Fig 1: FeTPPS selectively inhibits recombinant HMGB1-mediated caspase-11 activity in vitro.A, B Production of IL-1a, IL-1ß, TNFa, and IL-6 (as measured by ELISA) of WT or Casp11-/- peritoneal macrophages stimulated with LPS alone (1 µg/mL) or LPS (1 µg/mL) + HMGB1 (400 ng/mL) in the absence or presence of indicated doses of FeTPPS for 16 h. Data presented as mean ± SD of technical replicates. ****P < 0.0001 (unpaired t-test). C Western blots for IL-1a, caspase-11, and caspase-1 in the supernatants or GSDMD-NT, caspase-11, IL-1a, IL-1ß, caspase-1 in the cell lysates from WT or Casp11-/- peritoneal macrophages stimulated with LPS alone (1 µg/mL) or LPS (1 µg/mL) + HMGB1 (400 ng/mL) in the absence or presence of indicated doses of FeTPPS for 16 h. D Necrotic cell lysate preparation is shown in the left panel. Western blots for HMGB1, IL-1a, caspase-11, and caspase-1 in the supernatants or GSDMD-NT, Caspase-11, IL-1a, IL-1ß, and caspase-1 in the cell lysates from WT or Casp11-/- peritoneal macrophages stimulated with LPS alone (1 µg/mL), LPS (1 µg/mL) + HMGB1+/+, or HMGB1-/- MEF cells in the absence or presence of FeTPPS for 16 h. E Primary hepatocytes isolated from mice of indicated genotypes co-cultured with WT mouse peritoneal macrophages are shown in the left panel. IL-1a and IL-1ß released from macrophages after the stimulation of LPS (1 µg/mL) in the absence or presence of FeTPPS are shown in the right panel. Data presented as mean ± SD of technical replicates. Two-way ANOVA was used (IL-1a hmgb1f/f Albcre- LPS vs. LPS + FeTPPS **P = 0.004; hmgb1f/f Albcre- + LPS vs. hmgb1f/f Albcre+ + LPS ****P < 0.0001; IL-1ß hmgb1f/f Albcre- LPS vs. LPS + FeTPPS **P = 0.0025; hmgb1f/f Albcre- + LPS vs. hmgb1f/f Albcre+ + LPS ****P < 0.0001). F Production of IL-1ß (as measured by ELISA), Western blots of supernatants and cell lysates to detected IL-1ß, Caspase-1 cleavage from WT or NLRP3-/- peritoneal macrophages primed with LPS 100 ng/mL and indicated stimulation. G Western blots for IL-1a, caspase-4 (Casp4), GSDMD-NT in the supernatants or cell lysates. LDH assay and ELISA for IL-1a in the supernatants of PMA-primed human monocytic THP-1 cells transfected with scrambled siRNA or CASP4-specific siRNA upon HMGB1 (400 ng/mL) and LPS (1 µg/mL) stimulation in the absence or presence of FeTPPS. Data presented as mean ± SD of technical replicates. An unpaired t-test (two-sided) was used (LDH LH vs. LH + FeTPPS 2.5 µM **P = 0.0085; LH vs. LH + FeTPPS 5 µM **P = 0.0071; LH scramble vs. LH casp4 siRNA **P = 0.0043; IL-1a LH vs. LH + FeTPPS 2.5 µM *P = 0.0328; LH vs. LH + FeTPPS 5 µM *P = 0.0198; LH scramble vs. LH casp4 siRNA ***P = 0.004). Graphs are representative of at least three independent experiments.