Fig. 5. Myc performs a role in self-renewal by blocking differentiation. (A) Myc^40-178ER D3 cells were grown in the presence of LIF and in the absence or presence of 4OHT (25 nM) for up to 6 days. Colonies were then stained for AP activity and photographed at 20x magnification. (B) At corresponding sampling times (see A) the percentage of alkaline phosphatase positive colonies was determined. Data are the average of assays performed in triplicate and expressed as standard error of the mean. (C) The proportion of stem cells decreases following inactivation of Myc. Myc^40-178ER D3 ES cells were grown for 6 days under the conditions indicated. Five-hundred cells were then plated at clonal density in ESC+LIF (1x10³ units/ml) and colony forming units (AP-positive colony number) scored after a further 4 days growth (-4OHT). Data are shown of an experiment performed in duplicate. (D) mRNA levels for Oct4, Nanog, brachyury, GATA4 and ß-actin were evaluated by RT-PCR analysis in D3 myc^40-178ER cells before and after addition of 4OHT.