SHIP deficient mice have increased number of functional MKP in hematopoietic organs.

<p>(A) BM, spleen, and PB MKP phenotype in SHIP^−/− (−/−) and SHIP^ΔIP/ΔIP (ΔIP/ΔIP) mice. Shown are representative flow cytometry dot plots of CD41 (<i>x</i>-axis) versus c-Kit (<i>y</i>-axis) after gating on live and Lin⁻ cells. MKP (Lin⁻c-Kit⁺CD41⁺) are represented in the right upper quadrants and MK (Lin⁻c-Kit⁻CD41⁺) are represented in the right lower quadrants. Percentages for each population are indicated on each plot. (B) Represented are the mean±standard error mean (SEM) (n = 4 for experimental groups WT C57Bl6 and SHIP^−/−, while n = 6 for experimental groups WT 129SvJ and SHIP^ΔIP/ΔIP) of the absolute numbers of MKP in BM, spleen and PB of SHIP deficient mice (black columns) compared to respective WT littermates (gray columns). Mice strains specified in the graphs. (C) Represented is the mean±SEM (n = 4 for experimental groups WT C57Bl6 and SHIP^−/−, while n = 6 for experimental groups WT 129SvJ and SHIP^ΔIP/ΔIP) of total body MKP numbers determined as the calculated sum of MKP in one whole spleen plus MKP in one femur ×16.6 (since one femur is estimated to contained 6% the total marrow) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003565#pone.0003565-Helgason1" target="_blank">[3]</a>. (D) Represented is the mean±SEM of CFU-MK colonies in BM and spleen of SHIP^−/− (n = 7) and WT (n = 7) littermates. Statistical analysis was established using the Mann-Whitney test. <i>p</i> values indicated on each graph.</p

Increased phosphorylation of signaling proteins in SHIP^−/− MKP in BM.

<p>(A) Histogram representing the level of phospho-STAT3, phospho-Erk1/2 and phospho-Akt in Lin⁻c-kit⁺CD41⁺ BM cells isolated from SHIP^−/− (black line) and WT (gray line) mice. Isotype antibody control indicated on the histogram. (B) Represented is the mean±SEM of the mean fluorescence (MFI) intensity of phospho-STAT3, phospho-Erk1/2 and phospho-Akt on SHIP^−/− (−/−) (n = 4) and WT (n = 4) Lin⁻c-kit⁺CD41⁺ in BM. Significance was established using the Mann-Whitney test. <i>p</i> values indicated on each graph.</p

SHIP deficient mice MK are redistributed within hematopoietic organs.

<p>(A) Represented are the mean±SEM (n = 4 for experimental groups WT C57Bl6 and SHIP^−/−, while n = 6 for experimental groups WT 129SvJ and SHIP^ΔIP/ΔIP) of the absolute numbers of MK (Lin⁻c-Kit⁻CD41⁺) in BM, spleen and PB of SHIP deficient mice (black columns) compared to respective WT littermates (gray columns). Mice strains specified in the graphs. (B) Represented is the mean±SEM (n = 4 for experimental groups WT C57Bl6 and SHIP^−/−, while n = 6 for experimental groups WT 129SvJ and SHIP^ΔIP/ΔIP) of total body numbers of MK determined as the calculated sum of MK numbers in one whole spleen plus MK in one femur ×16.6 (since one femur is estimated to contain 6% of the total marrow) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003565#pone.0003565-Helgason1" target="_blank">[3]</a>. Significance was established using the Mann-Whitney test. <i>p</i> values indicated on each graph.</p

SHIP deficient MK precursors preserve endomitotic function.

<p>BM and splenocytes from SHIP^−/− and WT mice were enriched for CD41⁺ on AutoMACS, stained with Lin-panel and CD41-biotin/SA-APC, fixed/permeated and stained with PI in the presence of RNase. (A) Representative density plots of ploidy distribution of megakaryocytic cells. Lin⁻CD41⁺ cells were first selected by gating forward and side scatter by area and width. Propidium iodide (<i>x</i> axis) versus forward side scatter (<i>y</i> axis) density plots were then generated to assess ploidy distribution in BM (top panels) and spleen (bottom panels) of SHIP^−/− and WT mice. (B) Represented is the mean±SEM (n = 4 for each experimental group) percentage of BM (top) and spleen (bottom) Lin⁻CD41⁺ cells in different ploidy stages from 2N to 32 N. Statistical analysis was done using the Mann-Whitney test. <i>p</i> values indicated on each graph.</p

SHIP^−/− MK have lowered CXCR-4 receptor expression.

<p>(A) Histogram representing the level of CXCR-4 receptor expression on SHIP^−/− (black line) and WT (gray line) Lin⁻c-kit⁻CD41⁺ cells in BM. Isotype antibody control indicated in the histogram. (B) Represented is the mean±SEM of the mean fluorescence (MFI) intensity of CXCR-4 expression on SHIP^−/− (−/−) (n = 4) and WT (n = 4) Lin⁻c-kit⁻CD41⁺ in BM. Significance was established using the Mann-Whitney test. <i>p</i> values indicated in each graph.</p