IDB induces megakaryocytic differentiation of human CD34+ progenitors whereas PMA does not.

<p><b>(A–C, upper panels)</b> Morphology of adult human CD34+ hematopoietic progenitors cultured for 7 days in medium containing TPO and SCF alone or with 10 nM IDB or 10 nM PMA on Wright-stained preparations. Note that relative to control cells, cells treated with IDB were larger and had polyploid nuclei. <b>(A–C, lower panels)</b> Adult human CD34+ progenitors were treated with TPO and SCF with or without 10 nM IDB or 10 nM PMA. After 7 days of culture, the cells were stained with a fluorescent antibody directed against CD41a (open profiles) or with an isotype matched control antibody (black profiles). All flow cytograms in this and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051059#pone-0051059-g002" target="_blank">Figures 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051059#pone-0051059-g003" target="_blank">3</a> are representative of at least 3 independent runs. (D) Adult human CD34+ progenitors were treated with TPO and SCF with 25 nM IDB, 10 nM PMA, 100 nM PMA, or 1 µM Mezerein. After 7 days of culture, the cells were stained with a fluorescent antibody directed against CD41a (open profiles) or with an isotype matched control antibody (black profiles).</p

IDB induces early expression of promegakaryopoietic transcription factors.

<p>(<b>A</b>) CD34+ cells cultured in TPO/SCF or EPO/SCF for 12 days and subjected to Western blot analysis. (<b>B</b>)TPO/SCF +/− 100 nM IDB for 72 hrs and subjected to western blot analysis. Blots were then stripped and reprobed for each of the indicated proteins.</p

Inhibitor studies support that CD9 induction by IDB is mediated by PKCε.

<p>(<b>A</b>) CD34+ cells were cultured in TPO/SCF for 3 days with or without 100 nM IDB +/− 5 µM of GF109203X or 1 µM Go6976 and analyzed for CD9 (open panel) or isotype control (shaded panel) by flow cytometry. (<b>B</b>) CD34+ cells were cultured in TPO/SCF for 2 hours with or without 100 nM IDB +/− GF109203X or Go6976 and subjected to western blot analysis for the indicated antigens. (<b>C</b>) CD34+ cells were cultured for 48 hours in media containing TPO/SCF. Cells were then nucleofected as per the manufacturer's protocol for CD34+ cells with either scrambled or PKCε siRNA. After a 2 hour recovery, the cells were treated with either vehicle or 25 nM IDB for an additional 24 hours and mRNA was extracted and analyzed by PCR for CD9, PKCε, and GAPDH expression.</p

Single i.p. injection of IDB increases platelet counts and mitigates thrombocytpoenia induced by irradiation.

<p>(<b>A</b>) A single i.p. injection of IDB increased platelet counts at day 7 compared with vehicle injected control animals (N = 5 per group; *p<0.01). No differences were observed in hemoglobin and white blood counts (p = NS). (<b>B</b>) Groups of BALB/c mice (N = 10/group/time point) were injected with a single i.p. dose of IDB (720 or 1080 µg/kg body weight) 3 hours before irradiation with 6 or 8 Gy. At two weeks the mice treated with 6 Gy were euthanized and complete blood counts were performed. At three weeks the mice treated with 8 Gy were euthanized and complete blood counts were taken (* showed statistical significance of at least p<0.01 or greater).</p

Single i.p. injection of IDB increases platelet counts and megakaryocyte content even when administered 24 hours after irradiation.

<p>Groups of BALB/c mice (N = 10/group/time point) were injected with a single i.p. dose of IDB (1080 µg/kg body weight) 24 hours after irradiation with 6 Gy. At 2 weeks, mice were euthanized and blood platelet counts (<b>A</b>) were performed and tissue for histologic examination for megakaryocyte content of bone marrow (<b>B</b>) and spleen (<b>C</b>) were obtained. Megakaryocyte baseline values were determined on 5 untreated mice, euthanized to obtain spleens and bone marrows for histologic examination.</p

IDB induction of ERK activation, egr-1, and CD9 in CD34+ cells.

<p>(<b>A&B</b>) CD34+ cells were thawed and recovered overnight in serum-free medium supplemented with SCF (25 ng/ml). Cells were stimulated with TPO (40 ng/ml) with 25 nM IDB or vehicle. mRNA for CD9 and egr-1 were quantitated using SYBR green and real time PCR. The values shown in A and B represent the mean of 2–3 replicate determinations. (<b>C</b>) CD34+ cells were recovered overnight as described above. Cells were stimulated with TPO with or without IDB as indicated. Immunoblot analysis of phospho-ERK and total ERK then were performed on whole cell lysates. (<b>D</b>) CD34+ cells were cultured in serum-free media and TPO/SCF with or without 100 nM IDB+/− 10 µM of the MEK inhibitor U0126 for 3 days and then analyzed for CD9 expression by flow cytometry.</p

IDB increases post-irradiation megakaryocyte production in both the spleen and bone marrow.

<p>Histologic sections of spleens (upper panels) or bone marrow (lower panels) 2 weeks after mice received 6 Gy irradiation alone (<b>A</b> and <b>C</b>) or 6Gy + 1080 µg/kg IDB 3 hours prior to irradiation (<b>B</b> and <b>D</b>).</p