Figure 3: Spontaneous differentiation of wildtype and Fubp1 knockout ESC clones upon aggregation in EBs. (a) mRNA levels of the stem cell markers Oct4 and Nanog decreased equally in Fubp1 knockout (KO) and NTC control clones during spontaneous differentiation in EBs. (b) mRNA expression of ectoderm markers (Nestin and GATA4) slightly increased during EB formation (day 3 and day 5), but the expression was not affected by the absence of FUBP1. The same was observed for endoderm marker expression (Sox17 and β-catenin) at days 3 and 5. (c) The analysis of the mesoderm markers Brachyury, Flk-1, SnaiI, FGFR1, and Bmp4 revealed significantly reduced mRNA expression levels in Fubp1 KO clones at day 3 of ESC differentiation. In addition, the Brachyury target gene Foxa2 was significantly reduced in the Fubp1 KO compared to NTC control clones at day 3 and the Brachyury target gene Snai2 at day 5 of EB differentiation. Three independent experiments were performed, each with 4 NTC and 5 Fubp1 knockout ESC clones. The qPCR data represent the mean values ± SD; data was normalized to Gapdh mRNA expression and calculated as relative mRNA expression level ( ; ).