Eosinophil differentiation is inhibited by Id1. (A) After a 17-day period of differentiation, protein lysates were prepared from eosinophil progenitors transduced with eGFP or Id1 and a Western blot was performed with an N-terminal antibody against Id1 (top panel) and, as a control for equal loading, an antibody against β-actin (bottom panel). CD34⁺ cells were transduced with eGFP or Id1 and cultured in the presence of IL-5 for 17 days. (B, left) The percentages of eGFP-positive cells were determined by FACS analysis after 7, 10, 14, and 17 days of differentiation. ♦ indicates eGFP; ▦, Id1. Results are expressed as an average of 3 independent experiments plus or minus SEM. (Right) The difference between the percentage of eGFP-positive cells after 7 and 17 days of differentiation is depicted. ▪ indicates eGFP; ▦, Id1. (C) Proliferation was determined by counting the eGFP-positive cells. ♦ indicates eGFP; ▦, Id1. Results are expressed as fold increase in cell numbers compared with day 7 as an average of 3 independent experiments plus or minus SEM. (D) Proliferation was determined by performing ³H-thymidine incorporation assays. Data were depicted as a ratio between cells transduced with empty vector alone and cells transduced with Id1. ▪ indicates eGFP; ▦, Id1. Data were expressed as an average of 3 independent experiments plus or minus SEM. (E) The percentage of apoptotic, transduced cells was determined with annexin V–PE by FACS analysis after 7, 10, 14, and 17 days of differentiation. Bar shading is as in panel D. Data were expressed as an average of 3 independent experiments plus or minus SEM. (F) CD34⁺ cells were transduced with eGFP or Id1. After 14 and 17 days of differentiation, transduced cells were separated from the nontransduced cells, and cytospins were made. Cytospins were stained with May-Grünwald-Giemsa solution. (G) Eosinophil differentiation was expressed as a percentage of differentiated eosinophils within the eosinophil lineage. Data were expressed as an average of 7 independent experiments plus or minus SEM. (H) Eosinophil differentiation was also analyzed by staining the granule protein eosinophil peroxidase after 17 days of culture. Data were depicted as the ratio of the levels (arithmetric mean) of eosinophil peroxidase between cells transduced with empty vector alone or cells transduced with Id1. Data were expressed as an average of 3 independent experiments plus or minus SEM. (G-H) Bar shading is as in panel D.