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Blood, Vol. 111, Issue 2, 790-799, January 15, 2008
Quantitative expression profiling guided by common retroviral insertion sites reveals novel and cell type–specific cancer genes in leukemia
Blood Sauvageau et al.
111: 790
Supplemental Materials for: Sauvageau et al
Files in this Data Supplement:
- Document 1. Supplemental Notes (PDF, 94KB)
- Document 2. Supplemental Materials and Methods (PDF, 120KB)
- Figure S1. Increased incidence of irradiation-induced lymphomas in Eedhypo/hypo and Eednull/+ mutant mice (PDF, 122KB) -
A) Schematic representation of the experimental design.
B) Survival curves showing increased incidence of irradiation-induced (600 cGy) lymphomas in Eednull/+ and Eedhypo/hypo mutant mice compared to control littermates. p, p-value.
C) Flow cytometry analyses of lymphomas arising in irradiated Eednull/+ and Eedhypo/hypo mutant mice. Numbers in the quadrants indicate the percentage of cells in the corresponding window. Representative examples are shown. BM, bone marrow; Th, thymus; Sp, spleen; LN, lymph nodes.
D) Irradiation-induced lymphomas in Eednull/+ (left panel) and Eedhypo/hypo (right panel) mice are monoclonal or oliglonal. Genomic DNA isolated from the hematopoietic organs of control wild-type, Eednull/+ and Eedhypo/hypo irradiated animals was digested with EcoRI/BamHI (upper panel) or HindIII (lower panel) and hybridized to probes capable of detecting rearrangements at the immunoglobulin (Ig) gene locus and T cell receptor (TCR) ( C probe, (Leder et al.) and TCR probe (Malissen et al.), respectively. Mouse IDs are indicated below each genotypes. BM, bone marrow; Th, thymus; Sp, spleen; N.A., not analyzed.
- Figure S2. Mapping of retroviral integration sites (RIS) identified in Eed mutant and wild-type MMLV-induced tumors (PDF, 122KB) -
The 321 RIS identified are mapped to 170 distinct loci onto the mouse genome assembly from NCBI and the Mouse Genome Sequencing Consortium. Red labels indicate MMLV insertions found exclusively in Eedhypo/hypo tumors. Blue labels indicate retroviral insertions found only in wild-type tumors. Black labels indicate insertions found in tumors from both genotypes. The gene closest to the integration site was chosen to designate a RIS.
- Figure S3. Gene expression analysis of Randomly-selected genes and statistical comparison with CIS-associated genes (PDF, 129 KB) -
A) Color gradient representation of the  Ct values of Randomly-selected genes in non-transformed CD4+, CD8+, CD4+CD8+ (T DP) or B200+IgM+ (B) lymphoid cells from healthy wild-type mice. Phenotype and genotype of the analyzed cells are shown on top. Genes analyzed are shown to the right. Reactions were done in triplicate and average values were calculated. Average Ct values were determined using the 18S rRNA as the internal endogenous control gene to normalize the levels of target gene expression. Low Ct values denote high expression levels. Average Ct values for the 18S rRNA endogenous control was ~9. Mutant, Eedhypo/hypo; WT, wild-type.
B) Relative fold differences in gene expression levels of Eedhypo/hypo mutant and wild-type MMLV-induced lymphomas compared to non-transformed wild-type lymphoid cells. The phenotype, genotype and ID of the analyzed tumor cells are shown on top. Genes analyzed are shown to the right. Genes are grouped by chromosomal cluster, indicated by a colored bar beside the genes name. Reactions were done in triplicate and average values were calculated. Relative fold differences were determined using the Ct method.
The corresponding wild-type non-transformed cells were used as the reference calibrators to assess relative fold differences in expression levels in tumors. Differences of <-5 or >5 folds were considered significant. Tumor tissues taken for quantitative RT-PCR contained 75-100% lymphoid blasts. T DP, CD4+CD8+; B, B220+IgM+; Mutant, Eedhypo/hypo; WT, wild-type.
C) Distribution of the Ct values for CIS-associated (blue) and randomly selected genes (black) in MMLV-induced lymphoid tumors. The p-value was obtained by applying the Kolmogorov-Smirnov test to determine if the two datasets differ significantly.
D) Effect of the threshold on the result of a binomial test evaluating the over/under-representation of CIS-associated vs. randomly selected genes.
- Table S1. Neoplastic cell infiltration in hematopoietic and peripheral organs of MMLV infected mice (PDF, 23KB)
- Table S2. Common integration sites (CIS) identified in Eedhypo/hypo and wild-type tumors by inverse-PCR (XLS, 149 KB)
- Table S3. Quantitative RT-PCR results of CIS-associated genes in Eed-/- and wild-type lymphoid tumors (XLS, 720 KB)
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