Fig. 3. Erk1/2 inhibition reversibly blocks ES cell differentiation at the primitive-ectoderm stage. (A) Real-time RT-PCR for Fgf5 and Nanog was performed at day 2 of differentiation. Expression of Sox1 was determined for the same conditions at day 4. Results are from duplicate determinations of three experiments+s.e.m. relative to the expression level in ES cells (Fgf5, Nanog) or day 4 of differentiation (Sox1). (B) Flow cytometry profile of 46C ES cells plated in N2B27 plus 3 µM PD184352 for 2 days before media was changed to either N2B27 (-PD184352; green line) or N2B27 containing fresh inhibitor (+PD184352; black line) for 24 hours. (C) Images of 46C cells at day 3 of differentiation under the conditions shown. Scale bar: 200 µm.