<i>Flt3</i> regulatory regions enable the recruitment of a combination of master regulators of haematopoiesis.

<p>Detection of in vivo transcription factor binding at the sites of hypersensitivity to nuclease digestion was achieved by ChIP. Relative enrichments from X-ChIP material were determined against the IgG control material by Q-PCR at the location of the hypersensitive regions and normalised against two internal control regions (located at -3.5kb and -0.27kb from the ATG). Error bars represent the standard error of the mean. All plots are representative of a minimum of three independent experiments.</p

MYB deletion affects KSL cell number and differentiation.

<p>(A) Flow cytometric analysis of bone marrow from <i>Myb</i>^<i>F/F</i>:<i>MxCre</i> mutant mice and <i>Myb</i>^<i>+/+</i>:<i>MxCre</i> control animals, 24 or 48 hours following in vivo induction of the <i>Myb</i>^<i>F/F</i> allele deletion by p(I:C) injection. The representative two-dimensional plots show the gating on lineage negative cells and the typical KIT⁺/SCA-1⁺/LIN^- (KSL) staining. Histograms represent the percentage of KSL cells within the total bone marrow population, with numbers presented as mean ± SEM, determined from 3 independent experiments (***, p<0.0001). (B) Control and <i>Myb</i>-deleted KSL were sorted and seeded in methylcellulose. Colony numbers and size were assessed after 6 days. The right histograms represent total colony numbers and the relative proportions of colonies based on size respectively. Pictures of the counted colonies are presented in the left panels. (C) Sorted KSL from control and <i>Myb</i>-deleted animals 24 hours post injection of poly(I:C) were cultured into liquid medium containing SCF, FL and TPO. After culturing for 2 days, the cells were re-stained and analysed by flow cytometry for expression of CD11 and CD41 (left panel). Cells from the <i>Myb</i>^<i>F/F</i>:<i>MxCre</i> bone marrow were sorted on the basis of CD11 and CD41. The sorted cells were spun onto glass slides and stained with Diff-Quick (upper right panel). The images show representative cells demonstrating monocytic (CD11b⁺) and megakaryocytic (CD11b⁺CD41⁺ and CD11b^-CD41⁺) morphologies.</p

<i>Flt3</i> intronic element hypersensitivity coincides with FLT3 expression in primary haematopoietic stem/progenitor cells.

<p>Upper panel gives a schematic representation of the murine <i>Flt3</i> promoter and first intron (filled box indicates exon1) and shows the level of cross-species sequence conservation and the position of repeated sequences. The underlined regions A B and C indicate the general location of the regions of hypersensitivity to DNAseI digestion in HPC7 cells. Lower panel plots show the assessment of DNaseI digestion sensitivity in the HPC7 cell line and in primary KSL FIT3⁺, KSL FITt3^- cells, CMP (LIN^-/KIT⁺/SCA-1^-/ CD34^-/CD16/32⁺), GMP (LIN^-/KIT⁺/SCA-1^-/CD34⁺/CD16/32⁺) and MEP (LIN^-/KIT⁺/SCA-1^-/CD34^-/CD16/32^-). For primary cells, the analysis are restricted to the digestion sites observed in HCP7 at -1.45 kb (HS-A), 0.15kb (HS-B) and +7.5kb (HS-C) from the murine <i>Flt3</i> initiation codon.</p

<i>Myb and Cebpa</i> knockdown in primary KIT⁺ enriched bone marrow cells.

<p>(A) Two-dimensional flow cytometry dot plot showing the analysis of the KSL compartment of a KIT⁺ enriched population transfected with siRNA control or undergoing siRNA-mediated silencing of <i>Myb</i> or <i>Cebpa</i> for 20 hours. (B) Grouping four independent experiments, boxplots depict the variations in percentage of FLT3⁺ cells within the KSL populations. (C) Quantitative PCR analysis of <i>Myb</i> and <i>Cebpa</i> RNA expression in sorted transfected KSL cells 20 hours post transfection. Expression is normalised to <i>β</i>_<i>2</i><i>-microglobulin</i> and standardised to the control samples. Error bars represent the standard error of the mean. Plots are representative of 4 independent experiments. Numbers are plotted as mean ± SEM (**, p<0.001 and *, p<0.05).</p

Induced <i>Myb</i> ablation results in FLT3 down-regulation in a population highly enriched in LMPP.

<p>(A) Representative histogram and two-dimensional flow cytometric plot analysis of cells in the KSL compartment of the bone marrow of the cKO and control mice 24 hours post poly(I:C) injection. (B) The cells were further analysed for surface expression of FIT3 and the proportion of different KSL sub-fractions were assessed based on the surface expression of VCAM-1 and CD62L. Regrouping 7 experiments, the right panel shows their relative contribution in percentage of the gated population, indicated as plot header. (C) Serial gating defined a population highly enriched in LMPP (KSL/CD62L⁺/VCAM^-) for which the percentage of FIT3^hi cells was measured. (D) Quantitative PCR analysis of <i>Myb</i>, <i>Flt3</i>, <i>Il7r</i>α, <i>Dntt and Notch1</i> RNA expression in KSL cells isolated from the bone marrow of poly(I:C)-induced <i>Myb</i> cKOs and litter-mate controls 24 hours post injection. Expression is normalised to <i>gapdh</i> and standardised the <i>Myb</i>^<i>+/+</i>:MxCre control samples, set as 1. Error bars represent the standard error of the mean. Plots are representative of 7 independent experiments. Numbers are plotted as mean ± SEM (***, p<0.0001 and **, p<0.01).</p