Fig2. H2AK119ub plays a critical role in immunosuppression during secondary infections in sepsis.

(A) Stimulation methods of the LPS-induced secondary infection cell model were applied toPMsderived from WT mice. Western blotting (WB) analysis was used to detect H3K27ac, H3K27me3, H3K9me3, H3K79me3, H3K36me3, and H2AK119ub levels. (B) WB analysis of H2AK119ub levels in peripheral blood PBMCs from healthy individuals and sepsis survivors. (C)PMsderived from WT mice were treated with different concentrations of the histone H2A ubiquitination inhibitor PRT4165 for 4 hours, followed by LPS stimulation for 4 hours. WB analysis was used to detect H2AK119ub and IL-1β levels. (D, E) Before the establishment of the LPS-induced secondary infection model inPMsfrom WT mice,PMswere treated with 10 μM PRT4165 for 4 hours. IL-6 and IL-1β mRNA expression levels were measured by RT-PCR, and the level of IL-6 protein in the cell supernatant was measured by ELISA. (F) Schematic representation of intraperitoneal injection of PRT4165 in WT mice. The experimental group (n=3) received intraperitoneal injections of PRT4165 (20 mg/kg) for 3 consecutive days, followed by intraperitoneal injection of an E. coli suspension (3 × 10⁸ cfu/mL, 1 mL/mouse) dissolved in PBS. The control group (n=3) received intraperitoneal injection of the E. coli suspension only. Peritoneal lavage fluid was collected 4 hours later. Fig 2F: Citation to Use: Created in BioRender. An, Q. (2026) https://BioRender.com/ss4krtx. (G, H) The collected peritoneal lavage fluid was centrifuged, and the cell pellet was analyzed for IL-1β and IL-6 mRNA expression levels using RT-PCR. The supernatant was analyzed for IL-1β and IL-6 protein levels using ELISA. All experiments were repeated at least 3 times with similar results. The data are expressed as means ± SDs. Statistical analysis was performed using two-sided Student’s t-test (B, G, H) and one-way ANOVA (A, D, E). Data were considered statistically significant when *p ≤ 0.05, **p ≤ 0.01,***p ≤ 0.001, and ****p ≤ 0.0001.https://doi.org/10.6084/m9.figshare.30581426.