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Blood, Vol. 109, Issue 11, 4907-4913, June 1, 2007
Endogenous bcl-2 is not required for the development of Eµ-myc–induced B-cell lymphoma
Blood Kelly et al.
109: 4907
Supplemental materials for: Kelly et al, Vol 109, Issue 11, 4907-4913
Files in this Data Supplement:
- Table S1. Classification of overall mortality in reconstituted mice of the indicated genotypes (PDF, 14.2 KB) -
Lymphoma type was determined by immunofluorescent staining with surface marker–specific monoclonal antibodies and flow cytometric analysis.
- Figure S1. Progressive decline of donor-derived leukocytes in Eµ-myc/bcl-2–/– reconstituted mice (JPG, 78 KB)
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Peripheral blood was harvested from (the same) lethally irradiated C57BL/6-Ly5.1 mice 8 to 10 weeks and again 20 weeks following reconstitution with fetal liver cells from wt, bcl-2–/–, Eµ-myc, or Eµ-myc/bcl-2–/– (E14.5) embryos. Red blood cells were removed, and the remaining leukocytes were stained with fluorochrome-conjugated monoclonal antibodies to Ly5.1, Ly5.2, B220, IgM, and IgD and analyzed by flow cytometry. Representative FACS profiles indicate the frequency of donor-derived (Ly5.1–) leukocytes in mice of the indicated genotypes.
- Figure S2. Eµ-myc/bcl-2–/– reconstituted mice have abnormally low numbers of donor-derived mature B cells in their lymph nodes (JPG, 63.4 KB)
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Total numbers of donor-derived leukocytes and mature B cells in lymph nodes from wt, bcl-2–/–, Eµ-myc, Eµ-myc/bcl-2+/–, and Eµ-myc/bcl-2–/– reconstituted mice were determined by cell counting in a hemocytometer and by immunofluorescent staining with antibodies to Ly5.2, B220, IgM, and IgD followed by FACS analysis. Values represent means ± SEM from 5 to 10 mice of each genotype. Statistically significant difference: **P < .001.
- Figure S3. Eµ-myc and Eµ-myc/bcl-2–/– pro-B and pre-B cells have similar size (JPG, 57.6 KB)
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Flow cytometric analysis of forward light scatter (FSC), which reflects cell size, on donor-derived pro-B cells (Ly5.2+ B220+ c-Kit+ sIg–) and pre-B cells (Ly5.2+ B220+ c-Kit+ sIg–) from the bone marrow of lethally irradiated mice reconstituted with fetal liver–derived stem cells from wt, Eµ-myc, or Eµ-myc/bcl-2–/– embryos. Immunofluorescent staining with surface marker–specific monoclonal antibodies was used to identify donor-derived pro-B and pre-B cells.
- Figure S4. Loss of 1 allele of bcl-2 has no effect on Eµ-myc–induced lymphoma development (JPG, 31.5 KB)
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Cumulative tumor incidence for nonmanipulated (ie, nonreconstituted) Eµ-myc and Eµ-myc/bcl-2+/– mice, demonstrating comparable tumor incidence for mice of these 2 genotypes.
- Figure S5. No difference in overall survival of Eµ-myc and Eµ-myc/bcl-2–/– reconstituted mice (JPG, 39 KB)
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Kaplan-Meier curve incorporating all causes of death (myc lymphoma and nontumor related, which was mostly due to insufficient hematopoietic reconstitution or recipient-derived thymic lymphoma) for Eµ-myc and Eµ-myc/bcl-2–/– reconstituted mice, revealing that the presence or absence of bcl-2 does not influence the overall mortality of reconstituted animals.
- Figure S6. Loss of endogenous Bcl-2 does not select for development of lymphomas expressing abnormal levels of antiapoptotic or proapoptotic members of the Bcl-2 family or other apoptosis regulators (JPG, 142 KB)
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(A) Protein expression profile of pro- and antiapoptotic Bcl-2 family members in Eµ-myc/bcl-2–/– and control Eµ-myc lymphomas. Lymphomas were harvested from 3 moribund Eµ-myc/bcl-2–/– and 3 control Eµ-myc reconstituted mice. Protein lysates were prepared and subjected to Western blot analysis. Information on antibodies used for probing will be provided on request. A faint Bcl-2 signal could be detected in Eµ-myc/bcl-2–/– tumor lysates. (B) FACS purification of B220+Ly5.2+ Eµ-myc/bcl-2–/– lymphoma cells confirmed that the faint Bcl-2 signal was most likely attributable to the presence of host-derived (wt) stromal tissue. Protein lysates from bcl-2+/+ and bcl-2–/– murine embryonic fibroblasts (MEFs) served as positive and negative controls, respectively. Sample (*) represents a lymphoma for which no FACS-purified cells were available for analysis. Black vertical lines indicate where lanes in a blot have been removed. Identical samples were used for all blots.
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