2006-PO directly inhibits BFU-E growth. (A) Limiting dilution analysis of BFU-E growth from purified CD34⁺ cells in semisolid medium. CD34⁺ cells were cultured in fibrin clots in 96-well flat-bottom plates in the presence of IL-3, SCF, and EPO, with (●) or without (○) 2006-PO. After 14 days, the clots were fixed, stained for hemoglobin, and the number of clots in which BFU-E colonies did not form counted as BFU-E colony-negative wells. These data were plotted against the number of CD34⁺ cells plated into the wells originally. Each point represents the values obtained from 60 wells. The results are representative of 3 independent experiments. (B) Morphology and size distribution of the generated cells. Purified CD34⁺ cells were cultured in erythroid medium, with or without 2006-PO for 7 days and subjected to May-Grünwald-Giemsa staining (×1000). Results are representative of 5 independent experiments. Scale bars represent 10 μm. (C) Top panel: Purified CD34⁺ cells were cultured in erythroid medium. At the indicated days, the cells were harvested and analyzed by fluorocytometry. Results are representative of 3 independent experiments. Bottom panel: CFSE dilution assay. Purified CD34⁺ cells were incubated with CFSE and cultured in erythroid medium. At the indicated days, the cells were harvested and analyzed by fluorocytometry. Results are representative of 2 independent experiments. (D-E) Purified CD34⁺ cells were cultured in erythroid medium with or without 2006-PO for 7 days. Results are representative of 2 independent experiments. (D) Cell-cycle analysis of the generated cells. (E) The cells were labeled with propidium iodide and annexin V and analyzed using fluorocytometry.