Inhibition of PKB with a pharmacological inhibitor inhibits neutrophil differentiation. (A) CD34⁺ cells were cultured in presence of G-CSF to induce neutrophil differentiation during 17 days. Cells were cultured either in absence or presence of 10 μM LY294002 or 20 μM 1L-6-Hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO), a PKB inhibitor. Expansion was determined by counting the trypane blue–negative cell population. (B) During the 17-day culture period the percentage of apoptotic cells was determined by annexin V staining. (C) After 17 days of neutrophil differentiation, cytospins were made and stained with May-Grunwald Giemsa solution. (D) Data were expressed as both the percentage of differentiated neutrophils (E) and as absolute numbers. (F) Lactoferrin, (G) CD14, and (H) CD49d expression was analyzed by FACS to determine neutrophil, monocyte, and eosinophil/monocyte development, respectively. Error bars represent SEM.