Activating Smad1 signaling after EryP-CFC specification has no effect on EryP numbers, but a timed pulse of Smad1 expands EryP colony formation. (A) The primitive EryP colony-forming potential of Ainv18, iSmad1 mixed, and iSmad1 sc5 EBs were examined. EBs were induced on day 4 and replated at day 6 in the presence of EPO (2 U/mL). EryP colonies were scored 5 days later. Ainv18 is the parental ES cell line and control. For each sample, n = 3. (B) Data from a single representative experiment analyzing the EryP colony-forming potential of iSmad1iresEGFP sc5 EBs examined after continuous induction initiated at different times. Developing iSmad1 EBs were untreated or treated with doxycycline on the indicated days and Smad1 expression was maintained as previously described (Figure 3C-F) until day 5.75 when the EBs were harvested and replated with EPO (2 U/mL). EryP colonies were scored 5 days after plating. Shown is the fold change compared to the untreated (no dox) control. The modest increases seen here with induction at day 2 or day 3 were not statistically significant in multiple experiments. (C) The iSmad1 EBs were untreated (no dox) or treated with the doxycycline by the washout (wo) procedure on the day indicated. EBs were harvested and replated with EPO (2 U/mL) on day 5.75. The data shown are from a single representative experiment. Shown is the fold change compared to the untreated control. (D) Statistical analysis of EryP colony formation from day 5.75 iSmad1 EBs comparing untreated and samples induced at day 2 to 2.25 with doxycycline. For each sample, n = 4. Error bars represent the SEM and the asterisk indicates P < .01 compared to the no dox samples.