Constitutive Id2 expression enhances eosinophil differentiation. (A) After a 7-day period of culture, protein lysates were prepared from eosinophil progenitors transduced with eGFP or Id2 and a Western blot was performed with an N-terminal antibody against Id2 (top panel) and, as a control for equal loading, an antibody against β-actin (bottom panel). CD34⁺ cells were transduced with eGFP or Id2 and cultured in the presence of IL-5 for 17 days. (B) The percentages of eGFP-positive cells were determined by FACS analysis after 7, 10, 14, and 17 days of differentiation. Results are expressed as an average of 3 independent experiments plus or minus SEM. The difference between the percentage of eGFP-positive cells after 17 and 7 days of differentiation was depicted in the right panel. (C) Proliferation was determined by counting the eGFP-positive cells. 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 are depicted as a ratio between cells transduced with empty vector alone and cells transduced with Id1. Data are 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. Data are expressed as an average of 3 independent experiments plus or minus SEM. (F) CD34⁺ cells were transduced with eGFP or Id2. 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 are expressed as an average of 3 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 Id2. Data are expressed as an average of 3 independent experiments plus or minus SEM.