Id2 knock-down in granulocyte progenitors inhibits eosinophil and neutrophil differentiation. (A) Protein lysates were prepared from COS cells cotransfected with pSG5-Id2 and pRetrosuper Renilla RNAi or pRetrosuper Id2 RNAi. 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 Renilla RNAi or Id2 RNAi and cultured in the presence of IL-5 or G-CSF for 17 days. Proliferation during eosinophil (B) and neutrophil (C) differentiation 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. CD34⁺ cells were transduced with eGFP or Id2. After 14 and 17 days of eosinophil (D) and neutrophil (E) differentiation, transduced cells were separated from the nontransduced cells, and cytospins were made. Cytospins were stained with May-Grünwald-Giemsa solution. Data are expressed as an average of 6 independent experiments plus or minus SEM.