Fig. 4. Cytokine requirements for EKLF and GATA1 expression. EBs were differentiated with SR1 and various combinations of cytokines, focusing on VEGF, T3 and erythropoietin (A), or BMP4, SCF and VEGF (B). Total RNA was analyzed by semi-quantitative RT/PCR at day 8 of differentiation. For comparison, samples from EBs differentiated in serum (FBS) or in SR1 and all five cytokines were also analyzed. ‘Water’ indicates a no-RNA negative control. (C) EKLF and GATA1 gene expression in embryoid bodies differentiated for 8 days with in SR1 and the indicated cytokines (top), and quantitation/average of three experiments (bottom) after normalization to HPRT levels from the same samples. A previously determined, optimal concentration of BMP4 was used for these analyses. Signal with FBS was given an arbitrary level of ‘1’ (lane 8). ‘Water’ indicates a no-RNA negative control (lane 9).