Reduced expression of primitive endoderm markers Gata4 and Gata6 and increased expression of Nanog was observed in embryoid bodies upon inhibition of Mek.

<p>Embryoid bodies were grown in different concentrations of PD-0325901 or 0.04% DMSO for 7 days. Expression levels of (A) Gata6, and (B) Gata4 were analysed using western blotting. A representative blot and quantification from 3 independent experiments is shown for each marker. A dose dependent decrease in expression of both proteins was observed. Statistical analysis is a one-way Anova with a Dunnett’s post-hoc test. Whole-mount immunostaining of (C) Gata6 and (D) Nanog after treatment of embryoid bodies with 4 µM PD-0325901. A reduction in the percentage of nuclei expressing Gata6 was observed whilst there was an increase in the percentage of nuclei expressing Nanog. A representative image from 3 independent experiments is shown. Scale bars 10 µm. Dotted lines represent position that the relevant orthogonal or aerial images were taken. Statistical test is a paired t-test. Data is from 3 independent experiments, error bars represent SEM. (*P = 0.1–0.5, **p = 0.001–0.01, ***p<0.001).