ALK-5 gain-of-function leads to defective hematopoiesis. iALK-5 ES cells were differentiated as EBs in the presence or absence of Dox, and assayed for hematopoietic colonies. (A) Representative images of noninduced and induced ALK-5 cultures. (B-C) Frequency of primitive erythroid (B) and definitive CFCs (C) at days 4.25 and 6.25 of EB differentiation, respectively. Error bars indicate SE of 3 independent experiments performed in duplicate. Gene expression analyses for embryonic globin (D) and β major globin (E) at days 4.25 and 6.25 of EB differentiation. Transcripts are normalized to GAPDH. Bars indicate SE from 3 independent experiments performed in duplicate. (F-G) Relative levels of Flk-1, Gata1, Gata2, Lmo2, and SCL in 4.25 and 6.25 EBs, respectively. Transcripts are normalized to GAPDH. (H) A representative FACS profile for CD41 and c-Kit in induced and noninduced iALK-5 cells at EB day 6.25. Fluorescence intensity for c-Kit is indicated on the y axis and CD41 on the x axis. *P < .05, **P < .01. (I) A representative FACS profile for BrdU incorporation in induced and noninduced iALK-5 cells at EB day 6.25. iALK-5 day 6.25 EBs were labeled with 10μM BrdU and then stained with c-Kit–APC and CD41-PE. The incorporation of BrdU was visualized using FITC conjugated anti-BrdU antibody.