Interaction of C/EBP-ϵ with p65RelA is important for neutrophil differentiation. (A) Northern-blot analysis of secondary granule B9 and NGal gene expression in murine 32Dcl3–derived cells. Twenty micrograms of total RNA derived from 32Dcl3 cells transduced with retroviral vectors expressing the wild-type (T75), mutant (D75 and A75) C/EBP-ϵ, or an empty vector control was analyzed with probes for the secondary granule B9 and NGal genes. Cells were transiently transduced with lentivirus targeting RelA (lanes 3, 5, 7, 9) or nonspecific siRNA (lanes 2, 4, 6, 8). Lane 1 contained 20μg total RNA derived from the wild-type 32Dcl3 cells that were terminally differentiated for 6 days in the presence of 100 ng/mL G-CSF. (B) Neutrophilic differentiation of 32Dcl3 cells transduced with C/EBP-ϵ expression vectors in the presence or absence of p65RelA. Maturation of 32Dcl3 cells transduced with the wild-type C/EBP-ϵ (T75), D75 and A75 mutants, or control vector was observed in the presence or absence or p65RelA. Following infection with the lentivirus expressing RelA-specific siRNA, cells were cultured for 5 days in the absence of IL-3. Graphs represent the percentage of band-neutrophil–like differentiated cells. Only living cells were counted. Graphs show the average and SD of 3 transduction experiments. (C) Schematic representation of RelA activation pathway leading to increased affinity of C/EBP-ϵ to its cognate DNA binding sites. The basic region of C/EBP-ϵ subunits involved in DNA recognition is indicated as B. TA denotes transcriptional activation domain, whereas the leucine zipper dimerization domain is indicated as LLLL. Phosphorylation of the Thr75 site on C/EBP-ϵ results in increased interaction with activated nuclear NFkappaB, which in turn may produce changes in the conformation of C/EBP-ϵ subunits within the dimer to improve DNA recognition.