activity are detected from days 3 to 24, reaching a maximum at day 3 and then slightly decreasing (Fig. 2C). Expression of PAI-1 (mRNA and protein) is almost undetectable in undifferentiated cells and till day 7 and correlates mainly with terminal adipocyte and skeletal myogenic differentiations (Fig. 3). Interestingly, at day 24 of the differentiation, PAI-1 expression is significantly higher in cultures without RA treatment than in RA- treated embryoid bodies, suggesting that PAI-1 expression could be higher in skeletal myotubes than in adipocytes. Since these cultures are not pure differentiated cell populations, one could hypothesize that PAI-1 would not be expressed in adipocytes. Therefore, we analyzed the expression of PAI-1 by immunocyto- chemistry at day 24 in well differentiated cells. In all tested conditions, we were able to detect PAI-1 protein in both adipocytes and myotubes (Data S1). Variations of expression of the receptor of uPA, uPAR, are very limited during the differentiation process (data not shown). Altogether these results show that uPA activity is mainly present during the early d0–d14 period, whereas tPA activity is still strongly detected in well differentiated cells; PAI-1 protein is present only in the latter steps of differentiation, from d14 to d24. uPA Inhibition by Amiloride Decreases ESC Adipogenesis These striking regulations of endogenous components of the PA system during ESC differentiation suggested important biologic roles for these molecules. In order to analyze their regulatory potential, we treated ESCs with amiloride, a potent specific chemical uPA inhibitor which does not affect the tPA protease [32]. Because the first week of the differentiation process is critical for the ESC commitment [26], 100 mM of the inhibitor were applied on CGR8 ESCs in four different conditions: i) all the time of differentiation, from days 0 to 24 [A], ii) only from days 0 to 3 [A(d0–d3)], iii) from days 3 to 7 [A(d3–d7)], iv) from days 7 to 24 [A(d7–d24)] (Fig. 4A). The inhibitory effect of amiloride on uPA enzymatic activity was measured between days 0 to 14 of the differentiation process. Whereas uPA activity is very low in control undifferentiated cells, this activity augments as early as day 3, reaching a maximum, at day 7 into the 24 hrs conditioned medium of RA-treated (adipogenic conditions) or not (skeletal myogenic conditions) cells, respectively (Fig. 4B and C, black curves). These activities are completely inhibited by the amiloride treatment all the time of differentiation, (red curves), inhibition at day 14 is also observed when amiloride is added at day 7 (blue curves). Interestingly, when amiloride is added only between days 0 to 3, uPA activity is inhibited at day 3 but is fully recovered at days 7 and 14 (green curves). Likewise, when amiloride is added between days 3 to 7, uPA activities decrease during this period and are partially recovered by day 14 (violet curves). Importantly, these results demonstrate that the uPA inhibition by amiloride is a reversible process. Although close to the detection limit of the assay, similar results were obtained by analyzing uPA activity in cellular lysates (Data S2). We then analyzed the effects of the inhibitor on ESC differentiation. All amiloride treatments of CGR8 cells signifi- cantly decrease by more than 60% the expression of adipogenic markers measured at day 24: adiponectin (Fig. 4D), aP2 (Fig. 4E) and PPARgamma (Fig. 4F). These inhibitions of adipocyte formation resulted in less lipids accumulated in cell cultures, as detected by Oil RedO staining (Fig. 4G). As a control, we found no effect on cells treated with di-methyl-amiloride, an amiloride- derived compound which no longer inhibits uPA (data not shown). Since uPA activities are detected at least until day 14, these results suggest that uPA plays important successive roles during the whole process of ESC adipogenesis. The PA System Modulates ESC Differentiation Figure 3. endogenous PAI-1 mRNA and protein expression during ESC differentiation. 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 (d0) to 24 (d24). (A) 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 6 S.E.M. of at least 3 independent experiments. Significance is given as: *P,0.05, **P,0.01 and ***P,0.001. (B and C) PAI-1 protein expression in total cell lysates (B) or conditioned medium (C) was quantified by ELISA technique at various time, as indicated, between days 0 (d0) to 24 (d24). Values are given in ng of PAI-1 amounts and expressed as means of at least three independent experiments 6 S.E.M. Significance between RA and NoRA treatments is given. doi:10.1371/journal.pone.0049065.g003 In order to check whether the amiloride treatment could affect the extracellular matrix (ECM) secreted by ESCs during the differentiation process, we characterized the collagen 1 ECM composition by immunofluorescence studies after acellularization of the cultures at days 12 and 24. Collagen 1 is detected in low amounts at day 12 of differentiation in control cells, treated (Data S3) or not (Data S4) by RA and its expression is slightly augmented by day 24. Interestingly, the amiloride treatment A(d0–d24) strongly increases collagen 1 deposition at day 24 in both RA and NoRA treated cells and this increase is visible as soon PLOS ONE | www.plosone.org 6 November 2012 | Volume 7 | Issue 11 | e49065