The survival or development of Pen^−/− erythroid progenitors is rescued by pen. (A) An outline of the experimental design. (B) Effect of pen on the survival of EPO-responsive cells at different concentrations of EPO. A total of 5 × 10⁵ EMX/EGFP or EMX/pen cells were stimulated with SCF+TPO+IL3 plus EPO (4 U/mL) for 8 days. Cell numbers after 8 days were 1.27 × 10⁷ for EMX/GFP and 1.81 × 10⁷ for EMX/pen. A total of 3 × 10⁵ EMX/GFP or EMX/Pen cells that had been stimulated with SCF+TPO+IL-3 plus EPO for 8 days were washed with PBS and restimulated with EPO alone (0 to 32 U/mL) for 2 days. Most surviving cells were erythroblasts. Some were undifferentiated blasts. Cells rescued with EPO alone were counted. Data represent the means of triplicates. For reference, 6.1% of EMX/Pen cells were rescued by EPO at 4 U/mL. (C) Effect of Pen on the numbers of BFU-Es rescued by EPO. A total of 3 × 10⁵ EMX/EGFP or EMX/Pen calls that had been stimulated with SCF+TPO+IL3 plus EPO (4 U/mL) for 8 days were washed with PBS and restimulated with EPO alone (4 U/mL) for 2 days and then plated in methylcellulose culture medium supplemented with SCF, IL-3, and EPO (4 U/mL). Each 35 mm dish contained the equivalent of 2 × 10⁴ starting cells (or 1224 EPO-rescued cells in the case of EMX/pen). The graph shows the numbers of erythroid progenitors per 3 × 10⁵ starting cells. Data represent means ± SD; n = 3. For reference, the frequencies of large, medium, and small BFU-Es among EPO-rescued cells were 1.0%, 5.0%, and 7.7%, respectively. (D) A schematic summary of the differentiation of Pen^−/− EMX C.1. The TER119 MAb recognizes erythroblasts,³⁷  which exhibit the highest level of Pen expression in BM. The survival or development of EMX-derived BFU-Es, CFU-Es/proerythroblasts, and erythroblasts is rescued by pen in the presence of EPO.