Intra-molecular interaction is required for expansion of hematopoietic progenitors.

<p>A. Cellularity of <i>de</i> mutant FLs. Error bars represent the standard deviations of cell numbers of FLs. For each genotype, at least 3 livers were analyzed. B. Population analysis of <i>^de</i> mutant FLs by flow cytometry. The fetal LKS compartment was defined as the Lin⁻cKit⁺Sca-1⁺Mac1⁺ population. C. Frequency of LKS populations in <i>^de</i> mutant FLs. Error bars represent the standard deviations of more than two littermate embryos for each genotype. D. Population analysis of HSCs from MPPs in <i>^de</i> mutant FLs by flow cytometry. Lin⁻Mac1⁺ cells were subdivided by Sca-1 and CD48. E. Frequencies of HSCs (top) and MPPs (bottom) in <i>^de</i> mutant FLs. Error bars represent the standard deviations of more than four embryos for each genotype. Frequency was expressed as the percentage of each fraction within the whole population. F. Ability of <i>^de</i> mutant FL cells to reconstitute the hematopoietic system. 5×10⁵ cells of <i>^de</i> homozygous FLs and 5×10⁴ cells of the wild type/heterozygous controls were transplanted into lethally irradiated syngenic mice. Previously published data for <i>^dC</i> homozygous FLs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073649#pone.0073649-Yokoyama1" target="_blank">[8]</a> obtained in the same experimental setting were included for comparison. N: the number of FLs analyzed.</p

Lack of processing does not constitutively activate MLL.

<p>A. Expression of posterior <i>Hoxa</i> genes and MLL complex components during myeloid differentiation. Each population was isolated by cell sorting and analyzed by RT-qPCR. Relative expression levels (normalized to <i>Actb</i>) and expressed relative to those of LKS arbitrarily set as 100%. Error bars represent standard deviations of triplicate PCRs. B. Experimental scheme of myeloid progenitor serial replating assay. <i>MLL-ENL</i> was transduced into wild type myeloid progenitors in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073649#pone-0073649-g005" target="_blank">Figure 5C</a>. Colony-forming activity of wild type and <i>uc</i> homozygous myeloid progenitors was analyzed without any gene transduction in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073649#pone-0073649-g005" target="_blank">Figure 5D</a>. C. Clonogenic potentials of <i>MLL-ENL</i> (or vector)-transduced myeloid progenitors in myeloid progenitor serial replating assay. Transduced cells were cultured in semi-solid media and subjected to serial replating. CFUs per 10⁴ plated cells were enumerated after each round. Error bars represent standard deviations of three independent samples. D. Clonogenic potentials of myeloid progenitors derived from <i>uc</i> homozygous mice in myeloid progenitor serial replating assay. Error bars represent standard deviations of three independent samples.</p

Intra-molecular interaction is required for MLL-dependent gene activation.

<p>A. MLL proteins produced by <i>dC</i> and <i>de</i> mutations are shown schematically. The characteristics of each mutant are shown on the right. The N-terminal intra-molecular interaction domain (NIID) includes PHD1, PHD4 and FYRN and the C-terminal intra-molecular interaction domain (CIID) is FYRC. Positions of the three taqman probes for <i>Mll</i> [<i>Mll</i>(N), <i>Mll</i>(M) and <i>Mll</i>(C)] used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073649#pone-0073649-g001" target="_blank">Figure 1G</a> are indicated by red bars. Genomic sequences around the exon 11 of the human MLL and its murine counterpart are shown at the bottom. B. Genomic structures of the wild type allele and the recombined allele are shown with the targeting vector. C. Diagnostic PCR identifies the recombined allele in genetically engineered mice. <i>+/+</i>: wild type. <i>de/+</i>: <i>de</i> heterozygous D. Sequence of the exon junction of <i>Mll de</i> transcript. The PCR product generated by RT-PCR of <i>de</i> homozygous MEFs was sequenced. E. Genotypes at various developmental stages. Viability of embryos was confirmed by presence of heart contractions. <i>de/de</i>: <i>de</i> homozygous F. Morphologies of E13.5 embryos of <i>de</i> mutants. G. Expression of MLL proteins and RNAs in MEFs. Expression of MLL^N, MLL^C, and actin was visualized by western blotting. Relative expression levels of <i>Mll</i> mRNAs (normalized to <i>Gapdh</i>) are expressed relative to those of wild type arbitrarily set as 1. RT-PCR analysis demonstrated expression of the <i>de</i> mutant mRNAs. qPCR using the <i>Mll</i>(M) and <i>Mll</i>(C) taqman probe demonstrated that <i>Mll</i> mRNAs were expressed at the same levels in wild type and <i>de</i> homozygous MEFs, whereas the <i>Mll</i>(N) taqman probe showed that the murine exon corresponding to the human exon 11 was not transcribed. Error bars represent the standard deviations of triplicate PCRs. H. Expression of MLL target genes in <i>de</i> homozygous MEFs. Wild type and <i>dC</i> homozygous MEFs were also analyzed for comparison. Relative expression levels of various MLL target genes (normalized to <i>Gapdh</i>) are expressed relative to those of wild type-1 arbitrarily set as 1. RT-qPCR demonstrates that expression of MLL target genes including <i>Hoxc8</i>, <i>Hoxc9</i>, <i>Cdkn1b</i>, <i>Cdkn2c</i> was impaired in <i>de</i> homozygous and <i>dC</i> homozygous MEFs. Error bars represent the standard deviations of triplicate PCRs. <i>dC/dC</i>: <i>dC</i> homozygous.</p

MLL processing is not required for proliferation of oncogene-transformed myeloid progenitors.

<p>A. Experimental scheme of myeloid progenitor serial replating assay. <i>Hoxa9</i> was transduced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073649#pone-0073649-g006" target="_blank">Figure 6B</a> and <i>MLL-AF9</i> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073649#pone-0073649-g006" target="_blank">Figure 6C and 6D</a>. The time points at which colony forming units and <i>Hoxa9</i> expression were measured are shown. B. Clonogenic potentials of <i>Hoxa9</i>-transformed myeloid progenitors of wild type or <i>uc</i> homozygous mutant origin. CFUs per 10⁴ plated cells were enumerated at third and fourth round. Error bars represent standard deviations of three independent samples. C. Clonogenic potentials of <i>MLL-AF9</i>-transformed myeloid progenitors of wild type or <i>uc</i> homozygous mutant origin. CFUs per 10⁴ plated cells were enumerated at third and fourth round. Error bars represent standard deviations of three independent samples. D. <i>Hoxa9</i> expression in the first round colonies of <i>MLL-AF9</i>-transformed cells. Relative expression levels (normalized to <i>Gapdh</i>) and expressed relative to those of <i>MLL-AF9</i>-transformed wild type progenitors arbitrarily set as 100%. Error bars represent standard deviations of triplicate PCRs.</p

MLL processing is not required for MLL-dependent gene activation.

<p>A. Schematic structures of MLL proteins produced by <i>dC</i> and <i>uc</i> mutations. The characteristics of each mutant are shown on the right. The mutated processing sites are highlighted in red. B. Sequences at the processing sites of the PCR fragments amplified from genomic DNAs of the recombined ES cell clones. C. Expression of MLL proteins in embryos. Western blotting was performed on whole embryo extracts of various genotypes. MLL proteins were visualized by anti-MLL^C antibody. Anti-menin blot serves as a loading control. <i>uc/+</i>: <i>uc</i> heterozygous. <i>uc/uc</i>: <i>uc</i> homozygous. <i>dC/+</i>: <i>dC</i> heterozygous. <i>dC/ dC</i>: <i>dC</i> homozygous. D. Genotypes at various developmental stages. Viability of embryos was confirmed by presence of heart contractions. E. Expression of <i>Hoxc8</i> transcripts in E10.5 embryos. Whole mount in situ hybridization was performed using the <i>Hoxc8</i> probe <i>(Hoxc8</i>). Arrows indicate sites of target gene expression. Previously published data for the wild type and <i>dC</i> homozygous embryos <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073649#pone.0073649-Yokoyama1" target="_blank">[8]</a> are shown here for comparison. F. Expression of various genes in mutant MEFs. Three independently established MEF lines of wild typ<i>e</i>, <i>uc</i> homozygous and <i>dC</i> homozygous genotypes were examined by RT-qPCR for genes indicated at the tops of respective panels. Relative expression levels (normalized to <i>Gapdh</i>) are expressed relative to those of wild type-1 arbitrarily set as 1. Previously published data for wild type and <i>dC</i> homozygous MEFs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073649#pone.0073649-Yokoyama1" target="_blank">[8]</a> obtained in the same experiment are included for comparison. Error bars represent the standard deviations of triplicate PCRs. G. Proliferative capacities of <i>uc</i> homozygous MEFs. Proliferation assay (top) and 3T3 senescence assay (bottom) were performed for three lines each of wild type, <i>uc</i> homozygous and <i>dC</i> homozygous genotypes at passage 3. Previously published data for wild type and <i>dC</i> homozygous MEFs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073649#pone.0073649-Yokoyama1" target="_blank">[8]</a> obtained in the same experiments are included for comparison.</p

Molecular purging of multiple myeloma cells by culture and retroviral transduction of mobilized-blood CD34cells-2

<p><b>Copyright information:</b></p><p>Taken from "Molecular purging of multiple myeloma cells by culture and retroviral transduction of mobilized-blood CD34cells"</p><p>http://www.translational-medicine.com/content/5/1/35</p><p>Journal of Translational Medicine 2007;5():35-35.</p><p>Published online 12 Jul 2007</p><p>PMCID:PMC1948885.</p><p></p>transduced cells. (B) Relative fold expansion in culture, and recovery after transduction and selection of total cells (open bars) and CD34+ cells (black bars), were measured, in comparison with the initial cell population (fresh cells)