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Fig. 5. PI3K-dependent signalling is required for formation of blast colonies. (A) EBs were formed in the presence of 0 or 5 µM LY294002 (LY). EB cells were harvested on day 3, 3.75 or 4 as indicated and identical cell numbers (5x10⁴/ml) plated under blast culture conditions. Examples of blasts cultured for 4 days; original magnification 100x (10x/0.4 objective lens was used). Bars, 200 µm. (B) Following 4 days in culture, the blast colonies were counted. Data represent the average of triplicate counts (± s.d.) and are representative of four independent experiments. (C) EBs were formed from either untreated wild-type or PDK1^-/- ES cells and cells were harvested following 3.75 days in culture. Identical cell numbers (3x10⁴/ml) were then plated under blast culture conditions. Cells from EBs generated from the wild-type ES cells were plated either in the absence (0 LY) or the presence of 5 µM LY294002. Examples of blasts cultured for 4 days; original magnification 100x (10x/0.4 objective lens was used). Bars, 200 µm. (D) Following 4 days in culture the blast colonies were dissociated and the total number of cells counted. Data represent mean (± s.e.m.) from three individual experiments. ^**P0.01 (E and F) RT PCR analyses of 2° blasts. Wild-type or PDK1^-/- day 3.75 EB-derived cells were plated under blast culture conditions for 2-4 days. Cells from EBs generated from untreated wild-type ES cells were plated in the absence (0 LY) or presence of 5 µM LY294002 (5 µM LY). RNA was isolated from the initial day 3.75 EB cells (EB) and from the resulting blasts (days of differentiation are indicated) and used for RT-PCR analysis of the marker genes depicted. Note that the EB sample for Scl in E appears in the second lane.

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