biosciences). Luciferase reporter assays We utilized three different luciferase reporter assays for the detection of oestrogen transcriptional activity (p17M-ERE-Luc, a gift from Dr. D. Metzger, IGBMC, Strasbourg, France) and for detection of E2F1 transcriptional activity (pGL2-3XE2F1-Luc and pGL3-CCNE-Luc, a gift from Dr. C. Sardet, IGM, Montpellier, France). MCF7 GFP and GFP- ZNF703 stable cell lines were electroporated (Amaxa) with 250 ng TK- R-Luc (pGL3; Promega, France) as an internal control and 1 mg of one luciferase reporter vector according to the manufacturer’s protocol. For ER-related transcriptional activity, cells were seeded overnight in 60-mm dishes containing phenol red-free RPMI 1640 (Invitrogen) supplemented with 10% charcoal dextran-treated fetal bovine serum (Hyclone). 16 h later, 10 nM of 17-b estradiol (E2) were added to the medium and luciferase activity was determined after 24 h. For luciferase activity detection, we prepared transfected cells lysate and utilized the Dual-luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. We normalized each luciferase activity value by protein concentration determined by bradford assay and TK-renilla luciferase activity value. Each experi- ment was done in duplicate. Tumoursphere assay MCF7 GFP-ZNF707 and MCF7 GFP cell lines were cultured in adherent conditions in the presence or absence of E2 for 3 days then plated as single cells in 1% agarose coated plates at a density of 1000 cells/ml and grown for 5 days. The number of primary spheres was counted under microscope. Subsequent cultures after dissociation of primary spheres were plated in 1% coated plates at a density of 1000 cells/ml. As for primary spheres, the number of secondary spheres was determined under microscope. Experiments were done in triplicate. Tumoursphere cultures were grown in a serum-free mammary epithelium basal medium as previously described (Ginestier et al, 2009). Cell cycle progression analysis Cell cycle synchronization was achieved by a non-pharmacological method using cell-to-cell contact inhibition in conjunction with serum depletion to induce cell quiescence. In brief, individual cells were spread out in 24-well plates and were grown to 80% confluence. Then FBS-rich medium (10%) was removed and cells were incubated in high density conditions with charcoal FBS-poor medium (0.5%) for 72 h at 378C in a 5% CO2 atmosphere. Cells were subsequently released from the G1/S arrest by addition of serum-rich medium in the presence or absence of E2. Subsequent synchronization was analysed by flow cytometry. Briefly, supernatant and adherent cells were harvested, washed and suspended in 0.5 ml medium containing propidium iodide (40 mg/ml) and RNase A (40 mg/ml). Analysis of the cell cycle was done on the FACScan (BD biosciences) using CellQuest analysis software. Cell cycle progression was evaluated every 2 h over 24 h after release. It is important to note that a large proportion of cells arrested in G1/S phase will not enter into cell cycle due to senescence induced by the starvation. Statistical analyses Correlations between sample groups and histoclinical factors were calculated with the Fisher’s exact test for qualitative variables with discrete categories, and the Mann-Whitney test for continuous variables. DFS from 510 luminal breast tumours collected from the 11 public datasets was calculated from the date of diagnosis until date of first relapse whatever its location (local, regional or distant) or 164 2011 EMBO Molecular Medicine EMBO Mol Med 3, 153–166 www.embomolmed.org