Constitutive Id1 expression enhances neutrophil differentiation. CD34⁺ cells were transduced with eGFP or Id1 and cultured in the presence of G-CSF for 17 days. (A, 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. (B) 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. (C) Proliferation was determined by performing ³H-thymidine incorporation assays. Data are depicted as a ratio between cells transduced with empty vector alone or cells transduced with Id1. ▪ indicates eGFP; ▦, Id1. Data are expressed as an average of 3 independent experiments plus or minus SEM. (D) 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 C. Data are expressed as an average of 3 independent experiments plus or minus SEM. (E) CD34⁺ cells were transduced with eGFP or Id1. After 14 and 17 days of neutrophil differentiation, transduced cells were separated from the nontransduced cells, and cytospins were prepared and stained with May-Grünwald-Giemsa solution. (F) Neutrophil differentiation was expressed as a percentage of differentiated neutrophils within the neutrophil lineage. Data were expressed as an average of 3 independent experiments plus or minus SEM. (G) Neutrophil differentiation was also analyzed by staining the granule protein lactoferrin after 17 days of culture. Data were depicted as the ratio of the levels (arithmetric mean) of lactoferrin 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. (H) CD34⁺ cells, cultured in the presence of the cytokines SCF, FLT-3 ligand, and TPO, were transduced with empty vector alone or with Id1. After 3 days of culture, cells were injected into β2-microglobulin^-/- NOD/SCID mice. Six weeks after injection, mice were killed, eGFP-positive human myeloid cells were sorted, and cytospins were made. Cytospins were stained with May-Grünwald-Giemsa solution. (F-H) Bar shading is as in panel C.