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Blood, Vol. 113, Issue 7, 1444-1454, February 12, 2009
Impaired function of primitive hematopoietic cells in mice lacking the Mixed-Lineage-Leukemia homolog Mll5
Blood Madan et al.
113: 1444
Supplemental materials for: Madan et al
Files in this Data Supplement:
- Figure S6. No significant differences in T-cell receptor (TCR) Vα and Vβ usage between Mll5−∕− mice and wild-type littermates (JPG, 88.6 KB)
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(A) CD3high thymocytes. (B) CD3+ splenocytes. (C) CD3+ lymph node cells.
- Figure S7. Perturbations in B-cell development in the absence of Mll5 (JPG, 108 KB)
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Bone marrow cells from 10–14 week-old Mll5−∕− mice (KO/KO) and wild-type littermates (WT/WT) were analysed by flow cytometry for distinct stages of B-cell development following the scheme of Hardy and colleagues.5 Dot plots to the left show data from one representative staining. The bar graphs to the right give the frequency of cells with the indicated immunophenotype; mean values ± SD are shown (n=11). Grey bars: Mll5 knockout mice; black bars: wild-type littermates. (A) Analysis of B220/CD43 subpopulations. (B) Analysis of BP1/HSA subsets within the B220+CD43+ pro–B-cell population. (C) Analysis of B220/IgM subpopulations.
- Figure S8. Analysis of myelo-/ erythropoesis in the absence of Mll5 (JPG, 161 KB)
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Bone marrow cells from 10−14 week-old Mll5−∕− mice (KO/KO) and wild-type littermates (WT/WT) were analysed by flow cytometry with antibodies against the indicated surface markers, and in in vitro methyl cellulose assays. Dot plots to the left show data from one representative staining. The bar graphs to the right give the percentage of cells with the indicated immunophenotype or the total number of cells per 2 femurs + tibias, respectively; mean values ± SD are shown (n = number of animals analysed). Grey bars: Mll5 knockout mice; black bars: wild-type littermates. (A) In the absence of Mll5, the percentage and total number of cells considered early myeloid progenitors (EMPs) is significantly reduced. Bone marrow cells were stained with a cocktail of anti-lineage antibodies (anti CD3, CD4, CD8, CD19, CD127, GR1, Mac1, TER119, and NK1.1) together with antibodies against Sca-1 and kit. Only Lin-negative cells are shown. Rectangular gates in the dot plots mark the population considered EMPs. (B) Percentage and total number of common myeloid progenitors (CMPs = CD34+FcγRII/IIIlow), megakaryocyte/erythrocyte progenitors (MEP = CD34−FcγRII/IIIlow) and granulocyte/macrophage progenitors (GMP = CD34+FcγRII/IIIhigh), as defined by Akashi and colleagues.7 Bone marrow cells were stained with a cocktail of anti-lineage antibodies together with antibodies against Sca-1, kit, FcγRII/III and CD34. Only cells electronically gated for a Lin-negative/Sca-1−/kit+ surface phenotype are shown (EMP gate in Fig. S7A). Numbers in dot plots give the average percentage of cells in the respective gate ± SD. (C) Analysis of differentiated myeloid cells. Bone marrow cells were stained with antibodies against GR1 and Mac1 (CD11b). (D) Analysis of erythropoietic development based on the scheme of Socolovsky and colleagues.8 (Top) Total bone marrow cells were stained with antibodies against the erythrocyte marker TER119 and myeloid marker Mac1. Arabic numbers in the dot plots give the average percentage of cells in the respective gate ± SD (n=4). (Bottom) Total bone marrow cells were stained with a cocktail of anti-lineage antibodies (anti CD3, CD4, CD8, CD19, GR1, Mac1 and NK1.1) together with antibodies against the erythrocyte marker TER119 and the transferrin receptor CD71. Only Lin-negative cells are shown. The percentage of cells in the distinct TER119/CD71 populations labelled with roman numbers I–IV are as follows (WT/WT versus KO/KO): I: 39.4 ± 4.5%/33.4 ± 6.8%; II: 18.9 ± 4.3%/19.2 ± 3.0%; III: 34.3 ± 6.8%/41.8 ± 8.2%; IV: 1.0 ± 0.2%/0.7 ± 0.2%; n=7. (E) In vitro methylcellulose assays do not detect any significant differences in frequency or type of myeloid progenitors between Mll5−∕− mice (KO) and wild-type littermates (WT). Unfractionated bone marrow cells were plated in methylcellulose with a combination of cytokines (see Supplementary Methods) and cultured for 8 days. The graphs summarise data from 3 independent experiments with 3 different Mll5−∕− mice and respective littermate controls. CFU-GM = colony forming unit granulocyte, macrophage; BFU-E = blast forming unit erythrocyte; CFU-GEMM = colony forming unit granulocyte, erythroid, macrophage, megakaryocyte.
- Figure S9. No evidence for homing deficits of primitive hematopoietic cells lacking Mll5 (JPG, 59.2 KB)
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(A) Representative FACS plots of nucleated BM cells from transplant recipients after gating on live cells. The two lower panels show cells from an irradiated recipient mouse 16 hrs after i.v. injection of a 1:1 mixture of Mll5−∕−Lin−Kit+ cells stained with DDAO (red) and wild-type Lin−Kit+ cells stained with CFSE (green). The two top panels show BM cells from lethally irradiated control mice, which did not receive any labeled donor cells. The bar graphs to the right summarize the results of two independent experiments with a total of four recipients and four pairs of Mll5−∕− and wild-type donors. (B) Similar colony forming cell (CFC) numbers from BM of recipients transplanted with 20 × 106 total BM cells from either Mll5−∕− mice (KO/KO) or wildtype littermates (WT/WT). Negative control = number of colonies fromBM of lethally irradiated control mice receiving no donor cells. The right panel gives the percentage of colonies that were obtained from reconstituted BM relative to the number of colonies obtained from an equivalent input population of donor BM.
- Figure S10. Expression analysis of candidate Hox target genes by RT-PCR (A) and quantitative real-time PCR (B) (JPG, 92.3 KB)
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(A) No consistent differences in Hox expression were detectable by RT-PCR. The data shown are representative of three independent experiments. Lin-ve = FACS-sorted lineage negative BM cells; KO = homozygous Mll5 knockout animal; WT = wild-type littermate; No RT = negative control lacking reverse transcriptase. (B) More sensitive quantitative real-time PCR (qPCR) revealed very modest increases (less than 2 fold) in steady-state RNA levels for some Hox genes in Mll5−∕− spleens, testes, epididymidi and ovaries, and no difference in FACS-sorted lineage-negative (Lin-ve) BM cells. In Mll5−∕− thymi, steady-state RNA levels for the tested Hox genes were up to 4 fold reduced. The bar graphs summarize data from three independent experiments. That is, data were obtained with RNA isolated from organs of three different animals. In the upper panels, results are plotted as CT values. One CT value corresponds to a doubling of signal and all values equal to or above a CT of 38 were assumed to indicate lack of expression (n.d. = non detectable). The only available TaqMan assay for HoxB3 (Mm00650701) gave in all samples consistenly CT values greater than 40. HoxB3 was therefore omitted from qPCR analysis. β-Actin (β-act) served as control for RNA input. The lower panels show “fold change” in transcript abundance in samples from Mll5−∕− mice (grey bars) compared to wild-type controls (black bars). Levels of expression were standardized to β-actin and expression levels in wild-type controls were given an arbitrary value of 1. Error bars indicate mean standard deviation.
- Figure S11. No significant changes in global histone lysine methylation in the absence of Mll5 (JPG, 244 KB)
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Quantitative immunoblot analysis of histones purified from spleen, thymus and testis of homozygous Mll5-deficient mice (KO) and wild-type (WT) controls. (A–M) Results are arranged according to the specificity of the anti–methyl-lysine antibody used.
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