Figure 3

(A) Result of peak detection analysis of FRET/CFP data in maintenance (+ LIF) condition added excess amount of (500 ng/ml) recombinant FGF4 and 1 μg/ml heparin at the time point between T = 40 and 41 (120–123 min) (N = 34, from more than 3 colonies). (B) Before-after graph for ERK activity in each active cell before and after addition of FGF4 in + LIF condition. Vertical axis indicates number of pulses/hour. Note that only results of active cells (showing more than one peak during 240 min live-imaging, 23 of 34 cells) are plotted. Significant differences between before and after were determined by t-test. p value of paired t-test (P = 0.003) is shown in the graph. (C) Comparison of frequency of ERK pulse under + LIF (N = 89, from 8 colonies) and -LIF (N = 37, from 11 colonies) conditions. Data of the first 120 min of active cells in both conditions were used for analysis. Vertical axis indicates number of pulses/hour. Means of pulses/hour in + LIF and -LIF conditions are 0.69 and 0.81, respectively. Result of t-test (P = 0.2804) is presented in the graph. (D) Result of peak detection analysis of FRET/CFP data in differentiating condition (1 day after withdrawal of LIF) added FGF4 and heparin at the time point between T = 40 and 41 (120–123 min) (N = 19, from more than 3 colonies). (E) Before-after graph for ERK activity in each active cell before and after addition of FGF4 in -LIF condition. Only results of active cells (13 of 19 cells) are plotted. Result of paired t-test (P = 0.171) for before and after addition of FGF4 is shown in the graph. (F) Swarm plot showing frequency of ERK pulse of individual active cell before (green dots) and after (red dots) addition of FGF4 in + LIF (N = 23) and -LIF (N = 13) conditions. Vertical axis indicates number of pulses/hour. (G) An example for analysis of Gata6 + PrE cells differentiated from a Gata6-EGFP reporter ESC line in the presence of excess amount of (500 ng/ml) FGF4 and heparin in -LIF condition. PI, Propidium Iodide. (H) Result of FACS analysis of Gata6 + PrE cells differentiated from a Gata6-EGFP reporter ESCs. Means and standard errors from 3 experiments are shown. One-way ANOVA with Dunnett's multiple comparisons test was performed to verify statistical significant differences. MEKi, MEK inhibitor PD0325901 (1 μM). Significance levels of statistical tests are indicated as *P < 0.05; **P < 0.01; ***P < 0.001. ns indicates not significant (P > 0.05).