In parallel, whole cell extracts were made from mouse embryonic fibroblasts infected with the indicated adenoviruses for controls. Protein was quantitated using the BCA Protein Assay Reagent Kit (Pierce). Samples (150 mg) were boiled in sample buffer and subjected to SDS-PAGE on 8.5% polyacrylamide gels for p53 and Mdm2 assessment and 15% gels for ARF assessment. Western analysis was performed as previously described [54]. The blots were probed with antibodies specific for p53 (monoclonal antibody Ab-1 OP03 at 1:1000, Calbiochem), p19ARF (polyclonal antibody Ab-1 PC435 at 1:10,000, Calbiochem), and Mdm2 (polyclonal antibody C-18 sc812 at 1:1000, Santa Cruz Biotechnology). Equal protein loading was verified by staining blots with Ponceau Red (0.2% ponceau red in 3% trichloroacetic acid). Southern analysis Genomic DNA was isolated from lymphomas, normal spleen cells, tail samples and MEFs of specified genotypes. DNA (10 mg) was digested with BamH1 (for the p53 locus), AflII (for the p19ARF locus) or EcoRI (for the heavy chain locus). The restricted DNA was separated by agarose gel electrophoresis (0.8% gels), transferred to Hybond N+ membrane, and probed. The p53 probe was a human cDNA fragment (686 base pair DrdI-StuI fragment extending from exon 4 to exon 10). The ARF probe was the exon 1B portion of the ARF cDNA (kindly provided by Charles Sherr). The heavy chain locus probe was the heavy chain J3-J4 joining region genomic fragment [37]. On occasion, to verify that weanling mice were essentially tumor-free, genomic DNA isolated from B lineage cells was assessed by Southern analysis for any emergence of clonal heavy chain rearrangements. FACS analysis Mononuclear cells were harvested from the bone marrow of 3- week-old littermates and from lymphomas that arose. Cells were stained with various combinations of antibodies to IgD (11- 26c.2a), IgM (R6-60.2), CD19 (1D3), B220 (RA3-6B2), CD43 (S7), BrdU, and active caspase-3. All antibodies and staining reagents were from BD Biosciences. Cell staining procedures were performed either manually or using a Biomek 2000 robotic fluid handler (Beckman Instruments, Schaumburg, IL using a series of mini-programs developed with BioWorks software (Beckman Instruments). FACS analysis was performed on a FACSCalibur device equipped with a 488 nm argon laser and a ,635 nm red dye laser (Becton Dickinson (BD), San Jose, CA). Data was analyzed using FlowJo Software (TreeStar, Palo Alto, Ca). Cell cycle analysis of B lineage cells from normal and pretumorous mice Three hours before analysis, mice were injected with 100 mg/ kg BrdU. Bone marrow mononuclear cells were collected and stained with the B220 and CD19 antibodies to identify B lineage cells, with 7-AAD, and with anti-BrdU antibodies. BrdU Flow Kit reagents and directions were followed (BD/Pharmingen). The proportion of cells that had proceeded through S-phase, or resided in G0/G1 or in G2/M phases was determined. Viability assessment in culture of B lineage cells from normal and pretumorous mice Hematopoietic cells were harvested from the bone marrow, spleen and mesenteric lymph node, combined and enriched for B lineage cells using negative selection (SpinSep Mouse B Cell Enrichment Cocktail, Stem Cell Technologies). The antibodies used to label unwanted cell types for depletion were directed against CD4, CD8, CD11b, CD49b, Gr-1, TER119 and CD43. The E2F and Myc-Induced Lymphoma approach yielded a subset of B lineage cells from the small pre-B stage through more mature stages. These cells were cultured at 37uC for eight hours in DMEM plus 10%FCS/100 mM L- aspargine/50 mM 2-mercaptoethanol at a concentration of 46106 cells/ml. At selected time points cells were removed and stained with 7-AAD and B220 antibody, fixed and permeabilized, stained with activated caspase-3 antibody and analyzed by flow cytometry. Viable cells were negative for both 7-AAD and activated caspase-3. CFU Pre-B assays Equivalent numbers of bone marrow cells from non-transgenic 4–6 week old mice were resuspended in Methocult M3630 (Stemcell Technologies) according to manufacturer’s specifications to assay for pre-B cell colonies. This media, formulated for the detection and counting of mouse pre-B progenitors in bone marrow, is comprised of methylcellulose in Iscove’s MDM supplemented with recombinant IL-7, 2-Mercaptoethanol, L- glutamine, and fetal bovine serum. All samples were assayed in duplicate. After seven days colonies were counted using an inverted microscope. The count was based on the manufacturer’s description of expected colony appearance - namely that colonies are composed of at least 30 cells and that individual cells are tiny and irregular to oval in shape. DNA microarray analysis RNA was extracted from lymphoma samples using Qiagen RNeasy Kits (Qiagen). RNA sample integrity was verified by agarose gel electrophoresis or using an Agilent 2100 Bioanalyser. We prepared the targets for DNA microarray analysis and hybridized to Affymetrix Mouse 430 2.0 GeneChip arrays according to the manufacturer’s instructions and as previously published. To allow merging of expression array results from samples arrayed independently, some duplicate samples were arrayed to provide reference samples and the expression values standardized using ComBat [55]. The method for cross-platform comparison across different versions of Affymetrix GeneChip arrays was described previously [56]. Statistical analyses of microarray data Hierarchical clustering and visualization were performed using Gene Cluster 3.0 (http://bonsai.ims.u-tokyo.ac.jp/,mdehoon/ software/cluster/) and Java TreeView (http://jtreeview.sourceforge. net/). Genes and tumors were clustered by average linkage using uncentered correlation as the similarity metric. Analysis of expression data was described previously [56]. In summary, we collected training sets consisting of gene expression values of samples where the pathway activity was known. We created gene expression signatures by choosing the genes whose expression profiles across the training samples most highly correlated with the phenotype. Then, to predict the status of the phenotype on a tumor expression dataset, we fit a Bayesian probit regression model that assigned the probability that a tumor sample exhibited evidence of the phenotype, based on the concordance of its gene expression values with the signature. The Supporting Materials and Methods are available in Text S1. Supporting Information Figure S1 Tissue-specific expression of E2F family members. (A) Comparison of the relative expression of i) E2f1 mRNA, ii) E2f2 mRNA, iii) E2f3a and E2f3b mRNAs, and iv) E2f4 mRNA across several tissue types with the expression in bone marrow set to one. Tissues: BM-bone marrow, S-spleen, T-thymus, M-mammary gland, O-ovary, U-uterus, Lu-lung, and Br-brain. (B) Western blot analysis of E2F2 protein in tissues from wild type and matched PLoS Genetics | www.plosgenetics.org 13 September 2009 | Volume 5 | Issue 9 | e1000640