of biotin-labeled cRNA – was done according to the instructions provided in the Affymetrix GeneChip Expression Analysis manual. Each biotin-labelled sample was hybridized onto a GeneChipH Rat Genome Probe Array RG-U34A from Affymetrix for 16 h at 45uC in a GeneChip Hybridization Oven - according to the manufacturer’s recommendation. The expression probe arrays were washed and stained through a GeneChip Fluidics Station 400 according to the Affymetrix GeneChip Expression Analysis manual. The probe arrays were scanned using an HP Gene Array Scanner, controlled by the GeneChip software. Data were processed with the GeneChip Microarray Analysis Suite version 4.0 software (MAS 4, Affymetrix) with default analysis settings; genes were sorted according to robust analysis rules. The Rat1-control cell (time 0) sample was taken as reference for calculating relative expression values as Fold Change (www. affymetrix.com/support/technical/whitepapers.affx: Statistical Al- gorithms Description Document). Fold Changes were computed for Rat1-control cells at 909 following serum stimulation as well as for the Rat1-Omomer cells at 0 and 90 minutes time points. Data were filtered by setting a Fold Change threshold of +3 for the activated genes and 23 for the repressed ones. All data are MIAME compliant and the raw data were deposited in the GEO database, with accession number GSE25039. Correspondence Analysis (CA) was also performed on the same data, via routine present in the SAS (Statistical Analysis Software) package. The results obtained through CA were compared with those of the selection through robust analysis via MAS 4 software. We observed that 89% of genes identified by the manual procedure as decreased at 90 min in the presence of Omomyc were also detected by the CA. The same comparison on genes increased by the induction of Omomyc at 90 min gave again a remarkable fit between the two methods, with about 87% genes in common. To determine overlap among genes downregulated by Omomyc and Myc target genes, we crossed the Omomyc gene list with those of the Myc cancer gene database and genes whose promoters were reported to be occupied by Myc in ES cells [36,37]. 623 of the genes listed in the Myc Cancer Gene database and 984 of those listed as Myc bound targets in ES cells were present in the U34A array; genes not represented in the U34A microarray were excluded from the analysis. Omomyc-associated genes were annotated using Panther software (http://www.pantherdb.org/). Genes not assigned to a biological process (biological process unclassified) were not included in the figures. Luciferase reporter assay 293T cells, seeded in 24-well tissue culture plates, were transfected with nucleolin or p21 reporters – respectively pNucL14 and p21Cip1- Luc – driving expression of firefly luciferase, the Renilla luciferase reporter – pRL-TK from Promega – and combinations of Myc (cbSFLAG-c-Myc) and Omomyc (pCS-Omomyc) expressing plas- mids. Cells were lysed 48 h later and luciferase activities were measured by the Dual Luciferase reporter assay (Promega). Quantitative Chromatin Immunoprecipitation 293T cells, transfected with pNucL14 or p21Cip1-Luc, together with different combinations of cbSFLAG-c-Myc, cbSFLAG- Omomyc, pCS c-Myc and pCS Omomyc DNA, were processed as described in [3]. The FLAG M2 antibody (Sigma) was used for immunoprecipitation. Real-time PCR was performed with 6 mL of DNA per reaction in iTAQTM SYBR Green Supermix (BioRad). Accumulation of fluorescent products was monitored using a GeneAmp 9700 Sequence Detector (ABI). Each PCR reaction generated only the expected specific amplicon, as shown by the melting-temperature profiles of final products. No PCR products The Action Mechanism of Omomyc were observed in the absence of template. The indicated regions of nucleolin promoter and luciferase coding sequence – used as control – were amplified by qPCR. Background subtraction was calculated from the amount of reporter construct immunoprecipitated by anti-FLAG antibody in absence of FLAG-c-Myc and from the no- antibody samples. Primer sequences. Nucleolin: (forward) 59-CCCTTTCCGGG- GTACTACAG-39, (reverse) 59-GGAAGAGAGGGCCAACCT- TA-39; p21: (forward) 59-ACCGGCTGGCCTGCTGGAACT-39, (reverse) 59-TCTGCCGCCGCTCTCTCA CCT-39; firefly lucif- erase coding sequence: (forward) 59-GGAAAGACGATGACG- GAAAA-39, (reverse) 59-CGGTACTTCGTCCACAAACA-39 Gene expression analysis by Real-Time PCR Real Time PCR for gene expression analysis was performed on cDNA retro-transcribed (kit from BIO-RAD) from total RNA isolated by TRIzol reagent. PCR reactions were performed by iTaq SYBR Green Supermix With ROX kit (BIO-RAD). Accumulation of fluorescent products was monitored using a GeneAmp 9700 Sequence Detector (ABI). At least two independent amplifications were performed for each probe, with triplicate samples. Cycle threshold values were determined by automated analysis. Each PCR reaction generated only the expected specific amplicon, as shown by the melting-temperature profiles of final products. No PCR products were observed in the absence of template. Gadd45a: (forward) 59-CAGAGCAGAAGATCGAAAGGA-39, (reverse) 59-GACTCCGAGCCTTGCTGA-39; Akt1 (forward) 59- AACGACGTAGCCATTGTGAA-39, (reverse) 59-CCATCATT- CTTGAGGAGGAAGT-39; Ccnd1 (forward) 59-GCACAACG- CACTTTCTTTCC-39, (reverse) 59-TCCAGAAGGGCTTCAA- TCTG-39; Erbb2 (forward) 59-AGCTCAGAGACCTGCTTTG- G-39, (reverse) 59-AGGAGGACGAGTCCTTGTAGTG-39; Eif 3s9 (forward) 59-ACTGGCCGCTATGTGGTTAC-39, (reverse) 59-CAGCCAATAAGCATTGTCCA-39; Mxi1 (forward) 59-CG- GATGATCAACGTGCAG-39, (reverse) 59-GCGTAGCCATG- TTCACACTC-39; Pgk1 (forward) 59-CCAGATAACGAATAAC- CAAAGGA-39, (reverse) 59-GACTTGGCTCCATTGTCCA-39 In vitro growth curves To determine growth curves, 1.56104 cells were plated in triplicate in 12-well plates. From the next day, cells were trypsinized and counted daily with a haemocytometer for four days. Cell death was assessed by trypan blue staining. Supporting Information Table S1 List of genes organized into two pages (UP Omomer and DOWN Omomer) according to whether they are activated or repressed by Omomyc upon 90 min of serum stimulation of Rat1 cells. Genes were selected according to the criteria described in Results. Gene ID, gene symbol and name are shown, together with the probe set ID of the Affymetrix U34A array. The last two columns denote, respectively, whether genes are listed as upregulated (UP) or downregulated (DOWN) by Myc in the Myc target gene database and whether their promoters are listed as Myc bound promoters in either one of two studies performed in embryonic stem cells [36,37]. (XLS) Acknowledgments We thank Bruno Amati, Martin Eilers and Robert Eisenman for providing plasmids and advice; Marco Scarno` for help in the statistical analysis; and Andrea Levi for reagents and critical reading of the manuscript. We acknowledge the technical assistance of Nicola Rizzo. PLoS ONE | www.plosone.org 14 July 2011 | Volume 6 | Issue 7 | e22284