Baseline Characteristics of The Patient Population.

<p>Baseline Characteristics of The Patient Population.</p

Results of the MMPs and TIMPs serum levels.

<p>Only MMP-9 serum level was significantly higher in patients with new-EE compared to controls and patients with no new-EE. Medians and interquartile ranges are shown.</p

Predictors of New-EE by Multivariable Analysis.

<p>OR = odds ratio; SD = standard deviation; CI = confidence interval.</p

Flow chart showing enrollment of study patients.

<p>Flow chart showing enrollment of study patients.</p

Receiver Operating Characteristic curves analysis using vegetation length and MMP-9 serum level as predictors of new-EE.

<p>The area under de curves are given with their 95% confidence interval.</p

Gelatinolytic activity of MMP-9 (92 kDa) evaluated by in-gel zymography.

<p><b>A</b>: Gelatinolytic activity is greater in New-EE patients. <b>B</b>: Densitometric analysis of the values of gelatinase activity expressed as arbitrary units (AR). Results are given as means ±SEM. *p<0.0001 <i>vs.</i> Controls; †p = 0.05 versus No new-EE patients.</p

inhibition of uPA by amiloride treatments interfere with ESC adipogenesis.

<p>EBs from wild type CGR8 ES cells were induced to differentiate and treated or not by 100 µM amiloride for different period of time: from days 0 to 24 <b>[A]</b>, from days 0 to 3 <b>[A(d0–d3)]</b>, from days 3 to 7 <b>[A(d3–d7)]</b>, from days 7 to 14 <b>[A(d7–d14)]</b> or 24 <b>[A(d7–d24)].</b> (<b>A</b>) Scheme of the different amiloride treatments. (<b>B and C</b>) ELISA assay quantification of mouse activated uPA into the 24hrs conditioned medium of 10⁶ cells RA-treated (<b>C</b>, adipogenic conditions) or not (<b>B</b>, skeletal myogenic conditions) and treated or not by amiloride. (<b>D to G</b>) Effects of amiloride treatments on ESC adipogenesis. mRNAs from retinoic acid-treated (adipogenic conditions) EBs were extracted and analyzed by real time RT-PCR for the expression of the adipogenic markers adiponectin (<b>D</b>), aP2 (<b>E</b>) and PPARgamma (<b>F</b>). Results are expressed in arbitrary units, with the values of untreated CGR8 at day 24 taken as 1, and are the means ± S.E.M. of at least 3 independent experiments. Significance to Ct values is given as: *P<0.05, **P<0.01 and ***P<0.001. (<b>G</b>) Adipocyte formation of CGR8 treated or not by amiloride, as indicated, was visualized by Oil RedO staining, representative fields are shown.</p

endogenous tPA mRNA, protein expression and enzymatic activities during ESC differentiation.

<p>Retinoic acid-treated (RA) or not (NoRA) EBs from wild type CGR8 ESCs were induced to differentiate and analyzed at various time, as indicated, between days 0 (<b>d0</b>) to 24 (<b>d24</b>). (<b>A</b>) mRNAs were extracted and analyzed by real time RT-PCR for the expression of tPA. Results are expressed in arbitrary units, with the values of wild type CGR8 at day 0 taken as 1, and are the means ± S.E.M. of at least 3 independent experiments. Significance is given as: *P<0.05, **P<0.01 and ***P<0.001. (<b>B and C</b>) tPA antigen (upper panels) or specific tPA enzymatic activity (lower panels) were quantified by ELISA technique in either total cell lysate protein extracts (<b>B</b>) or in 24 hr. conditioned medium of cells cultivated without serum (<b>C</b>). Mean values of at least three independent experiments are given.</p

inhibition of uPA by amiloride treatments regulates ESC skeletal myogenesis.

<p>EBs from wild type CGR8 ES cells were induced to differentiate to skeletal myotube and treated or not by 100 µM amiloride for different period of time as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049065#pone-0049065-g004" target="_blank">figure 4</a>. (<b>A</b>) mRNAs were extracted and analyzed by real time RT-PCR for the expression of the skeletal myogenic markers MyH1 (left panel) and Myogenin (right panel). Results are expressed in arbitrary units, with the values of untreated CGR8 at day 24 taken as 1, and are the means ± S.E.M. of at least 3 independent experiments. Significance to Ct values is given as: *P<0.05, **P<0.01 and ***P<0.001. (<b>B</b>) MyH1 protein expression at day 24 of differentiation of CGR8 cells treated or not by amiloride, as indicated, was analyzed by Western blotting with anti-MyH1 (<b>α-MyH1</b>) antibodies. Membranes were reprobed with <b>α-ERK</b> antibodies as loading control. (<b>C</b>) Myotube formation of CGR8 treated or not by amiloride, as indicated, was analyzed by immunofluorescence experiments with anti-MyH1 antibodies (right panels) and nuclei were counterstained with DAPI (left panels), representative field are shown. (<b>D</b>) Differentiated cells at day 24 were analyzed for Myogenin expression by flow cytometry, two different experiments are shown, controls giving either 28.7% (upper panels) or 16.2% (lower panels) myogenin positive cells.</p

human PAI-1 expression regulates ESC skeletal myogenesis of A2lox.cre mESC clone3 after doxycycline induction.

<p>EBs from A2lox.cre mESC clone3 cells were induced to differentiate to skeletal myotube and treated or not by doxycycline for different period of time as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049065#pone-0049065-g005" target="_blank">figure 5</a>. (<b>A</b>) mRNAs were extracted and analyzed by real time RT-PCR for the expression of the skeletal myogenic markers MyH1 (left panel) and Myogenin (right panel). Results are expressed in arbitrary units, with the values of untreated A2lox.cre mESC clone3 cells at day 24 taken as 1, and are the means ± S.E.M. of at least 3 independent experiments. Significance is given as: *P<0.05, **P<0.01 and ***P<0.001. (<b>B</b>) MyH1 protein expression at day 24 of differentiation of A2lox.cre mESC clone3 cells treated or not by doxyxcycline, as indicated, was analyzed by Western blotting with anti-MyH1 (<b>α-MyH1</b>) antibodies. Membranes were reprobed with <b>α-ERK</b> antibodies as loading control. (<b>C)</b> Myotube formation of A2lox.cre mESC clone3 cells treated or not by doxycycline, as indicated, was analyzed by immunofluorescence experiments with anti-MyH1 antibodies (right panels) and counterstained with DAPI (left panels), representative fields are shown. (<b>D</b>) Differentiated cells at day 24 were analyzed for Myogenin expression by flow cytometry.</p