Fig 2: Effects of CBP suppression on HMGB1-mediated inflammatory response. THP-1 cells (A) and primary MPM cells (B) were pretreated with SGC-CBP30 (4 µM) for 2 h and then stimulated with rhHMGB1 (500 ng/ml) for 12 h; the production of TNF-a, IL-1ß, and IL-6 were measured by ELISA. (C) THP-1 cells and primary MPM cells were pretreated with or without SGC-CBP30 (4 µM) for 2 h followed by stimulation with rhHMGB1 (100 ng/ml) for 1 h. Cell lysates were prepared and the levels of phospho-MAPKs, phospho-IKKa/ß and phospho-I?Ba were determined by Western blot analysis. (D) THP-1 cells were transfected with CBP shRNA or negative control shRNA, and after 72 h, stimulated with rhHMGB1 (100 ng/ml) for 1 h. The cell lysates were subjected to immunoblotting analysis using indicated antibodies. Data shown were representative of three independent experiments. Error bars indicate mean ± SD. **p < 0.01; ***p < 0.001.

Fig 3: Schematic illustration of CBP bromodomain inhibitor prevents sepsis-related HMGB1 secretion and blocks HMGB1 pro-inflammatory activity. The potent and selective CBP inhibitor, SGC-CBP30, significantly rescues mice from LPS-/CLP-induced sepsis model. In addition, SGC-CBP30 suppresses LPS-induced HMGB1 expression and cytoplasmic translocation. Furthermore, by triggering the reactivation of PP2A and the stabilization of MKP-1, CBP bromodomain inhibition prevents rhHMGB1-stimulated activation of MAPKs and NF-?B pathways and production of proinflammatory cytokines.

Fig 4: Inhibitory effect of SGC-CBP30 on HMGB1 release and expression in LPS-stimulated cells. THP-1 cells (A) and primary MPM cells (B) were treated with SGC-CBP30 (4 µM) at 8 h after stimulation with LPS (500 ng/ml). After LPS stimulation for the indicated time, HMGB1 release was measured by ELISA. THP-1 cells were treated with LPS (500 ng/ml) for 8 h and then incubated with SGC-CBP30 (4 µM). After the indicated time of LPS stimulation, nuclear and cytoplasmic fractions were analyzed by Western blot using anti-HMGB1 antibody (C). Cells were incubated with mouse anti-HMGB1 antibody and then incubated with Alexa Flour 555-conjugated anti-mouse (red) secondary antibody. The nuclei were counterstained with DAPI (blue). The location of HMGB1 was observed under a confocal laser microscope. Scale bar: 10 µm (D). The cell lysates were immunoprecipitated with anti-HMGB1 antibody, followed by immunoblotting with anti-acetyl lysine and anti-HMGB1 antibodies (E). Quantification of HMGB1 transcripts by real-time PCR with GAPDH as the internal control (F). Whole cell lysates were subjected to immunoblotting with anti-HMGB1 and anti-ß-actin antibodies (G). Data shown were representative of three independent experiments. Error bars indicate mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 5: Kakonein inhibits NLRP3 inflammasome activation of cardiac vascular endothelium in hyperglycaemic mice. (A) Fluorescence indicating the effect of kakonein or metformin hydrochloride on NLRP3 (green) with caspase-1 (red) co-localization in cardiac endothelial cells. (B) Quantitative analysis of the co-localization of NLRP3 with caspase-1 (n = 8). (C) Fluorescence indicating the effect of kakonein or metformin hydrochloride on HMGB1 (green) with VWF (red) co-localization. (D) Quantitative analysis of the co-localization of HMGB1 with vWF (n = 8). (E) IL-1ß content in serum was detected by ELISA kit (n = 8). ## P < .01 compared with the control. **P < .01 compared with hyperglycaemia