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Blood, Vol. 113, Issue 7, 1455-1463, February 12, 2009
Mll5 contributes to hematopoietic stem cell fitness and homeostasis
Blood Zhang et al.
113: 1455
Supplemental materials for: Zhang et al
Files in this Data Supplement:
- Table S1. Sequences of oligonucleotide primers used in RT-PCR assays (PDF, 57.8 KB)
- Figure S1. Hematopoietic lineage development in Mll5−∕− mice (JPG, 178 KB)
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(A) Left panel: representative FACS data showing neutrophils (Gr-1hi, CD11b+) and monocyte/macrophages (Gr-1−/int, CD11b+) in the spleen and bone marrow of an Mll5−∕− and control littermate. Right panel: the graph shows the relative frequencies of the indicated populations of cells, normalized to the wild-type average values, and expressed as percentages (5 mice per group). Neutrophils were increased in the spleens (p < 0.05, Student’s t-test) but not the bone marrow. (B) Left panel: representative FACS data showing NK (NK1.1+, CD3−) and NK T (NK1.1+, CD3+) cells in the liver and spleen of an Mll5−∕− and control littermate. Right panel: as in A, 5 mice per group. Mll5−∕− mice showed significant differences for all of the graphed populations (p < 0.05, Student’s t-test).
- Figure S2. Thymocytes, lymphocytes and dendritic cells in Mll5−∕− mice (JPG, 184 KB)
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(A) Left panel: representative FACS data showing CD4 and CD8 expression on thymocytes from an Mll5−∕− mouse and a control littermate. Right panel: the graph shows the relative frequencies of the indicated populations of cells, normalized to the wild-type average values, and expressed as percentages (4 mice per group). SP: single positive, i.e., CD4+CD8− or CD4−CD8+; DP: double positive, i.e., CD4+CD8+; DN: double negative, i.e. CD4−CD8−. (B) Left panel: representative FACS data showing CD4+ and CD8+ T cells, B cells (B220+, IgM+) and dendritic cells (MHC-II+, CD11c+) in the spleen of an Mll5−∕− mouse and a control littermate. Right panel: as in A, 5 mice per group. DC: dendritic cells.
- Figure S3. Mll5 expression pattern in mouse tissues and in hematopoeitic stem or progenitors cell populations (JPG, 81 KB)
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(A) Relative expression of Mll5 in various tissues as determined by Taqman quantitative RT-PCR. Levels of expression were determined relative to that of hypoxanthine-guanine phosphoribosyl transferase (HPRT) and normalized to the expression of Mll5 in the liver. Values shown are means ± SD. Six samples of each tissue were analyzed in triplicate, except for the testes and ovary, for which there were three samples each. (B) Left panel: relative expression of Mll5 in HSC and progenitor populations as determined by SYBR Green quantitative RT-PCR. Levels of expression were determined relative to that of β-actin, and normalized to the expression of Mll5 in LT-HSCs. Values shown are means ± SD for groups of three mice. Right panel: absolute expression of Mll5 in the indicated cells as determined through use of a standard curve.
- Figure S4. Impaired repopulation of irradiated recipients by Mll5−∕− bone marrow cells (JPG, 168 KB)
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The graphs show repopulation ratios calculated as in Fig. 3C for the indicated cell types at four weeks (A) or three months (B) after transplantation.
- Figure S5. Impaired repopulation of irradiated recipients by Mll5−∕− fetal liver cells (JPG, 59.6 KB)
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The graph shows repopulation ratios calculated as in Fig. 3C for the indicated cell types three months after transplantation. N/D: not done.
- Figure S6. Analysis of bone marrow homing (JPG, 118 KB)
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(A) Experimental strategy for the short-term homing assay. (B) Left panel: Representative FACS data showing equivalent accumulation of CFSE-labeled lin− bone marrow cells from Mll5−∕− mice and wild-type littermates twelve hours after transfer into irradiated recipients (as in A). Right panel: relative frequencies of transferred CFSE-labeled lin− bone marrow cells (5 mice per group) in the recipients twelve hours after transfer. (C) Representative FACS data showing expression of CXCR4, VLA-4 and VLA-5 on Mll5−∕− LSK cells. Solid line: +/+; dashed line: −∕−; shaded histogram: isotype control.
- Figure S7. Expression of selected genes known to regulate HSC homeostasis in Mll5−∕− LSK cells (JPG, 68 KB)
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Expression of the indicated genes was determined by SYBR Green quantitative RT-PCR. Levels of expression were standardized to that of β-actin. Values shown are means ± SD.
- Figure S8. Myeloproliferative disease in Mll5-deficient, Mx1-cre, KrasG12D mice (JPG, 80.5 KB)
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(A) Survival of Mx1-cre, KrasG12D mice of the indicated Mll5 genotypes after polyI:C treatment to induce Cre recombinase expression from Mx1-cre. (B) White blood cell counts from individual mice of the indicated Mll5 genotypes at the time of euthanasia due to disease symptoms. (C) Hemoglobin levels in the blood of mice of the indicated Mll5 genotypes at the time of euthanasia. (D) Spleen weights for mice of the indicated Mll5 genotypes at the time of euthanasia. (E) Representative FACS data showing CD11b and Gr-1 expression on cells from the bone marrow and spleens of mice of the indicated genotypes.
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