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 ± 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 (α-MyH1) antibodies. Membranes were reprobed with α-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.