Retrovirus production and BM transplantation. Retroviral superna- tants were produced by transient transfection of 293T cells with the retro- viral construct and appropriate packaging plasmids (provided by H. Cantor, Dana-Farber Cancer Institute, Boston, MA). Retroviral infection and BM reconstitution experiments were performed as previously described (20, 36, 40). In brief, lineage-negative (CD3e, TCR- , NK1.1, DX5, CD19, Ter- 119, Mac-1, and Gr-1) BM cells were sorted from BALB/c or CD4-Cre c- Mycflox/flox and C57BL/6 mice and cultured in the presence of Flt3L, SCF, IL-6, and IL-7 (all from R&D Systems). On day 2 and 4 after sorting, cells were subjected to retroviral transduction by centrifugation at 2,300 rpm for 1.5 h at room temperature with 8 μg/ml polybrene. On day 5, 1–2 × 106 cells were injected intravenously into irradiated (550 rad) 5–8-wk-old syn- geneic BALB/c or RAG 2 / c / recipients. Flow cytometry and cell sorting. Monoclonal antibodies specific for CD4 (RM4-5, GK1.5), CD8 (53–6.7), TCR- (H57-597), CD3e(145- 2C11) Gr-1 (RB6-8C5), erythroid cell marker (Ter-119), CD19 (1D3), CD11b (M1/70), pan-NK (DX5), and NK1.1 (PK136) were purchased from BD and were used as biotin, FITC, PE, peridinin chlorophyll protein (PerCP), PerCP-Cy5.5, PE-Cy7, allophycocyanin (APC), or APC-Cy7 con- jugates. Single-cell suspensions were prepared from peripheral blood, BM, spleen, lymph node, thymus, lung, and liver of ICN1-transplanted animals and related controls at the indicated time points. Cells were analyzed by FACSCalibur cytometer (BD) equipped with CellQuest Software (BD Bio- sciences) and FlowJo (Tree Star, Inc.) and were sorted by a FACSAria (BD) or a MoFlo cell sorter (Cytomation). Sorted cells were of >98% purity, as determined by postsort analysis. BrdU labeling. BrdU incorporation procedure was performed as previ- ously described (13). In brief, mice were injected intraperitoneally with 1.8 μg freshly resuspended BrdU in water every day. Sorted cells from BrdU-treated mice were fixed, permeabilized, and stained intracellularly using the BrdU labeling kit from BD. CFSE labeling. Sorted CD4+8+ cells were incubated with 10 μM CFSE (In- vitrogen) at 5 × 106 cells/ml PBS, 0.1% BSA for 10 min at 37°C. Cells were washed two times with RPMI 1640 medium to eliminate the remaining CFSE and injected intravenously into nude mice. Cell division of CFSE-labeled cells was analyzed by flow cytometry on day 2, 4, 6, and day 8 after injection. SKY. Metaphase chromosomes were prepared from tumor CD4+8+ cells. In brief, cells were treated with colcemid for 2 h, and then incubated in 0.075 M KCl for 20 min at 37°C. Chromosomes were subsequently fixed in 3:1 methanol/acetic acid. Metaphases were dropped on slides, dried overnight, and stained with DAPI before microscopic examination. SKY was done us- ing the SkyPaint DNA kit for mouse samples (Applied Spectral Imaging) ac- cording to the manufacturer’s instructions. Spectral images were captured using a microscope (E800; Nikon) equipped with ASI spectral cube, 60× objective, and analyzed using SKYView software from Applied Spectral Imaging. At last 20 metaphases were analyzed per sample. Array CGH Profiling. Genomic DNA from sorted cells was extracted us- ing DNeasy Tissue kit (QIAGEN) and processed for hybridization by the Belfer Cancer Genomics Center, Dana-Farber Cancer Institute. The DNA was hybridized to 244 K Whole Mouse Genome Chip (G4122A; Agilent Technologies) according to the manufacturer’s instructions. Tail DNA from the same mouse as reference. The median spatial resolution for this chip is 8.05 Kb; probes cover all known genes. Data were assembled and analyzed using CGH Analytics software (Agilent Technologies). Southern blot analysis. 10 μg of genomic DNA was digested with EcoRI overnight at 37°C, separated on a 1.0% agarose gel, transferred onto nitro- cellulose membrane. After UV cross-linking, the blot was hybridized with a p32-labeled probe from the J 2 region of the TCR- gene. Electrophoretic Mobility Shift Assays (EMSA). Nuclear extracts were prepared from cells using nuclear and cytoplasmic extraction reagents (PIERCE). 2860 Protein concentration of each extract was measured by OD 595 nm. 5 μg of each nuclear extract was used in each EMSA. To generate probes for EMSAs, 5 end biotin-labeled double-stranded oligonucleotides containing the bind- ing sites for E2A and NF- B were synthesized from Integrated DNA Tech- nologies. The following oligonucleotides were used for EMSA: E2A E-box consensus sequences, CCCCAACACCTGCTGCCTGAG; NF- B binding motif, AGTTGAGGGGACTTTCCCAGGC. The probes were purified with PAGE. EMSA was performed using LightShift chemiluminescent EMSA kit (Thermo Fisher Scientific), following the manufacturer’s instructions. Immunoprecipitation and Western blot. Cells were lysed in radioim- munoprecipitation assay (RIPA) buffer supplemented with Protease Inhibi- tor Cocktail (Sigma-Aldrich). A 1,000-μg aliquot of total cell lysates was incubated with 10 μg of specific antibodies or control IgG for 2 h or over- night at 4°C. A 40-μl aliquot of 50% protein A–agarose slurry was added and incubated for another hour, followed by several washes with 0.1% NP-40 in PBS. The precipitates bound to the beads were analyzed by immunoblotting with Ubiquitin antibody (Santa Cruz Biotechnology, Inc.). For Western blot analysis, cells were lysed in RIPA buffer supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich). Lysates (40 μg) were sepa- rated on 4–20% SDS-PAGE and transferred onto PVDF membrane (Milli- pore). Nonspecific binding was blocked by incubation in blocking buffer (5% nonfat milk in TBST), followed by incubation with the primary anti- bodies and the appropriate horseradish peroxidase–conjugated secondary anti- bodies diluted in blocking buffer. Immunoreactive proteins were detected using enhanced chemiluminescence (ECL) reagents (GE Healthcare). The E2A, Mdm2 antibodies were purchased from BD. ERK1/2, phosph-ERK1/2 antibodies, and MEK1/2-specific inhibitor PD98059 were purchased from Cell Signaling Technology. Cyclin D3 antibody was obtained from Santa Cruz Biotechnology. Cyclin D1 antibody was purchased from NeoMarker, and the p19Arf and c-Myc antibodies were purchased from Abcam. p53 antibody was provided by R. Depinho (Dana-Farber Cancer Institute, Boston, MA). Quantitative RT-PCR. cDNA was prepared by oligo-dT primer using the Superscript II Reverse Transcription kit (Invitrogen). Quantitative RT-PCR was performed using an ABI7300 machine (Applied Biosystems). c-Myc, E47, Bmi-1, p19Arf, Mdm2, and p53 RNA was determined relative to GAPDH ex- pression using the TaqMan Gene Expression Assay (Applied Biosystems). All quantitative RT-PCR reactions were done in triplicate in 20 μl volume. Microarray analysis. Sorted cells were homogenized for RNA isolation using the TRIZOL reagent (Invitrogen) and purified using QIAGEN RNeasy minicolumns. RNA samples were processed for hybridization on the Mouse Expression-Array-430.2.0 Genechips (Affymetrix) by the Micro- array Core Facility of the Dana-Farber Cancer Institute. Data were loaded onto the DNA-Chip Analyzer (dChip; http://www.dchip.org) program for normalization and quantification. Normalization was performed using the default settings of the dChip software, genes were selected for expression differences higher than 1.5-fold by Student’s t test (P < 0.05) or for signifi- cant changes (P < 0.05). The microarray data have been deposited in the GEO database under accession no. GSE12948. Online supplemental material. Fig. S1 shows global gene expression profil- ing in control, abnormal nonmalignant cells, and tumor cells. Table S1 provides tumorigenicity assays in nu/nu homozygous mice. The online version of this article is available at http://www.jem.org/cgi/content/full/jem.20081561/DC1. The authors wish to thank Dr. Scott J. Rodig (Brigham & Women’s Hospital) for histopathological analysis, Xiaoyan Li for technical assistance with BMT, and Rui Chang and Marei Dose for their help with the work of CD4-Cre c-MycFL/FL mice. We would also like to thank Frances Lundblad and Linnea Benson for editorial help. This work was supported by National Institutes of Health (NIH) grants P01 CA109901 and R01 AI45846. X. Li is supported by the Lymphoma Research Foundation, Lee Fellowship, and NIH training grant T32-CA70083. The authors have no conflicting financial interests. NOTCH-INITIATED T-ALL | Li et al.