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Blood, Vol. 113, Issue 6, 1304-1314, February 5, 2009
Oncogenic Kras-induced leukemogeneis: hematopoietic stem cells as the initial target and lineage-specific progenitors as the potential targets for final leukemic transformation
Blood Zhang et al.
113: 1304
Supplemental materials for: Zhang et al
Southern analysis
Genomic DNA samples were prepared using the Puregene® Genomic DNA Purification Kits (Qiagen) per manufacturer’s instructions. Rearrangement at the T-cell receptor β locus was detected as previously described.1
REFERENCES
1. Roberts CW, Leroux MM, Fleming MD, Orkin SH. Highly penetrant, rapid tumorigenesis through conditional inversion of the tumor suppressor gene Snf5. Cancer Cell. 2002;2:415-425.
Files in this Data Supplement:
- Table S1. Assessment of T-cell development (PDF, 15.1 KB) -
Thymocytes were isolated from individual recipient mice transplanted with either control cells or cells expressing oncogenic K-ras. Thymocytes were simultaneously stained for CD4, CD8, and a T-cell marker, and analyzed on a BD FACS Calibur. DP cells and CD8 SP cells were defined based on their expression of CD4 and CD8. Expression of T-cell markers was analyzed on gated population of cells. The results were presented as average percentages of positive cells out of total gated cells ± standard deviation. Student t test was performed and the results that showed a significant difference from control cells were indicated with asterisks.
- Table S2. CGH analysis of malignant thymic T cells and bone marrow myeloid cells isolated from the same animal (A922) (PDF, 22 KB) -
Genomic DNA was extracted from A922 lymphoma cells and A922 bone marrow myeloid cells. CGH analysis was performed by Nimblegen using mouse 4-array whole genome set. Clear regions of amplifications (+: single copy; ++: double copies) and deletions (-: single copy; --: double copies) are seen in the two cell types. The observable karyotype changes seen in the lymphoma cells by G-banding analysis (Fig. 6) were confirmed by CGH analysis.
- Figure S1. Survival of recipient mice transplanted with control bone marrow cells or bone marrow cells expressing oncogenic K-ras (JPG, 78.4 KB)
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Lethally irradiated mice were transplanted with 2.5 × 105 control bone marrow cells (10 mice) or bone marrow cells expressing oncogenic K-ras (52 mice) along with same amount of competitor cells. Over a period of 24 weeks, ~60% of recipient mice transplanted with bone marrow cells expressing oncogenic K-ras succumbed to hematopoietic malignancies, including T-cell leukemia, T-cell lymphoblastic lymphoma, and JMML. The lethal window for each malignancy was labeled above the time scale.
- Figure S2. Oncogenic K-ras induces monoclonal or oligoclonal lymphomas (JPG, 105 KB)
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Southern blot of DNA obtained from JMML myeloid cells (A922 Mye) and cell lines established from thymic lymphomas developed in recipient mice transplanted with HSCs expressing oncogenic K-ras (A922 Thy, 158 Thy, and C158 Thy). The blot was hybridized with a probe to the Vβ region of the T-cell receptor. A922 Mye sample contains only the germline band. Both of alleles in A922 Thy sample underwent recombination, confirming its clonality established by mouse G banding analysis (see Results). In contrast, 158 Thy and C158 Thy are oligoclonal with one clone dominant in the whole populations.
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