Figure 2. Expression of C/EBPβ in C/EBPβ-deficient Gr1⁺CD11b⁺ cells from late septic mice reactivates miRNA expression and attenuates cell differentiation.

Bone marrow Gr1⁺CD11b⁺ cells were isolated from control or C/EBPβ conditional knockout mice during late sepsis. Cells were transfected with a C/EBPβ expression plasmid or an empty vector. After 36 hr, portions of the cells were harvested and used for ChIP assay and PCR. (A) Transcription factor binding to the miR-21 promoter. Enrichment of the miR-21 promoter sequences in the ChIP DNA was measurec as described in Fig. 1. Right panel show western blotting of C/EBPβ protein levels after transfection with C/EBPβ. *, compare with IgG. (B) miR-21 expression was determined by real-time PCR as in Fig. 1. (C) The remainder of the cells (2×10⁶) was differentiated for 6 days with M-CSF plus rIL-4 (10 ng/ml/each), and numbers of differentiated cells were determined by flow cytometry. Percentages of differentiated cells (left) and absolute numbers of cells (right) in the culture after 6-day incubation are shown. Data are expressed as mean ± s.d. (*^/#p < 0.05) of 7 mice per group and represent one of two experiments. *, compared with cells from C/EBPβ control mice; ^#, compared with cells from C/EBPβ cKO mice and transfected with vector. cKO, conditional knockout.