Journal: Nature Metabolism

Article Title: iNOS modulates inflammatory responses in an NO-independent manner through direct interaction with IRG1 in mitochondria

doi: 10.1038/s42255-026-01492-1

Figure Lengend Snippet: a , Itaconate levels were measured in the cell lysate or media from BMDM culture using HPLC at specific time points following LPS/IFNγ stimulation (n = 6 mice per group). Nitrite was measured in media by Griess assay, and superoxide anion in cell pellets by HPLC from WT (n = 7 mice) and iNOS KO (n = 4 mice) BMDMs. All data is expressed as mean values ± SEM. Statistical differences were calculated as a two-way ANOVA with multiple comparisons test and indicate the variation between genotypes over time. b , Pearson correlation (r) comparing nitrite production from WT vs. intracellular itaconate at each time point (black dot). c , Representative Western blot of n = 3 mice per group; BMDMs following 6, 18 and 24 h exposure to LPS/IFNγ, probed with anti-iNOS, anti-IRG1 and anti-GAPDH (loading control). d , Membrane integrity was assessed using CellTox™ Green Cytotoxicity Assay (Promega). Cells were seeded at 50,000 cells in a 96-well plate and cultured for 24 h with LPS/IFNy or 100 µM H202 or lysed 30 min prior the assay as indicated. Data are presented as mean values of n = 4 mice per group ± SD. Statistical differences were calculated as one-way ANOVA with Dunnett’s multiple comparisons. e , Heatmap of fold change of protein abundance linked with itaconate catabolism / anabolism of LPS/IFNγ from Bailey et al. (2019). Data are expressed as fold-change of M( LPS/IFNγ ) versus unstimulated macrophages M( unstimulated ) and a scale indicating the fold change is shown (n = 4 mice per group); two-way ANOVA followed by Tukey HSD method to create a set of confidence intervals based on the sample means. Proteins having a P-value < 9×10 −6 (based on Bonferroni correction = 0.05/5704) were considered significant. f , Heatmap of normalised abundance values for metabolites for both genotypes from Bailey et al. (2019); Gch1, iNOS KO and WT following 18 h stimulation with LPS/IFNγ. Scale indicate metabolites abundance (n = 6 mice per group), a two-way ANOVA was performed between genotype group. g , Glucose levels were assessed in cell media at 24 h from samples used in Fig. using glucose strips ranging from 0 g/L to >20 g/L. h , Intracellular itaconate measured by HPLC 24 h following media change at 22 h (pink bar) or not (grey bar). Data are presented as mean values of n = 3 or 4 mice per group ± SD. Statistical differences were calculated as one-way ANOVA with Tukey’s multiple comparisons. Statistical significance is indicated as ****p < 0.0001, ***p < 0.001, **p < 0.005, *p < 0.05, ns= non-significant.

Article Snippet: Mouse iNOS was acquired from Cayman chemicals (60864), human iNOS from Origene (TP311819) and both human and mouse IRG1 were purified as previously described (see ‘Purification of Mouse Irg1’).

Techniques: Griess Assay, Western Blot, Control, Membrane, CellTox Assay, Cytotoxicity Assay, Cell Culture, Quantitative Proteomics

Journal: Nature Metabolism

Article Title: iNOS modulates inflammatory responses in an NO-independent manner through direct interaction with IRG1 in mitochondria

doi: 10.1038/s42255-026-01492-1

Figure Lengend Snippet: a , Itaconate levels measured in cell lysate (pink) or media (purple) from BMDM culture using HPLC at specific time points following LPS/IFNγ stimulation ( n = 6 mice per group). Nitrite (orange) measured in media by Griess assay, and superoxide anion (blue) in cell pellets by HPLC from WT ( n = 7 mice) and iNOS-KO ( n = 4 mice) BMDMs. Data are presented as mean values; error bars, s.d. 2-OH-E + , 2-hydroxyethidium. b , Data at 6 h and 18 h were extracted and plotted as a bar chart. Statistical differences were calculated using a two-way analysis of variance (ANOVA) with Šídák’s multiple comparisons test. ND, not detected. c , Densitometry of IRG1/GAPDH is shown ( n = 3 mice). Data are expressed as mean values; error bars, s.e.m. Statistical differences were determined by a two-way ANOVA with Šídák’s multiple comparisons test for each time point; see Extended Data Fig. for a representative western blot. d , HEK cells transfected with iNOS and IRG1 cDNAs and treated with iNOS inhibitors (1400W (10 µM) or aminoguanidine (AG; 1 mM)) or NO donors (NOC18 (50 µM) or SIN-1 (100 µM)). Itaconate was measured in cell pellets and nitrite production in media. Data are represented in bar charts as means of n = 5 independent experiments; error bars, s.e.m. Statistical differences were calculated using one-way ANOVA with Dunnett’s multiple comparisons test against the IRG1 condition for itaconate measurement and against the iNOS condition for nitrite measurement. e , Representative western blot of n = 3 independent experiments showing protein levels of IRG1 and iNOS compared to the loading control GAPDH following co-transfection of HEK cells with IRG1 and iNOS cDNAs. f , Intracellular itaconate and nitrite measured in HEK cells transfected with human IRG1 (ACOD1) cDNA. Data are expressed as the mean of n = 3 independent experiments; error bars, s.e.m. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test. g , Relative gene expression of ACOD1 , NOS2 and GCH1 in human bone marrow organoids compared with GAPDH control. Data are represented in bar charts as the means of n = 3 biological replicates; error bars, s.d. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test against the CTRL condition. h , Intracellular levels of itaconate in human bone marrow organoids measured by mass spectrometry. Data are represented in bar charts as the mean values of n = 3 biological replicates; error bars, s.d. Statistical differences were calculated using an unpaired t -test. Statistical significance is indicated as **** P < 0.0001; *** P < 0.001; ** P < 0.005; * P < 0.05; NS, not significant.

Article Snippet: Mouse iNOS was acquired from Cayman chemicals (60864), human iNOS from Origene (TP311819) and both human and mouse IRG1 were purified as previously described (see ‘Purification of Mouse Irg1’).

Techniques: Griess Assay, Western Blot, Transfection, Control, Cotransfection, Gene Expression, Mass Spectrometry

Journal: Nature Metabolism

Article Title: iNOS modulates inflammatory responses in an NO-independent manner through direct interaction with IRG1 in mitochondria

doi: 10.1038/s42255-026-01492-1

Figure Lengend Snippet: a , Representative Western blot of 3 showing proteins levels of IRG1 and iNOS measured in HEK cells transfected with human IRG1 (hIRG1) cDNA. b , Table of cycle threshold (Ct) values for each gene of interest ( NOS2, ACOD1 , and GCH1 ) in unstimulated (CTRL) or 24 h LPS/IFNγ stimulated of n = 4 human monocyte-derived macrophages (Human MDMs) or n = 3 bone marrow organoids (BM organoids). c , Relative gene expression of ACOD1 , NOS 2 and GCH1 in human MDMs compared to control. Data is represented in bar charts as mean of n = 4 biological replicates ± SD. Statistical differences were calculated as an unpaired t-test. d , Intracellular levels of itaconate in human MDMs measured by HPLC. Data is represented in bar charts as mean of n = 4 biological replicates ± SD. Statistical differences were calculated as an unpaired t-test. Statistical significance is indicated as ****p < 0.0001, ***p < 0.001, **p < 0.005, *p < 0.05, ns= non-significant.

Article Snippet: Mouse iNOS was acquired from Cayman chemicals (60864), human iNOS from Origene (TP311819) and both human and mouse IRG1 were purified as previously described (see ‘Purification of Mouse Irg1’).

Techniques: Western Blot, Transfection, Derivative Assay, Gene Expression, Control

Journal: Nature Metabolism

Article Title: iNOS modulates inflammatory responses in an NO-independent manner through direct interaction with IRG1 in mitochondria

doi: 10.1038/s42255-026-01492-1

Figure Lengend Snippet: a , Hydrogen peroxide levels were determined in media following HEK cell transfection with IRG1 or iNOS cDNA, in the presence or absence of NOC12. Data are expressed as mean values of n = 4 technical replicates; error bars, s.e.m. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test. b , Activity of purified mouse IRG1 (detailed in ) measured by HPLC following incubation with different NO and nitrite donors: 1 mM nitrite, 1 mM SIN-1, 1 mM NOC12 and NOC18, 1 mM GSNO (+10 mM of GSH or GSSG), 1 mM H 2 O 2 , 1 mM DTT, 50 or 500 µM of citrulline and 50 or 500 µM of arginine. Data are expressed as mean values of n = 3–4 independent experiments; error bars, s.e.m. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test against the CTRL condition. c , HEK cells were transfected with IRG1 WT or its mutants cDNA (C184A, C340A, C387A, C432A, C452A, ALA5) and both intracellular itaconate and nitrite levels were measured in the presence or absence of iNOS cDNA. Data are expressed as mean values of n = 4 independent experiments; error bars, s.e.m. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test against WT or WT + iNOS, respectively. d , Volcano plot of proteins pulled down by Co-IP and identified by mass spectrometry in M(LPS/IFNγ) showing proteins significantly associated with IRG1 in WT (black dots) or iNOS-KO (blue dots) ( n = 4 mice per group) following a two-tailed Student’s t -test set at 0.5. e , IRG1 interactome from WT BMDMs was exposed to STRING analysis to exhibit known or predicted interactions using medium-confidence (0.400) settings. f , iNOS intensity data from mass spectrometry were extracted and expressed as a bar chart of mean values of n = 4 independent experiments; error bars, s.e.m. g , Densitometry of iNOS/IRG1 in IP elute in the iNOS-KO model ( n = 3 mice per group). IRG1 was precipitated from the cell pellet. Western blots (Extended Data Fig. ) were pre-incubated with red fluorophore secondary anti-IgG rabbit and then re-incubated with rabbit anti-IRG1 (using a green secondary antibody), allowing visual separation of IRG1 (53 kDa) from IgG heavy chains present in the immunoprecipitate. Data are expressed as the mean of n = 3 mice; error bars, s.e.m. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test against WT. h , Densitometry of iNOS/IRG1 in IP elute in the Gch1 -KO model ( n = 3 mice). IRG1 was precipitated from the cell pellet. Western blots (Extended Data Fig. ) were pre-incubated with red fluorophore secondary anti-IgG rabbit and then re-incubated with rabbit anti-IRG1 (using a green secondary antibody), allowing visual separation of IRG1 (53 kDa) from IgG heavy chains in the immunoprecipitate. Data are expressed as the mean of n = 3 mice; error bars, s.e.m. Statistical differences were calculated using Dunnett’s multiple comparisons test against WT. Statistical significance is indicated as **** P < 0.0001; ** P < 0.005; * P < 0.05.

Article Snippet: Mouse iNOS was acquired from Cayman chemicals (60864), human iNOS from Origene (TP311819) and both human and mouse IRG1 were purified as previously described (see ‘Purification of Mouse Irg1’).

Techniques: Transfection, Activity Assay, Purification, Incubation, Co-Immunoprecipitation Assay, Mass Spectrometry, Two Tailed Test, Western Blot

Journal: Nature Metabolism

Article Title: iNOS modulates inflammatory responses in an NO-independent manner through direct interaction with IRG1 in mitochondria

doi: 10.1038/s42255-026-01492-1

Figure Lengend Snippet: Mouse IRG1 was purified as detailed in Material & Methods. a , IRG1 purification quality is assessed by Coomassie and Western blotting. b , The activity of purified mouse IRG1 was determined by UV, using HPLC at 210 nm and measured 4 h following addition of cis-aconitate at 37 °C in a 0.2 M sodium phosphate solution. c , HPLC chromatograph example of cis-aconitate and itaconate standard detected by HPLC at 210 nm. d , Corresponding nitrite levels of Fig. measured for each condition involving NO production; data is expressed as mean of n = 4 independent experiments ± SEM. e , Effect of increasing concentration of GSH on itaconate standard set at 50 mM. Data are expressed as mean of n = 4 independent experiments ± SEM; Statistical differences were calculated as a one-way ANOVA with Dunnett’s multiple comparisons test against Itaconate standard with no GSH. f , GSH/GSSH ratio were extracted from metabolomic analysis performed in Gch1 WT and Gch1 KO BMDMs and iNOS KO and WT following 18 h stimulation with LPS/IFNγ . Data is plotted as mean values of n = 6 mice per group ± SEM and show the ratio of GSH/GSSG in both M( LPS/IFNγ ) versus unstimulated macrophages (M( unstimulated )) for both genotypes. A one-way ANOVA with Tukey’s mutliple comparisons test was performed. g , Each cysteine of mCAD was mutated individually and replaced with alanine. A mutant was also created with all cysteines mutated to alanines (ALA5). Mismatches are shown by a small white bar and indicated with arrows. h , Protein levels of IRG1 WT and mutants, iNOS and GAPDH (loading control) following transfection were assessed by Western blotting (n = 3 independent experiments). Statistical significance is indicated as ****p < 0.0001, ***p < 0.001, **p < 0.005, *p < 0.05, ns= non-significant.

Article Snippet: Mouse iNOS was acquired from Cayman chemicals (60864), human iNOS from Origene (TP311819) and both human and mouse IRG1 were purified as previously described (see ‘Purification of Mouse Irg1’).

Techniques: Purification, Western Blot, Activity Assay, Concentration Assay, Metabolomic, Mutagenesis, Control, Transfection

Journal: Nature Metabolism

Article Title: iNOS modulates inflammatory responses in an NO-independent manner through direct interaction with IRG1 in mitochondria

doi: 10.1038/s42255-026-01492-1

Figure Lengend Snippet: a,b Proteins identified in the WT ( a ) or iNOS KO IRG1 ( b ) -interactome were subjected to Gene Set Enrichment Analysis in DAVID using Gene Ontology (Biological Process (BP), Molecular Function (MF) and Cellular Component (CC)) and KEGG pathways databases. Categories obtained were filtered by ranking P-values and the ten first are shown here (See Extended Data Table for full list of proteins). c , Volcano plot of M( unstimultaed ) (Fold change (FC) / Log P value) from mass spectrometry data shows proteins significantly associated with IRG1 in WT or iNOS KO following a a two-tailed student T-test set at 0.5. d , Nitrite and extracellular itaconate levels in samples from iNOS KO, WT and WT in the presence of AG from BMDMs all stimulated with LPS/IFNγ. Data are presented as mean values of n = 4 mice per group ± SEM. Statistical differences were calculated as a one-way ANOVA with Dunnett’s multiple comparisons test. e , IRG1 was precipitated from cell pellet in the iNOS KO model. Input (Pre and post-IP lysate) and IP elute were subjected to Western blotting using rabbit anti-iRG1, mouse anti-GAPDH and mouse anti-iNOS . f , Densitometry of IRG1/GAPDH is shown as mean values of n = 3 mice ± SEM. g , For the IP elute, Western blot (n = 3 mice) was pre-incubated with red fluorophore secondary anti-IgG rabbit and then re-incubate with rabbit anti-IRG1 (using a green secondary antibody) allowing to separate visually IRG1 (53 kDa) to IgG heavy chains from IP. h , IRG1 was also precipitated out of cell pellets from Gch1 WT (n = 3 mice) and Gch1 KO BMDMs (n = 3 mice) stimulated with LPS/IFNγ for 18 h, and IP elute directly subjected to Western blotting as detailed previously. i , Densitometry of IRG1/GAPDH is shown as mean values of n = 3 mice ± SEM. j , Input (Pre and post-IP lysate) from Gch1 KO model (n = 3 mice per group) were subjected to Western blotting using rabbit anti-iRG1, mouse anti-GAPDH and mouse anti-iNOS. Statistical significance is indicated as ****p < 0.0001, ***p < 0.001, **p < 0.005, *p < 0.05, ns= non-significant.

Article Snippet: Mouse iNOS was acquired from Cayman chemicals (60864), human iNOS from Origene (TP311819) and both human and mouse IRG1 were purified as previously described (see ‘Purification of Mouse Irg1’).

Techniques: Mass Spectrometry, Two Tailed Test, Western Blot, Incubation

Journal: Nature Metabolism

Article Title: iNOS modulates inflammatory responses in an NO-independent manner through direct interaction with IRG1 in mitochondria

doi: 10.1038/s42255-026-01492-1

Figure Lengend Snippet: IRG1 interactome in LPS/IFNγ iNOS KO versus WT BMDMs

Article Snippet: Mouse iNOS was acquired from Cayman chemicals (60864), human iNOS from Origene (TP311819) and both human and mouse IRG1 were purified as previously described (see ‘Purification of Mouse Irg1’).

Techniques:

Journal: Nature Metabolism

Article Title: iNOS modulates inflammatory responses in an NO-independent manner through direct interaction with IRG1 in mitochondria

doi: 10.1038/s42255-026-01492-1

Figure Lengend Snippet: a , Representative western blot of two mitochondria isolation preparations (total n = 4 mice); 7 µg of protein for each cell compartment (mitochondria, cytosol or whole lysate) from 18 h LPS/IFNγ-stimulated murine BMDMs (WT iNOS BMDMs treated or not with AG and iNOS-KO BMDMs) was loaded into an SDS–PAGE and further probed with anti-iNOS, anti-IRG1, anti-GAPDH (cytosol control) and anti-VDAC (mitochondria control). b , Immunofluorescence of fixed BMDMs following 18 h LPS/IFNγ stimulation from WT and iNOS-KO mice after incubation with mouse anti-iNOS (AF488), anti-Hsp60 (AF680) and DAPI (blue). Single-channel images, as well as the superposition of channels (merge; yellow), are shown. Three regions of interest (ROIs) for each image were used for colocalization analysis. White indicates colocalization of iNOS and Hsp60 on Hsp60 staining (red). A table summarizing the mean of the colocalization factor and Manders’ coefficient from three ROIs for each animal ( n = 3 mice) is also shown. c – f , Computational predictions of the (IRG1) 2 –(iNOS) 2 heterotetramer using AlphaFold-Multimer for the murine ( c ) and human ( e ) heterotetramer, respectively. The two snapshots are related by a 90 °C rotation around the z axis. The predictions show a well-established interface between the two dimers. c , Predicted structure of the murine IRG1–iNOS heterotetramer (iNOS monomers are in red and pink and IRG1 monomers in blue and lavender) in the presence of calmodulin (orange) and haem and flavins (green). d , Molecular dynamics simulations of the murine IRG1–iNOS heterotetramer. The solid lines represent the number of heavy atom contacts between the (IRG1) 2 and (iNOS) 2 homodimers, and the different colours represent three different 300 ns replicas. The stable conformation observed over the 300 ns trajectory supports the reliability of the predicted protein–protein interface. e , Predicted structure of the human IRG1–iNOS heterotetramer (iNOS monomers are in red and pink and IRG1 monomers in blue and lavender) in the presence of calmodulin (orange) and haem and flavins (green). f , Molecular dynamics simulations of the human IRG1–iNOS heterotetramer. The solid lines represent the number of heavy atom contacts between the (IRG1) 2 and (iNOS) 2 homodimers, and the different colours represent three different 300 ns replicas. The stable conformation observed over the 300 ns trajectory supports the reliability of the predicted protein–protein interface. g – i , Binding kinetics of IRG1 and iNOS measured by surface plasma resonance. g , Multi-cycle kinetics analysis of human IRG1 binding to human iNOS. Surface plasma resonance sensograms show the average response curves from duplicate injections of human IRG1 over a human iNOS-immobilized CM5 sensor chip (25–200 nM). Fitted constants of k a = 6.8 × 10 4 M −1 s −1 and k d = 0.013 s −1 , which results in a K D = 189 nM. h , Multi-cycle kinetics analysis of mouse IRG1 binding to mouse iNOS. Surface plasma resonance sensograms show the average response curves from duplicate injections of mouse IRG1 over a mouse iNOS-immobilized CM5 sensor chip (25–200 nM). Fitted constants of k a = 4.5 × 10 4 M −1 s −1 and k d = 0.008 s −1 , which results in a K D = 174 nM. i , k a , association constant; k d , dissociation constant; K D , equilibrium constant of the IRG1–iNOS interaction for both human and mouse proteins; Cyt., cytosol; Col., colocalization; WColCoef., weighted colocalization coefficient; Unst., unstimulated.

Article Snippet: Mouse iNOS was acquired from Cayman chemicals (60864), human iNOS from Origene (TP311819) and both human and mouse IRG1 were purified as previously described (see ‘Purification of Mouse Irg1’).

Techniques: Western Blot, Isolation, SDS Page, Control, Immunofluorescence, Incubation, Staining, Binding Assay, Clinical Proteomics

Journal: Nature Metabolism

Article Title: iNOS modulates inflammatory responses in an NO-independent manner through direct interaction with IRG1 in mitochondria

doi: 10.1038/s42255-026-01492-1

Figure Lengend Snippet: a , Western blot of two mitochondria isolation (total n = 4 mice per group, 1 mitochondria isolation per mice, 2 shown by Western blot); 7 µg of protein for each cell compartment (mitochondria, cytosol or whole lysate) from 18 h LPS/IFNγ stimulated murine BMDMs (WT iNOS BMDMs treated or not with AG and KO iNOS BMDMs) was loaded into an SDS-PAGE and further probed with anti-iNOS, anti-IRG1, anti-GAPDH (cytosol control) and anti-VDAC (mitochondria control). b , Immunofluorescence of fixed BMDMs following 18 h LPS/IFNγ stimulation from WT and iNOS KO mice (n = 3 mice per group) after incubation with mouse anti-iNOS (AF488), anti-Hsp60 (AF680) and DAPI (blue). Single channel images as well as superposition of channels (Merge; yellow) are shown. 3 regions of interest (ROI) for each image were used for colocalisation analysis. White indicate colocalisation of iNOS and Hsp60 on Hsp60 staining (red).

Article Snippet: Mouse iNOS was acquired from Cayman chemicals (60864), human iNOS from Origene (TP311819) and both human and mouse IRG1 were purified as previously described (see ‘Purification of Mouse Irg1’).

Techniques: Western Blot, Isolation, SDS Page, Control, Immunofluorescence, Incubation, Staining

Journal: Nature Metabolism

Article Title: iNOS modulates inflammatory responses in an NO-independent manner through direct interaction with IRG1 in mitochondria

doi: 10.1038/s42255-026-01492-1

Figure Lengend Snippet: a , Predicted structure of the murine IRG1 iNOS heterotetramer (IRG1 monomers are in red and pink and iNOS monomers in sky blue and blue) in the presence of calmodulin (orange and sand colour). c , Molecular dynamics simulations of the murine IRG1-iNOS heterotetramer. The solid lines represent the number of heavy atom contacts between the (IRG1)2 and (iNOS)2 homodimers, and the different colours represent three different 300 ns replicas. The stable conformation observed over the 300 ns trajectory supports the reliability of the predicted protein-protein interface. b , Predicted structure of the human IRG1-iNOS heterotetramer (IRG1 monomers are in red and pink and iNOS monomers in sky blue and blue) d , Molecular dynamics simulations of the human IRG1-iNOS heterotetramer in the presence of calmodulin. The solid lines represent the number of heavy atom contacts between the (IRG1)2 and (iNOS)2 homodimers, and the different colours represent three different 300 ns replicas. The stable conformation observed over the 300 ns trajectory supports the reliability of the predicted protein-protein interface. e , Representative multi-cycle kinetics curves for mouse iNOS on a mouse IRG1 immobilised chip (10 – 80 nM). f , Representative multi-cycle kinetics curves for human iNOS on a human IRG1 immobilised chip (10 – 80 nM). g , Comparison sensorgram of BSA injection (100 nM) on iNOS chip compared to IRG1 (100 nM). h , Average sensograms from injections of IRG1 (200 nM) over iNOS- and eNOS-immobilized chips show differential binding responses.

Article Snippet: Mouse iNOS was acquired from Cayman chemicals (60864), human iNOS from Origene (TP311819) and both human and mouse IRG1 were purified as previously described (see ‘Purification of Mouse Irg1’).

Techniques: Comparison, Injection, Binding Assay

Journal: Nature Metabolism

Article Title: iNOS modulates inflammatory responses in an NO-independent manner through direct interaction with IRG1 in mitochondria

doi: 10.1038/s42255-026-01492-1

Figure Lengend Snippet: Following transfection of HEK cells with iNOS cDNAs (WT and mutants W457A and W457F), alone or in combination with IRG1 cDNA, the ability of each iNOS to regulate IRG1 was tested. a , b , Nitrite production by Griess assay ( a ) and itaconate in cell pellets by HPLC ( b ) were measured in n = 7 independent experiments. Data are presented as mean values; error bars, s.e.m. Statistical differences were calculated using one-way ANOVA with Dunnett’s multiple comparisons test against the IRG1 condition for itaconate measurement and against the iNOS condition for nitrite measurement. Protein levels are shown in Extended Data Fig. . c , A 0.5 µg aliquot of IRG1 was incubated with 25 µM of cis -aconitate (substrate (S)) with increasing concentration of intact ( n = 4 independent experiments) or denaturated ( n = 3 independent experiments) iNOS in the absence of arginine for 18 h at 37 °C. Itaconate levels were determined by HPLC using an itaconate standard to normalize the data. Data are expressed as mean values; error bars, s.e.m. Statistical differences were calculated using one-way ANOVA with Dunnett’s multiple comparisons test against the 0.5 µg IRG1 + S condition. d , Nitrite levels of intact IRG1 in the absence or presence of l -arginine and other iNOS cofactors (BH4, NADPH, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN)). Data are represented as mean values of n = 3 independent experiments; error bars, s.e.m. e , iNOS native form in M(LPS/IFNγ) stimulated for 18 h in WT, iNOS-deficient, Gch1 WT and Gch1 -KO BMDMs using blue native gels and non-denaturing conditions; n = 4 mice for the Gch1 -KO model and n = 5 mice for the iNOS-KO model (details in ). Statistical significance is indicated as **** P < 0.0001; *** P < 0.001; ** P < 0.005.

Article Snippet: Mouse iNOS was acquired from Cayman chemicals (60864), human iNOS from Origene (TP311819) and both human and mouse IRG1 were purified as previously described (see ‘Purification of Mouse Irg1’).

Techniques: Transfection, Griess Assay, Incubation, Concentration Assay

Journal: Nature Metabolism

Article Title: iNOS modulates inflammatory responses in an NO-independent manner through direct interaction with IRG1 in mitochondria

doi: 10.1038/s42255-026-01492-1

Figure Lengend Snippet: a , Representative Western blot of n = 3 independent experiment following transfection of HEKs cells with iNOS cDNAs (WT and mutants W457A and W457F), alone or in combination with IRG1 cDNA. GAPDH was used as a loading control. b , Example of HPLC chromatograph measuring itaconate produced by IRG1 for 18 h at 37 °C in HEPES buffer and incubated with intact iNOS or denatured iNOS.

Article Snippet: Mouse iNOS was acquired from Cayman chemicals (60864), human iNOS from Origene (TP311819) and both human and mouse IRG1 were purified as previously described (see ‘Purification of Mouse Irg1’).

Techniques: Western Blot, Transfection, Control, Produced, Incubation

Journal: Nature Metabolism

Article Title: iNOS modulates inflammatory responses in an NO-independent manner through direct interaction with IRG1 in mitochondria

doi: 10.1038/s42255-026-01492-1

Figure Lengend Snippet: a , AlphaFold Multimer v2.3 prediction of the IRG1-iNOS dimer (IRG1 monomer with iNOS monomer). b , Molecular dynamics simulations at different temperatures showing that the interaction between the units is stable. c , MM/GBSA free energy calculations of three snapshots of the molecular dynamics simulations.

Article Snippet: Mouse iNOS was acquired from Cayman chemicals (60864), human iNOS from Origene (TP311819) and both human and mouse IRG1 were purified as previously described (see ‘Purification of Mouse Irg1’).

Techniques: