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. 2014 Mar 21;9(3):e92471. doi: 10.1371/journal.pone.0092471

Figure 5. Human and murine C/EBP-ε induces expression of HNP-1 in primary bone marrow (BM) cells from transgenic HNP-1 mice.

Figure 5

Murine BM cells from seven transgenic HNP-1 mice were isolated and early granulocyte precursors isolated by density centrifugation on a discontinuous Percoll 1.072 gradient. Cells were retrovirally transduced with an empty expression vector (pMIG) or with a vector expressing either human or murine C/EBP-ε (pMIG-CEBPE or pMIG-Cebpe respectively). Cells were incubated for 48 hours. (A) Green fluorescent protein (GFP) was used as reporter gene in the vectors and transduction efficiency evaluated by flow cytometry. (B–G) Comparative quantification of mRNA for CCAAT/enhancer binding protein-ε (human CEBPE or murine Cebpe), human neutrophil peptide-1 (DEFA1), cathelicidin antimicrobial peptide (Camp), and lipocalin-2 (Lcn2) was done by real-time PCR using Gapdh as normalizer. Error bars depict standard deviation. (B, E–G) Levels are shown as fold induction by either murine Cebpe (mCebpe) or human CEBPE (hCEBPE) compared to levels from negative control transduction (pMIG). (C) Relative quantification of human CEBPE in murine bone marrow cells from four transgenic HNP-1 mice transduced with control vector (pMIG) or human CEBPE. (D) Expression of murine Cebpe in Cebpe transduced cells were compared to human CEBPE in CEBPE transduced cells by comparing Delta Ct between the transduced gene and Gapdh. The transduced mouse with the lowest expression of C/EBP-ε was used as calibrator. (H) Western blotting of C/EBP-ε, 24p3, and beta-actin in transduced cells from two mice. (I–J) Cells were fixed in formaldehyde. Cell and nuclear membranes were lysed before fragmentation of DNA by sonication. Chromatin was immunoprecipitated using protein A/G magnetic beads and an antibody against C/EBP-ε, C/EBP-α, or negative control rabbit IgG. After washing procedures, immune complexes were eluted and reversed and DNA recovered. DNA was used as a template for quantitative PCR. Primers used were specific for putative C/EBP sites in the DEFA1 promoter and promoters of the specific granule protein cathelin-related antimicrobial peptide (Camp). Levels are depicted as fold enrichment compared to negative control IgG immunoprecipitation.