Early or late depletion of Smad1 has opposite effects on primitive erythroid potential. (A) The primitive EryP colony-forming potential was analyzed for EBs derived from a representative miSmad1 ES-cell clone. Smad1 knockdown was induced by 1 μg/mL doxycycline treatment starting either 2 or 4 days after initiation of growth in EB culture medium permissive for differentiation. The EBs were harvested at day 5.75 and recultured in 2 U/mL EPO in semisolid methylcellulose medium. Erythroid colonies were counted 4 days later, and the EryP potential of induced cells was compared with untreated cells. Results are shown as total mean colony number counted in each experimental condition, and each graph is a representative result from a pool of at least 4 independent experiments, each done in triplicate. (B) The EryP colony assays were repeated with the addition of doxycycline to the methylcellulose, to rule out potential confounding effects of doxycycline on cell differentiation.