Fig. 4. Myc^T58AER-dependent self-renewal is independent of cell cycle and apoptotic effects and is reversible by withdrawal of 4OHT. (A) ES cells were plated at 5x10⁵/ml in duplicate wells and scored at 12-hour intervals for 72 hours. (B) Flow cytometry analysis of PI stained cells. Top panel, myc^T58AER cells + LIF (1x10³ units/ml); middle panel, vector alone + LIF (1x10³ units/ml); bottom panel, myc^T58AER + 4OHT (5 nM). The percentage of cells in different cell cycle phases was determined using MultiCycle AV software (Phoenix Flow Systems). (C) Cell viability was determined by counting 1000 cells for Trypan Blue exclusion. (D) Myc^T58AER ES cells were maintained in the presence of 4OHT for 14 days (-LIF) then differentiated as embryoid bodies in either the absence or presence of 4OHT (-LIF). Cells were harvested for each of 7 days (1-7) and analysed for differentiation status as indicated. Upper panel, northern blot analysis: RNA samples probed for Oct4, brachyury, Fgf5, ornithine decarboxylase (Odc) and GAPDH. Lower panel, immunoblot (blot) analysis (blot): 40 µg total protein was resolved by SDS PAGE, transferred to a membrane then probed with anti-Myc and anti-Oct4 antibodies. (E) Colony-forming assays (performed in duplicate) on R1 and D3 ES cells passaged for 30 days in ESC media plus LIF (1x10³ units/ml) or ESC media with 10 nM 4OHT. Cells were then plated at clonal density and after 6 days colony forming units (AP-positive colonies) were scored after growth under the conditions indicated (either maintained in ESC+LIF, or switched to ESC±LIF in the absence of 4OHT). Assays are shown as the mean of duplicate experiments.