Fig. 3.

Signalling pathway inhibitors regulate emergent properties during the ESC-to-EPL cell transition. (A) A five-level factorial experimental design showing all combinations of the five signalling pathway inhibitors. 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). The eight combinations containing both L and R, for which cells were non-viable from day 2, are shown with greyed-out letters. (B-E) Colony morphology was scored on day 6 (B), whereas cell number (C), proliferation (D) and apoptosis (E) were recorded at days 2, 4 and 6. Data were averaged across biological replicates where n≥3. To correct for non-normal distributions, morphology and apoptosis data were log transformed, and proliferation data were raised (x⁶). 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). Coefficients for each variable±s.e.m. for MLR with two-way interaction terms (iii). *P<0.05.