The PA System Modulates ESC Differentiation Figure 7. human PAI-1 expression regulates ESC skeletal myogenesis of A2lox.cre mESC clone3 after doxycycline induction. 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 figure 5. (A) 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 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) 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 (a-MyH1) antibodies. Membranes were reprobed with a-ERK antibodies as loading control. (C) 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. (D) Differentiated cells at day 24 were analyzed for Myogenin expression by flow cytometry. doi:10.1371/journal.pone.0049065.g007 the identification of important adipogenic genes differentially regulated during this precise timing. Though PAI-1 ectopic overexpression during ESC terminal adipocyte differentiation gave similar inhibition than amiloride treatment, intriguingly, early (d0–d7) overexpression during differentiation has no effect on adipogenesis. To explain this lack of effect one can hypothesize that, during the early steps of ESC commitment, PAI-1 protein would lead to both adipogenic inhibitory effects, via uPA enzymatic inhibition, and adipogenic stimulatory effects, independently of uPA; resulting in the net absence of effect on adipogenesis. Additional works are necessary to further explore this hypothesis. Finally the analysis of PAI-12/2 iPSCs constituted a third converging argument to demonstrate the implication of the PA system in ESC adipogenesis. Indeed, these cells are prone to form adipocytes spontaneously and, contrarily to wild type cells, do not respond anymore to the retinoic acid treatment. Furthermore, these results fit with the observation that knock-out PAI-1 mice are prone to obesity [27]. Regarding skeletal myogenesis and similarly to adipogenesis, we found a decrease of differentiation resulting from uPA inhibition or PAI-1 ectopic overexpression during the terminal differentia- tion of ESCs. These results are in agreement with the previous roles described for uPA and PAI-1 in in vitro muscle cell differentiation [15] and in vivo skeletal muscle regeneration [16,17]. Surprisingly, EB treated between days 0 to 3 presented a significant increase in skeletal myogenesis compared to untreated cells. The two approaches, uPA inhibition or PAI-1 overexpres- sion, gave similar results. Because of the low expression level of the proteases during this period, we did not expect an effect by interfering with the PA. However, our results are consistent with the fact that a robust enzymatic activity of uPA is effectively detected at day 3. During this period uPA activity seems to specifically repress skeletal myogenesis, demonstrating that the commitment towards this particular mesodermal lineage can be PLOS ONE | www.plosone.org 11 November 2012 | Volume 7 | Issue 11 | e49065