The PA System Modulates ESC Differentiation Figure 2. endogenous tPA 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 tPA. 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) tPA antigen (upper panels) or specific tPA 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.g002 Upon differentiation, with or without RA treatment, tPA protein expression in supernatants and tPA mRNAs increase gradually, reaching a maximum at day 24 (Fig. 2A and B). tPA activities in supernatants, although globally following an identical profile to uPA activities do not decrease significantly after day 14, staying at high levels until day 24. Intracellular tPA protein and PLOS ONE | www.plosone.org 5 November 2012 | Volume 7 | Issue 11 | e49065