Fig. 4.

Signalling pathway inhibitors regulate gene expression during the ESC-to-EPL cell transition. (A-G) Naïve ESCs were cultured over 6 days in 330 U/ml LIF+400 μM L-proline with combinations of five inhibitors (U, U0126; S, SU5402; L, LY294002; R, rapamycin; P, PF-4708671). Quantification of changes in expression of pluripotency genes Rex1 (A) and Oct4 (B), primitive ectoderm markers Dnmt3b (C), Fgf5 (D), Lefty2 (E) and Otx2 (F), and mesendoderm gene Mixl1 (G) at day 6. All samples were normalised to Actb and then to cells grown in 1000 U/ml LIF. Data were averaged across biological replicates where n≥3. Data were modelled using either MLR, MLR with two-way interaction terms or a BRANNGP, with scatter of each model comparing the actual fit with the prediction from the model with adjusted R² (i). Coefficients for each variable±s.e.m. for standard MLR (ii). *P<0.05.