Tie-2 expression during Mk differentiation of cord blood or peripheral blood cord blood or peripheral blood CD34⁺ cells.

<p>Kinetics of Tie-2 expression during TPO-driven megakaryocytic differentiation of cord blood (CB, <i>Left Panels</i>) or cord blood (PB, <i>Right Panels</i>) CD34⁺ cells. Purified CB or PB CD34⁺ cells have been grown in serum-free medium in the presence of TPO and, at various days of culture, cell aliquots have been harvested and and analyzed for cell growth (<i>top panels</i>), for Tie-2 expression at mRNA and protein level (mean percentage of positive cells ± SEM observed in three separate experiments, <i>middle panels</i>) and for the expression of membrane differentiation markers CD34, CD41, CD61 and CD42b (<i>bottom panels</i>). In the <i>inset</i> the Tie-2 WB analysis on Mks grown from PB is reported. Mean values ± SEM observed in three separate experiments.</p

Angiopoietin and TPO-induced cell signaling in HUVEC cells.

<p>HUVEC cells, grown under standard endothelial cell culture conditions, have been starved overnight of growth factors and stimulated as indicated in panel B of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039796#pone-0039796-g010" target="_blank">Fig.10</a>. <i>A –</i> Flow cytometry and Western Blot analysis of Tie-2 expression in HUVEC cells starved of growth factors and then stimulated for 15 min either in the absence (C ) or in the presence of either TPO or Ang-1 or Ang-2. <i>B and C</i> - WB and densitometric quantification (<i>top</i> and <i>bottom</i> panels, respectively).</p

Effect of exogenous angiopoietins on the growth of UT7/mpl cells.

<p><i>Top Panels</i>: UT7/mpl cells have been grown either in the absence (C ) or in the presence of either GM-CSF, TPO, Ang-1, Ang-2, TPO+Ang-1 or TPO+Ang-2 and the number of living cells was determined at each day of culture. <i>Bottom Panel</i>, left: At day 4 and 7 of culture cell aliquots were harvested and analyzed for cell cycle by PI labeling and flow cytometry. The proportion of S phase cells (mean values ± SEM observed in three separate experiments) is reported. * p<0.05 <i>Bottom panel</i>, right: UT7/mpl cells have been plated in methylcellulose either in the absence (control) or in the presence of either TPO, TPO+Ang-1, TPO+Ang-2 or TPO+Ang-1+Ang-2 and after 10 days of <i>in vitro</i> culture the number of colonies was evaluated under an inverted microscope. Mean values ± SEM observed in three independent experiments are shown. ** p<0.01.</p

Effect of exogenous Ang-1 and Ang-2 on TPO-driven Mk differentiation of UT7/mpl cells.

<p>UT7/mpl cells have been grown in the presence of TPO alone or in combination with either Ang-1 or Ang-2 and at various days of culture the expression of CD61 (<i>Top Panel</i>) and CD41 (<i>Middle Panel</i>) antigens by flow cytometry and the percentage of polylobated (i.e., with 2 or 4 nuclear lobes) nuclei was evaluated by microscopy inspection of individual MGG-stained cells (<i>Bottom Panel</i>). The data represent mean values ± SEM observed in three separate experiments. In the top and middle panels the difference between TPO and TPO+Ang-1 or TPO+Ang-2 was significant (p<0.05). * p<0.05; ** p<0.01.</p

Growth, differentiation, Tie-2 and Tie-1 expression in UT7/mpl cells. A:

<p>Cell growth curve of UT7/mpl cells grown in the presence of GM-CSF or TPO. <b>B</b>: CD41 and CD61 (mean values ± SEM in three separate experiments) expression in UT7/mpl cells grown for 7 days either in the presence of GM-CSF or TPO. <b>C</b>: Proportion of cells with polylobated nuclei (mean values ± SEM in three separate experiments) at day 7 of either GM-CSF or TPO treatment. <b>D</b>: Flow cytometric analysis of Tie-2 expression in UT7/mpl cells grown either in the absence of growth factors (starved) or in the presence of either GM-CSF or TPO or Ang-1 or Ang-2 for 36 h. <b>E</b>: Mean Fluorescence Intensity (MFI) values of Tie-2 expression as evaluated by flow cytometry analysis. The data represent the mean values ± SEM observed in three separate experiments. <b>F</b>: Western blotting analysis of Tie-2 and TPO-R expression in UT7/mpl cells grown as above. <b>G</b>: Quantification of western blot results by band densitometry analysis (Mean Absorbance Units ± SEM from three separate experiments). <b>H</b>: Tie-1 expression in UT7/mpl cells. Flow cytometric analysis of Tie-1 expression in UT7/mpl cells deprived for 36 h of growth factors (starved) or grown for 36 h either in the presence of either GM-CSF, TPO, Ang-1, Ang-2, TPO+Ang-1 or TPO+Ang-2. In the first seven panels, from the top to the bottom a representative flow cytometry analysis of Tie-1 is shown. Mean Fluorescence Intensity (MFI) values (mean values ± SEM observed in three separate experiments) are reported in <b>I</b>. *, **, ***: p<0.05, p<0.01, p<0.001, respectively.</p

Angiopoietin and TPO-induced cell signaling in UT7/mpl cells and in normal megakaryocytes.

<p><i>A –</i> Flow cytometry and Western Blot analysis of Tie-2 expression in UT7/mpl cells starved for 16 h of growth factors and then stimulated for 15 min either in the absence (C ) or in the presence of either TPO or Ang-1 or Ang-2. <i>B</i> – UT7/mpl cells have been starved for 16 h of growth factors and then stimulated for various times (15, 90 and 180 min) with either TPO or Ang-1 or Ang-2 or TPO+Ang-1 or TPO+Ang-2. The cells were then harvested, lysed and analyzed by western blotting for the expression of p-ERK-1/−2, p-38, p-STAT-5A and p-AKT and their non-phosphorylated forms. Beta-actin was used to normalize for sample loading. A representative experiment out of three performed is shown. <i>C</i> – Day 7 megakaryocytes derived from unilineage cell cultures of normal CB CD34⁺ cells have been first starved for 6 h of exogenous growth factors and then stimulated for various times (15, 30 or 60 min) with either TPO or Ang-1 or Ang-2. Cells were then washed, lysed and processed as above. Beta-actin was used to normalize for sample loading. <i>D-E</i> Densitometric analysis of WB autoradiograms, performed on UT7/mpl (<i>panel D</i>) or d7 PB MKs (<i>panel E</i>) expressed as absorbance arbitrary units. The results represent the mean values observed in three separate experiments.</p

Effect of endogenous angiopoietin neutralization on Mk differentiation of UT7/mpl cells.

<p><b>A-</b> Effect of endogenous angiopoietin neutralization by Tie-2/Fc on Mk polyploidization in UT7/mpl cells. UT7/mpl cells have been grown for 7 days either in the presence of GM-CSF or TPO and either in the absence or in the presence of Tie-2/Fc; after 7 days of culture the cells have been harvested and evaluated for CD41 and CD61 expression by flow cytometry (<i>top</i> and <i>middle panels</i>) and for the proportion of cells with polylobated nuclei (<i>bottom panel</i>). Mean values ± SEM observed in three separate experiments. ** p<0.01; *** p<0.001. <b>B-</b> Effect of siAng-1 RNA or siAng-2 RNA on Mk differentiation of UT7/mpl cells. UT7/mpl cells have been transfected with either a control scramble siRNA (C ) or siAng-1 RNA or siAng-2 RNA or siAng-2 RNA or both siAng-1+ siAng-2 RNAs and then grown for four days in the presence of TPO and assayed for Ang-1 mRNA (<i>left, top panel</i>) or Ang-2 mRNA (<i>right, top panel</i>), for Mk membrane CD41 (<i>left, middle panel</i>) or CD61 (<i>right, middle panel</i>) antigens and for the presence of polylobated nuclei (<i>bottom panel</i>). Mean values ± SEM observed in three independent experiments are shown. *, **, ***: p<0.05, p<0.01, p<0.001, respectively.</p

Release of angiopoietins by differentiating megakaryocytes.

<p>PB (<i>top panel</i>) or CB (<i>middle panel</i>) CD34⁺ cells have been grown in unilineage Mk cultures and culture supernatants have been harvested at various days of culture and evaluated for Ang-1 and Ang-2 content per 10⁶ cells. Mean values ± SEM observed in three separate experiments are shown. In the <i>bottom panel</i> the ratio Ang-1/Ang-2 at various days of culture is plotted.</p

Effect of exogenous angiopoietins on TPO-driven Mk differentiation of HPCs.

<p>CD34⁺ HPCs purified either from CB (<i>top panel</i>) or PB (<i>bottom panel</i>) have been triggered to Mk differentiation with TPO either in the absence of growth factors (TPO ) or in the presence of either 100 ng/ml Ang-1 (TPO+Ang-1) or 100 ng/ml Ang-2 (TPO+Ang-2) and then assayed for Mk ploidy by evaluating the percentage of polylobated Mks. The data reported in the Figure represent mean values ± SEM observed in three separate experiments. * p = <0.05 when compared to the corresponding TPO-treated sample. *** p = <0.001 when compared to the corresponding TPO-treated sample.</p

Release of angiopoietins during differentiation of UT7/mpl cells.

<p>UT7/mpl cells have been grown for 7 days in the presence of either GM-CSF (<i>Top Panels</i>) or TPO (<i>Middle Panels</i>) and the concentration of Ang-1 and Ang-2 in the culture supernatants was evaluated by a sensitive and specific ELISA assay. The levels of angiopoietins reported in the figure correspond to the accumulated amount of angiopoietins during the culture. Mean values ± SEM observed in three separate experiments. In the bottom panel the ratio Ang-1/Ang-2 at various days of culture is plotted.</p