Disturbance of erythrocyte maturation by forced expression of c-Myb. (A) VE-cadherin⁺CD31⁺cells were sorted from differentiating wild-type ES cells and c-mybTet/KO ES cells as described in Figure 2. Cells were recultured for 9 days with OP9 cells and cytokines (SCF, IL3, Epo) in the presence of different concentrations of Tet. Cells were harvested and analyzed by flow cytometry for expression of Ter119 and CD71. Numbers indicate the percentage of cells in rectangular area. (B) CD71^highTer119^– and CD71^highTer119⁺ cells were sorted from wild-type cell cultures and subjected to RT-PCR analysis for c-myb expression (left panels). CD71^highTer119⁺ cells were sorted from wild-type and c-Myb–rescued (0 ng/mL Tet) cultures and subjected to RT-PCR analyses for expression of c-myb, Gata1, and Gata2 genes (right panels). Results shown are representative of at least 3 independent experiments. (C) VE-cadherin⁺CD31⁺ cells sorted from differentiating wild-type ES cells and c-mybTet/KO ES cells were recultured for 5 days with OP9 cells and cytokines in the absence of Tet. Tet (1 μg/mL) was then added in some cultures (Tet–> Tet+) and incubation continued, whereas others were kept in the absence of Tet (WT and Tet– > Tet–). After 3 days of incubation, cells were harvested and analyzed by FACS for expression of Ter119 and CD71. CD71^highTer119⁺ cells were further sorted and subjected to RT-PCR analyses for expression of c-myb and Gata2 genes. Numbers indicate the percentage of cells in rectangular area.