Quantitative image analysis of Bra, Nanog and Oct4 or Sox2 following treatment with mesendodermal-inducing factors. (A,B) Confocal images of WT mESCs following 48 h Act/Chi treatment stained for brachyury (Bra; green) and Nanog (red) with either Oct4 (A) or Sox2 (B, yellow). Merged images are shown with the corresponding magnified regions denoted by a white box (i). (C-F) Time evolution of the distributions of the expression of brachyury (C), Nanog (D), Oct4 (E) and Sox2 (F) during differentiation. WT E14-Tg2A mESCs treated with Act, Chi or Act/Chi for 24, 48, 72 and 96 h were stained as described (A,B). The nuclei were segmented based on Hoechst staining and the average pixel intensity for each fluorescent channel was quantified. The intensities for brachyury (C), Nanog (D), Oct4 (E) and Sox2 (F) are displayed as histograms for each time point. The bisecting orange lines in each histogram correspond to the mean fluorescence levels. (G) Pearson correlation coefficient for the correlations between Bra and Nanog (top left), Bra and Oct4 (bottom left) and Bra and Sox2 (top right) for the different time points. The horizontal line represents the correlation for LB. Scale bar represents 100 μm. Hoechst stain is used for the nuclei. AC, activin A + chiron; Act, activin A; AFU, arbitrary fluorescence units; Bra, brachyury; Chi, chiron CHIR99021; LB, leukaemia inhibitory factor and bone morphogenetic factor; mESC, mouse embryonic stem cell; WT, wild type.