Fig 1: Inhibition of KDM6A attenuates PU.1 binding and Hotairm1 transcription in MDSCs from septic patients. PBMCs were first purified and depleted of the HLA-DR+ cells using biotin-coupled anti-HLA-DR antibody and anti-biotin microbeads, followed by the positive selection of CD33+LOX1+ cells with biotin-coupled anti-CD33 and anti-LOX1 antibodies. These CD33+LOX1+HLA-DR- cells were incubated with 4 µM of GSK-J4 or 0.1% DMSO for 12 h. (A–D) Chromatin was isolated and immunoprecipitated with anti-KDM6A, anti-H3K27me3, anti-H3K4me3, anti-PU.1, or anti-IgG control antibody. The ChIPed DNA was analyzed by real-time qPCR. Samples values were normalized to the “input” DNA and are presented relative to the IgG-immunoprecipitated samples (1-fold). (E) Total RNA was isolated and Hotairm1 levels were determined by real-time RT-qPCR. Sample values were normalized to GAPDH RNA as an internal control and are presented relative to HS (1-fold). Data represent mean ± SD for 3-4 patients or HS per group. *p < 0.05, early or later sepsis vs. HS; **p < 0.05, GSK-J4 vs. DMSO. (F) Western blotting of KDM6A. The results are representative of two Western blots. HS, healthy subjects.