Fig. 3. Inhibition of PI3K-dependent signalling does not block haemopoiesis in early development but, rather, enhances it. (A) The number of haemopoietic colonies formed by day 6 EB-derived cells differentiated in the absence (EB 0LY/HCA 0LY) or presence of 5 µM LY294002 (EB 5LY/HCA 0LY). Colonies were defined as follows: primitive erythroid (Ery/P), definitive erythroid (Ery/D), myeloid (including macrophage and granulocyte/macrophage colonies), mixed lineage (including erythroid-macrophage colonies and multi-lineage colonies), and secondary EBs (EB). Data are the mean of quadruple counts (± s.e.m.) from two independent experiments, ^*P<0.05; ^**P0.01. (B) Images of day 6 erythroid colonies (left panels) and day 12 haemopoietic colonies (right panels) formed by day 6 WT or PDK1^-/- EB cells [original magnification 100x (left panels) or 20x (right panels)]. Bars, 400 µm. Arrow indicates erythroid colony. (C) FACS analysis of Kit (c-kit) and Flk-1 expression of cells derived from EBs formed in the absence (0 µM LY) or presence (5 µM LY) of 5 µM LY294002 or formed from PDK1^-/- ES cells. The days of differentiation are indicated. Numbers in each quadrant represent the percent of the total live cells in each sample. Similar trends were observed in three independent experiments.