Journal: Frontiers in Immunology

Article Title: Fumarate inhibits the formation of neutrophil extracellular traps (NETs) in a Nrf2-controlled and Annexin-A1-dependent manner associated with mitochondrial fusion

doi: 10.3389/fimmu.2026.1770063

Figure Lengend Snippet: Impact of dimethyl fumarate on the regulated cell death of neutrophils. The analyses were performed with (A) PrestoBlue (cell viability; spectrophotometry), (B) Caspase 3/7 activity assay (apoptosis; flow cytometry) and (C) quantification of ZBP-1 (PANoptosis indicator; confocal microscopy) expression respectively. Neutrophils isolated from the bone marrow were treated with DMF (25 µM) for 1h or they were left unstimulated (CTR). Some of the cells were subsequently stimulated with lipopolysaccharide (LPS) at a concentration of 50 μg/mL. To determine necroptosis (D) and pyroptosis (E) extDNA was quantified after DMF, application of specific inhibitors: Nec-1, DIS treatment (1 h) or their combination. Additionally some cells were pretreated with DMF (DMF→Nec-1/DIS) incubated with or without LPS. The results are expressed as the mean values ± SD; n≥3. Values significantly different between the groups (p < 0.05) according to one-way ANOVA ( post hoc Bonferroni test) are designated by letters, where the same letter indicates no differences between groups (different letters indicate statistical differences. Explanation of abbreviations: ZBP-1, Z-DNA-binding protein 1; extDNA, extracellular DNA; Nec-1, necrostatin-1; DIS, disulfiram.

Article Snippet: The following inhibitors were used: nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor – ML385 (10 μM, MedChemExpress), formyl peptide receptor 2 (Fpr2) antagonist - WRW4 (10 μM, MedChemExpress), NADPH oxidase (NOX) inhibitor - Diphenyleneiodonium chloride (DPI, 10 μM, Merck), necroptosis inhibitor - Necrostatin-1 (50 μM, Torcis Bioscence, Bristol, UK), pyroptosis inhibitor - Disulfiram (30 μM, MedChemExpress) and mitochondrial complex I inhibitor - Rotenone (5 μM, Sigma-Aldrich, Saint Louis, MO, USA).

Techniques: Spectrophotometry, Activity Assay, Flow Cytometry, Confocal Microscopy, Expressing, Isolation, Concentration Assay, Incubation, Binding Assay

Journal: Frontiers in Immunology

Article Title: Fumarate inhibits the formation of neutrophil extracellular traps (NETs) in a Nrf2-controlled and Annexin-A1-dependent manner associated with mitochondrial fusion

doi: 10.3389/fimmu.2026.1770063

Figure Lengend Snippet: Effect of CCCP possibly leading to lethal NETosis. mitochondria fragmentation inducer on PANoptosis of neutrophils. (A) Quantification of ZBP-1 (PANoptosis indicator) expression (confocal microscopy) and (B) Caspase 3/7 activity assay (apoptosis; flow cytometry) respectively. Neutrophils were left unstimulated (CTR), stimulated with LPS (50 µg/mL), pre-treated with CCCP (5 µM, 1 h), or pre-treated with CCCP followed by LPS stimulation (CCCP→LPS). To determine pyroptosis (Ci, Cii) and necroptosis (Di, Dii) extDNA was quantified after CCCP, application of specific inhibitors: Nec-1, DIS treatment (1h) or their combination. Additionally some cells were pretreated with CCCP (CCCP→Nec-1/DIS) incubated with or without LPS. The results are expressed as the mean values ± SD; n≥3. Values significantly different between the groups (p < 0.05) according to one-way ANOVA ( post hoc Bonferroni test) or Kruskal-Wallis test with Dunn’s post hoc are designated by letters, where the same letter indicates no differences between groups (different letters indicate statistical differences. Explanation of abbreviations: ZBP-1, Z-DNA-binding protein 1; extDNA, extracellular DNA; Nec-1, necrostatin-1; DIS, disulfiram.

Article Snippet: The following inhibitors were used: nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor – ML385 (10 μM, MedChemExpress), formyl peptide receptor 2 (Fpr2) antagonist - WRW4 (10 μM, MedChemExpress), NADPH oxidase (NOX) inhibitor - Diphenyleneiodonium chloride (DPI, 10 μM, Merck), necroptosis inhibitor - Necrostatin-1 (50 μM, Torcis Bioscence, Bristol, UK), pyroptosis inhibitor - Disulfiram (30 μM, MedChemExpress) and mitochondrial complex I inhibitor - Rotenone (5 μM, Sigma-Aldrich, Saint Louis, MO, USA).

Techniques: Expressing, Confocal Microscopy, Activity Assay, Flow Cytometry, Incubation, Binding Assay