Acetylation of C/EBPε K121 and K198 is functionally important during neutrophil differentiation. CD34⁺ cells were differentiated toward neutrophils in the absence or presence of 5 mM NAM. (A) In UCB-derived cells, neutrophil differentiation was determined by cytospin analysis of neutrophil morphology and fluorescence-activated cell sorter analysis of intracellular lactoferrin expression (mean fluorescence intensity [MFI]). (B) In SCN patient BM-derived cells, neutrophil development was determined by cytospin analysis of the percentage mature neutrophils and metamyelocytes. (C) At day 7 of differentiation, intracellular lactoferrin expression (MFI) was analyzed and protein lysates were prepared, followed by western blot analysis of C/EBPε expression. (D) CD34⁺ cells from UCB were retrovirally transduced with eGFP-PMX-C/EBPε, eGFP-PMX-C/EBPε eGFP-PMX-C/EBPε K121/198R, eGFP-PMX-C/EBPε K15xR, and C/EBPε R121/198K and differentiated toward neutrophils. As control, an empty vector was used. Sorted cells were analyzed after 14 days. Neutrophil differentiation was determined by cytospin analysis of morphology and fluorescence-activated cell sorter analysis of lactoferrin expression (MFI). (E) mRNA expression of lactoferrin and collagenase was analyzed by real-time quantitative polymerase chain reaction. Data represent the expression of lactoferrin and collagenase relative to the empty vector. Data are representative of 3 (or more) UCB donors or patient samples. Error bars represent standard error of the mean (between donors). *P < .05; **P < .01.