Supplemental Data 1 Supplemental Data 1: Materials and Methods Patients, methods and quantification of gene expression Procedure concerning the selection of patients, tissue resections and QPCR analysis are described in (Pillaire et al., submitted). Briefly, 74 patients who came from many parts of France and underwent surgery for primary colorectal adenocarcinoma resection were selected. Exclusion criteria were patients treated with adjuvant therapy and with tumor microsatellite instability (MSI) since MSI positive cancers are known to be associated with mismatch repair deficiency. The study was approved by the National Institute of Cancer (INCa) following the recommendations of the National Agency of Agreement and Evaluation for Health (ANAES) and in agreement with the 2004 French Bioethics law. For RNA extraction, 40 to 80 frozen sections (5 to 10µm thick) from tumor and adjacent normal tissues were at once homogenized using RNeasy extraction kit and total RNA was extracted according to the manufacturer’s instruction (QIAGEN SA, Courtaboeuf, France). The quality of total RNA was assessed with the Agilent 2100 ® bio-analyzer (Agilent Technologies, Massy, France) and 1 to 2µg of total RNA were reverse transcribed using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). All studied genes and four control genes (18S, GAPDH, HPRT, YWHAZ) were amplified in triplicate from tumor and normal samples using the TaqMan Universal PCR Master Mix and the TaqMan Low Density Array technology (Applied Biosystems). All PCR amplifications were performed with the TaqMan Low Density Array technology. To normalize gene expression in the matched tumor and normal samples, the QBase software ( was used to test the expression stability of the control genes. For each sample pair, the two most stable control genes were used by QBase to normalize the expression of all transcripts. Then the T(tumor) / N(normal) ratios of normalized values were calculated. Pillaire MJ, Selves J, Gouraud PA, Danjoux M, Negre V, Do C, Gordien K, Bieth A, Guimbaud R, Trouche D, Pasero P, Méchali M, Hoffmann JS, Cazaux C A "3R" signature of progression, genetic instability and negative outcome in colorectal cancer. Submitted for publication. Statistical Analysis The major criteria of gene expression analysis in tumors were the individual ratio between standardized tumor and adjacent normal tissue gene expressions. In order to take into account the non Gaussian distribution of gene expression, non parametric statistics analyses were used. Binomial exact tests evaluated the significance of gene over- and under-expression. Correlation between genes was assessed with a non parametric Spearman’s correlation (Spearman’s rho). All computations were performed using Stata 9.0 SE at the Clinical Data Management and Statistical Analysis Platform of the Hospitals and School of Medicine of Toulouse (Tiermips). Samples preparation and western-blotting For nuclear extracts preparation, cells were harvested using a hypotonic lysis buffer containing 10mM Tris (pH8.0), 10mM NaCl, 2mM MgCl2 and protease inhibitors and incubated 5 min on ice. 50µl/ml of 10% NP40 were added to each sample that were incubated 10 min on ice. After centrifugation, the pellet was re-suspended using a hypertonic buffer containing 20mM Hepes (pH7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA and 10% glycerol (all from Sigma-Aldrich). Total cell extracts were obtained by harvesting the cells in a buffer containing 1% Triton X-100, 2% SDS, 150mM NaCl and phosphatases/proteases inhibitors in 100mM Tris-HCl (pH 7.4). After centrifugation of nuclear or total extracts, the supernatants were collected and samples were quantified using a Dc Protein Assay kit (from Bio-Rad). For Tip60 analysis, nuclear extracts from each cell lines were prepared and submitted to classical SDS-PAGE. To analyze p400 protein amounts and PARP cleavage, total cell lysates were prepared and 10 to 60µg of proteins per lane were separated by NuPAGE® Novex 3-8% Tris-acetate gel (from Invitrogen SARL). Gels were transferred on a PolyVinylidine DiFluoride (PVDF) membrane. Specific primary antibodies as well as peroxidase-conjugated secondary antibodies were used according to standard western blot procedure and peroxidase was then detected by using the (Roche Diagnostics, Meylan, France).