The PA System Modulates ESC Differentiation Figure 1. endogenous uPA mRNA, protein expression and enzymatic activities during ESC differentiation. 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 (d0) to 24 (d24). (A) 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 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) uPA antigen (upper panels) or specific uPA enzymatic activity (lower panels) were quantified by ELISA technique in either total cell lysate protein extracts (B) or in 24 hr. conditioned medium of cells cultivated without serum (C). Mean values of at least three independent experiments are given. doi:10.1371/journal.pone.0049065.g001 enzymatic inhibition could be due to the presence of high amounts of PAI-1 proteins during this period (Fig. 3B). Intracellular uPA protein and activity are detected only transiently during the differentiation process, at days 3 and 7, being almost undetectable by day 14 (Fig. 1C). PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e49065