Fig 2: Prdx4 is secreted upon activation of the NLRP3 inflammasome and co‐localizes with caspase‐1 in MVBs Western blot analysis of Prdx4, pro‐caspase‐1, Gapdh, and E‐Cadherin from the cytosolic and insoluble cell fraction of LPS and/or ATP‐stimulated BMDMs or untreated controls.Prdx4 concentration in supernatants of Prdx4 WT or KO BMDMs, primed for 6 h with LPS, and pulsed for indicated time points with 5 mM ATP. Each circle represents a mean of n = 3 mice; vertical lines indicate SD. **P < 0.01; n.s. not significant (two‐tailed t‐test).Concentration of Prdx4 in the serum of WT mice, injected with LPS (4.5 mg/kg BW) for the time points indicated. Each dot represents an individual mouse. Horizontal lines indicate mean. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. not significant (two‐tailed t‐test).Concentration of Prdx4 in supernatants of Prdx4 WT and KO BMDMs. Cells were primed with LPS (100 ng/ml) for 6 h, followed by pretreatment with 20 μM YVAD or DMSO as control for 30 min and stimulated with 5 mM ATP for 4 h or no further stimulation. Each bar represents a mean of n = 3 mice; vertical lines indicate SD. **P < 0.01; n.s. not significant (two‐tailed t‐test).Schematic illustration of selected mechanisms that were targeted by either glycine, necrosulfonamide (NSA), or GW4869 to study LPS+ATP‐induced Prdx4 secretion.Relative levels of Prdx4 secretion in response to LPS+ATP stimulation and pretreatment with either glycine, NSA, or GW4869 and relative levels of caspase‐1 secretion in response to LPS+ATP stimulation and pretreatment with in response to LPS+ATP stimulation and pretreatment with GW4869. Each bar represents a mean of n = 3 biological with two technical replicates; vertical lines indicate SD. *P < 0.05; **P < 0.01; n.s. not significant (two‐tailed t‐test).Western blot analysis of subcellular organelle fractions. OptiPrep density gradient ultracentrifugation was used to fractionate subcellular organelles from Prdx4 WT BMDMs that were primed with LPS (100 ng/ml) for 12 h and stimulated with 5 mM ATP for 4 h.Data information: Data are representative of two (A, B, D, E) or three (G) independent experiments.Source data are available online for this figure.

Fig 3: Oxidation-resistant, decamer-promoting, and decamer-resistant mutants alter PRDX4 HMW species.A, reducing and nonreducing Western blots for PRDX4 in C10 cells expressing PRDX4 mutant constructs treated with vehicle control (C) and 500 μM TBuOOH (T) for 2 min. B, reducing and nonreducing Western blots for PRDX4 in the media from C10 cells expressing PRDX4 mutants. C, reducing and nonreducing Western blots for PRDX4 and ERO1a in C10 cell lysates expressing PRDX4 mutant constructs and either pCMV (C) or ERO1a (E). D, reducing and nonreducing Western blots for PRDX4 in the media from C10 cells expressing PRDX4 mutants and either pCMV (C) or ERO1a (E) for 24 h. Right panels, quantification of the three distinct PRDX4 species in the bracketed regions in the nonreducing Western blots, compared with total PRDX4 detected in these fractions combined. Results are mean plus SEM values derived from multiple pooled experiments. ERO1a, endoplasmic reticulum oxidoreductase alpha; HMW, high molecular weight; PRDX4, peroxiredoxin-4; TBuOOH, tertbutyl hydroperoxide.

Fig 4: Oxidation of PRDX4 alters its binding partners.A, Coomassie blue gel of the pulldown of PRDX4-SBP from C10 cell lysates treated with vehicle control or 500 μM TBuOOH for 2 min. Bands cut out for further analysis by MS are indicated. B, peptide spectral matches for PRDX4 identified in each band compared with total PRDX4 peptide spectral matches identified in each sample. C, MS/MS results showing the proteins found in each band. MS/MS, tandem MS; PRDX4, peroxiredoxin-4; TBuOOH, tertbutyl hydroperoxide.

Fig 5: Role of IL‐1 receptor blockade and myeloid‐specific ablation of Prdx4 in the endotoxin‐shock model Percent body weight of male Prdx4 WT and KO mice over the 48 h course of LPS (4.5 mg/kg BW) injection (i.p.) and treatment with IL‐1 receptor antagonist (IL‐1RA) Anakinra (200 μg/mouse) or control. Arrows indicate time point of Anakinra injection. Each circle represents a mean of n = 5 mice; vertical lines indicate SEM. ***P < 0.001 (two‐way‐ANOVA, Bonferroni post‐test).Serum concentration of Cxcl1, TNF‐α, and IL‐1β in Prdx4 WT and KO mice injected with LPS, LPS, and IL‐1RA or control. Each dot represents an individual mouse. Horizontal lines indicate mean. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. not significant (two‐tailed t‐test).Percent body weight of male Prdx4‐flox and Prdx4‐ΔLysMCre mice over the 48 h course of 4.5 mg/kg BW LPS (i.p.). Each circle represents a mean of n = 7 mice; vertical lines indicate SEM. *P < 0.05; ***P < 0.001 (two‐way‐ANOVA, Bonferroni post‐test).Serum concentration of Cxcl1, TNF‐α, and IL‐1β in Prdx4‐flox and Prdx4‐ΔLysMCre mice injected with LPS. Each dot represents an individual mouse. Horizontal lines indicate mean. *P < 0.05; ***P < 0.001; n.s. not significant (two‐tailed t‐test). Data are representative of two independent experiments.