Fig. 6. PI3K-dependent signalling is required for optimal development of erythroid and myeloid lineages. (A) Day 3.75 EB-derived cells generated in the absence of inhibitor were plated into blast colony culture conditions in the absence (EB 0LY/blast 0LY) or presence (EB 0LY/blast 5LY) of 5 µM LY294002. In addition, cells from day 3.75 EBs formed in the presence of 5 µM LY294002 were plated under blast culture conditions without inhibitor (EB 5LY/blast 0 LY). After 4 days, blast-colony-derived cells were harvested and identical numbers of cells for each condition plated into HCAs (in the absence of LY294002). Numbers of each type of haemopoietic colony generated are shown. Data represent the average of triplicate counts (± s.d.) and are representative of three individual experiments. ^***P<0.005 (B) Day 6 EB-derived cells, differentiated in the absence of LY294002, were plated into HCAs in the absence (EB 0LY/HCA 0LY) or presence of 5 µM LY294002 (EB 0LY/HCA 5LY). Data represents the mean (±s.e.m.) from two independent experiments (n=3-5). Examples showing the size differential of Ery/D colonies that developed in the absence or presence of LY294002 are shown in the inserts; original magnification 100x (10x/0.4 objective lens was used). Bars, 200 µm. These results are representative of five individual experiments, ^*P<0.05; ^***P<0.005. (C) Mouse bone marrow cells were plated into HCAs in the absence (0 LY) or presence of 5 or 10 µM LY294002 (5 µM LY or 10 µM LY, respectively). Data represent the mean (± s.e.m.) of three independent experiments. Examples of overall colony formation are depicted above the appropriate bar graph; original magnification 20x (10x/0.4 objective lens). Bars, 1 mm. In addition, colony morphologies are shown in the inserts; original magnification 100x (10x/0.4 objective lens). Bars, 200 µm. ^***P<0.005 for GM colonies compared with 0 LY.