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Figure 4: Quantification of the mesoderm marker Brachyury and FLK-1 and cell cycle analysis in cells of differentiating EBs. (a) Intracellular staining of single cells prepared from EB structures for the mesoderm marker Brachyury showed a significant reduction in the proportion of Brachyury+Fubp1 KO cells at day 3 of EB differentiation compared to control cells derived from NTC EBs. This reduction was still visible at day 4, but not any longer at day 5 of EB differentiation, when 12% of Fubp1 KO and NTC cells derived from EBs were Brachyury+. (b) During EB differentiation, the percentage of FLK-1+ NTC cells increased from 3% (day 3) to 28% (day 4) and 39% (day 5). In comparison, the proportion of FLK-1+Fubp1 KO cells was clearly decreased. The data represent the mean values ± SD. In each of two independent experiments, 3 (a) or 6 (b) NTC and 3 (a) or 6 (b) Fubp1 knockout ESC clones were used ( ; ). (c) Quantification of Ki-67+ cells in FLK+ and FLK cells in 3 NTC and 5 Fubp1 KO ESC clones by flow cytometry (left panels). FACS analysis was performed after 5 days of EB aggregation and differentiation in medium without LIF. Right panels: 3 NTC and 5 Fubp1 KO ESC clones were used for cell cycle analysis of FLK+ and FLK cells following 5 days of differentiation in EBs. For all experiments, data represent the mean values ± SD.