FIG 4.
Increased histone deacetylation of PU.1 gene in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, the AMs were treated with 0.1% formaldehyde to cross-link histone proteins to DNA, lysed, and sonicated to generated chromatin fragments. Chromatin immunoprecipitation was performed using anti-H3K4me3, anti-H3ac, and anti-H3K27me3 antibodies in separate reactions. DNA in the precipitated chromatin was isolated and used as the template for real-time PCR to amplify the 3′URE, 5′URE, and promoter regions of the PU.1 gene. The CT values obtained were used to determine the ratios of percent input of H3K4me3 to H3K27me3 and H3Ac to H3K27me3. Data are presented as means ± SD from three independent experiments.