Figure 1: Lack of S100A9 expression prevents assembly of the pStat3-C/EBP β protein complex at the miR-21 promoter in Gr1⁺CD11b⁺ cells in late sepsis.

Detection of transcription factor binding at the miRNA promoters. Gr1⁺CD11b⁺ cells were isolated from the bone marrow of sham and septic mice (n = 3–4 mice per group). Formaldehyde-fixed cells were lysed, and the pelleted nuclei were digested with chromatin shearing enzymatic cocktail. The chromatin was immunoprecipitated with antibodies specific to pStat3 (Tyr⁷⁰⁵), C/EBP β, or IgG isotype control antibody. Next, chromatin cross-links were reversed to recover the protein-bound DNA. The purified DNA was amplified by quantitative real-time PCR to measure the level of enrichment of miR-21 and miR-181b promoter DNAs in chromatin, using promoter-specific primer/probe sets. PCR reactions were performed in triplicate. Samples values were normalized to the “input” DNA (DNA isolated before immunoprecipitation) and are presented as fold enrichment relative to the IgG-immunoprecipitated samples (set at 1-fold). Data are mean ± s.d. (n = 3–4 mice per group). *p < 0.05 vs. sham; **p < 0.05 vs. early sepsis. The results are representative of three independent experiments. KO, knockout.