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

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

characterization and adipogenic capacities of PAI-1^−/− pluripotent cells.

<p>(<b>A</b>) In comparison to primary MEFs and wt CGR8 ESCs, the degree of reprogrammation of wt and PAI-1 KO, clones 1 and 3, iPSCs was characterized by the mRNA levels of Oct4 and Nanog genes and by the extinction of the expression of GFP gene. Results are expressed in arbitrary units, with the values of CGR8 mESCs 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>) Characterization, during the differentiation process of iPSCs, of the expression of ESC commitment master genes. Retinoic acid-treated (RA) or not (NoRA) EBs from wild type CGR8 ESCs and iPSC clones were induced to differentiate. mRNAs were extracted at various time, as indicated, and analyzed by real time RT-PCR for the expression of Pax6, Sox17 and Brachyury. Results are expressed in arbitrary units, with the values of wild type CGR8 taken as 1, and are the means ± S.E.M. of at least 3 independent experiments. (<b>C</b>) Retinoic acid-treated (<b>RA</b>) or not (<b>NoRA</b>) EBs from either wild type (<b>WT</b>) pluripotent cells or PAI-1^−/− iPSCs (<b>PAI-1 KO</b>) were induced to differentiate to adipocytes. mRNAs were extracted and analyzed by real time RT-PCR for the expression of the adipogenic markers PPARgamma and adiponectin. Results are expressed in arbitrary units, with the values of WT NoRA condition at day 24 taken as 1, and are the means ± S.E.M. of at least 3 independent experiments. Significance is given as: N.S. non significant, *P<0.05, **P<0.01 and ***P<0.001.</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

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

endogenous PAI-1 mRNA and protein expression during ESC differentiation.

<p>Retinoic acid-treated (RA) or not (NoRA) EBs from wild type CGR8 ES cells 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 PAI-1. 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 </b><b>and </b><b>C</b>) PAI-1 protein expression in total cell lysates (<b>B</b>) or conditioned medium (<b>C</b>) was quantified by ELISA technique at various time, as indicated, between days 0 (<b>d0</b>) to 24 (<b>d24</b>). Values are given in ng of PAI-1 amounts and expressed as means of at least three independent experiments ± S.E.M. Significance between RA and NoRA treatments is given.</p

Doxycycline-induced human PAI-1 expression inhibits ESC adipogenesis of the A2lox.cre mESC clone3.

<p>(<b>A and B</b>) Retinoic acid-treated (<b>RA</b>, panel <b>B</b>) or not (<b>NoRA,</b> panel <b>A</b>) EBs from A2lox.cre mESC clone3 cells treated (<b>Dox</b>, red lines) or not (<b>Ct</b>, black lines) by 0.5 µg/ml of doxycycline were induced to differentiate and analyzed at various time, as indicated, between days 0 (<b>d0</b>) to 24 (<b>d24</b>). Endogenous mouse (mPAI-1, blue lines) and ectopic human (hPAI-1) PAI-1 protein expressions in conditioned medium were quantified by ELISA technique. Values are given in ng of PAI-1 amounts and expressed as means of at least three independent experiments ± S.E.M. (<b>C</b>) Evaluation of the turnover of Dox-induced intracellular human PAI-1 by ELISA after doxycycline removal. ESCs were treated with doxycycline either all the time (blue line) or between days 0 to 3 (red line) and hPAI-1 protein was analyzed until day 7. Values are given in ng of human PAI-1 amounts/mg of total proteins. Retinoic acid-treated EBs from A2lox.cre mESC clone3 cells were induced to differentiate to adipocyte and treated or not by doxycycline for different period of time: from days 0 to 24 <b>[Dox]</b>, from days 0 to 3 <b>[Dox(d0–d3)]</b>, from days 3 to 7 <b>[Dox(d3–d7)]</b>, from days 7 to 24 <b>[Dox(d7–d24)].</b> (<b>D</b>) Scheme of the different doxycycline treatments. (<b>E to G</b>) mRNAs were extracted and analyzed by real time RT-PCR for the expression of the adipogenic markers adiponectin (<b>E</b>), aP2 (<b>F</b>) and PPARgamma (<b>G</b>). 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 to Ct values is given as: N.S. non significant, *P<0.05, **P<0.01 and ***P<0.001. (<b>H</b>) Adipocyte formation of A2lox.cre mESC clone3 cells treated or not, as indicated, by doxycycline was visualized by Oil RedO staining, representative fields are shown.</p

endogenous uPA 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 uPA. 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>) uPA antigen (upper panels) or specific uPA 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