Journal: Nature Communications

Article Title: An inherited mitochondrial DNA mutation remodels inflammatory cytokine responses in macrophages and in vivo in mice

doi: 10.1038/s41467-025-65023-4

Figure Lengend Snippet: a Schematic of m.5019A > G mt-Ta mutation model. b Pyrosequencing results of pre-differentiation bone marrow and post-differentiation bone marrow-derived macrophages (BMDMs) from m.5019A > G mice ( n = 6). Heteroplasmy range between 70% and 87%. Colours indicate matched bone marrow and BMDMs. c Mitochondrial DNA (mtDNA) copy number in non-stimulated (non-stim) and lipopolysaccharide (LPS)-stimulated wildtype (WT) and m.5019A > G BMDMs ( n = 3; LPS 6 h). d 35 S-methionine labelling and quantification of mitochondrial proteins in non-stim WT and m.5019A > G BMDMs ( n = 3). e Seahorse XFe24 oxygen consumption rate (OCR) trace in non-stim WT ( n = 3) and m.5019A > G BMDMs ( n = 4). f Coenzyme Q (CoQ) redox measurements with or without antimycin A (Ant A) in non-stim WT and m.5019A > G BMDMs ( n = 3). g Heatmap of all identified complex I (CI), CIII and CIV subunits and assembly factors in non-stim WT ( n = 6) and m.5019A > G ( n = 7) BMDMs. h CV-ATP5A, CIII-UQCRC2, CIV-MT-COI, CII-SDHB and CI-NDUFB8 protein levels in non-stim and LPS-stimulated WT and m.5019A > G BMDMs ( n = 3; LPS 6 h & 24 h). Representative blot shown. i Comparison of log 2 FC values of CI structural subunits from proteomics ( n = 6; WT and n = 7; m.5019A > G ) and RNA sequencing ( n = 3) data with Pearson r correlation and two-tailed statistical analysis applied. j Mitochondrial mass ( P = 0.0000000322) and ( k ) normalised mitochondrial membrane potential (MMP) measurements in non-stim m.5019A > G vs WT BMDMs using MitoTracker Green (MTG) and tetramethyl rhodamine methyl ester (TMRM) ( n = 8). Data are scaled log 2 intensities, log 2 FC or mean ± s.e.m. n number represents independent biological replicates (mice) from a minimum of two independent experiments. P- values calculated using two-tailed Student’s t test for two group comparisons or multiple two-tailed unpaired t tests corrected for multiple comparisons using Benjamini, Krieger and Yekutieli method. a Created in BioRender. Dwane, L. (2025) https://BioRender.com/to6x4hj .

Article Snippet: Where stated, pre-treatment of BMDMs with Ruxolitinib (HY-50856; MedChemExpress; 1 μM) or an anti-IFNAR monoclonal blocking antibody (MAR1-5A3; ThermoFisher Scientific; 20 μg/mL) prior to LPS stimulation were used to inhibit IFNAR signalling.

Techniques: Mutagenesis, Derivative Assay, Comparison, RNA Sequencing, Two Tailed Test, Membrane

Journal: Nature Communications

Article Title: An inherited mitochondrial DNA mutation remodels inflammatory cytokine responses in macrophages and in vivo in mice

doi: 10.1038/s41467-025-65023-4

Figure Lengend Snippet: a , b Overrepresentation analysis (ORA) using KEGG terms of all differentially expressed genes from RNA sequencing ( n = 3) and differentially abundant proteins ( n = 6; WT and n = 7; m.5019A > G ) increased in non-stimulated (non-stim) m.5019A > G vs wildtype (WT) BMDMs. c , d Proton efflux rate (PER) measurements in non-stim WT and m.5019A > G BMDMs ( n = 3; WT and n = 4; m.5019A > G )( P = 0.000036). e Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements in non-stim and lipopolysaccharide (LPS)-stimulated WT and m.5019A > G BMDMs ( n = 6; WT and n = 7; m.5019A > G ). f Lactate/pyruvate ratio in cell culture medium (CCM) from metabolomics in non-stim and LPS-stimulated WT and m.5019A > G BMDMs ( n = 3; LPS 6 h & 24 h). g Heatmap of hypoxia-inducible factor 1-α (HIF-1α) targets and glycolytic enzymes from proteomics in LPS-stimulated WT and m.5019A > G BMDMs ( n = 4; WT and n = 5; m.5019A > G ; LPS 6 h). h Schematic of U- 13 C-glucose tracing into lactate and the tricarboxylic acid (TCA) cycle, indicating the first round labelling pattern. i m + 3 labelling in lactate and m + 2 labelling in citrate from U- 13 C-glucose in non-stim and LPS-stimulated WT and m.5019A > G BMDMs ( n = 3; LPS 6 h). Data are scaled log 2 intensities or mean ± s.e.m. n number represents independent biological replicates (mice) from a minimum of two independent experiments. P- values calculated using two-tailed Student’s t test for two group comparisons or multiple two-tailed unpaired t tests corrected for multiple comparisons using Benjamini, Krieger and Yekutieli method.

Article Snippet: Where stated, pre-treatment of BMDMs with Ruxolitinib (HY-50856; MedChemExpress; 1 μM) or an anti-IFNAR monoclonal blocking antibody (MAR1-5A3; ThermoFisher Scientific; 20 μg/mL) prior to LPS stimulation were used to inhibit IFNAR signalling.

Techniques: RNA Sequencing, Cell Culture, Two Tailed Test

Journal: Nature Communications

Article Title: An inherited mitochondrial DNA mutation remodels inflammatory cytokine responses in macrophages and in vivo in mice

doi: 10.1038/s41467-025-65023-4

Figure Lengend Snippet: a Heatmap comparing metabolite levels in non-stimulated (non-stim) (α-KG, P = 0.001812; Fumarate, P = 0.006172; Malate, P = 0.000451) and lipopolysaccharide (LPS)-stimulated (α-KG, P = 0.005704; Succinyl-CoA, P = 0.025485; Aspartate, P = 0.047568) wildtype (WT) and m.5019A > G BMDMs ( n = 5; LPS 6 h; left), and comparing metabolite levels in non-stim WT and m.5019A > G BMDMs in the presence or absence of glutamine (Gln) ( n = 3; LPS 4 h; right) (Citrate, P = 0.008855; Isocitrate, P = 0.000054; α-KG, P = 0.0000001; Succinate, P = 0.0128505; Fumarate, P = 0.0000541; Malate, P = 0.0000116; Aspartate, P = 0.0297496). b α-Ketoglutarate (α-KG)/succinyl-CoA and α-KG/succinate ratio in non-stim WT and m.5019A > G BMDMs ( n = 5). c Oxoglutarate dehydrogenase complex (OGDHC) and pyruvate dehydrogenase complex (PDHC) E3 subunit (DLD) levels from proteomics in non-stim WT ( n = 4), non-stim m.5019A > G ( n = 5), LPS-stimulated WT ( n = 6; 6 h) and LPS-stimulated m.5019A > G BMDMs ( n = 7; 6 h) ( P = 0.000047). d Schematic of U- 13 C-glutamine tracing into the tricarboxylic acid (TCA) cycle, indicating oxidative versus reductive labelling patterns. e m + 5 labelling from U- 13 C-glutamine in isocitrate and citrate in non-stim and LPS-stimulated WT and m.5019A > G BMDMs ( n = 5; LPS 6 h). f m + 5 labelling in L-2-hydroxyglutarate (L-2-HG) from U- 13 C-glutamine in non-stim and LPS-stimulated WT and m.5019A > G BMDMs ( n = 3; LPS 6 h). g Schematic of U- 13 C-glucose tracing into the TCA cycle, indicating oxidative versus reductive labelling patterns. h m + 3 labelling from U- 13 C-glucose in citrate, malate ( P = 0.000405; P = 0.000405) and succinate in non-stim and LPS-stimulated WT and m.5019A > G BMDMs ( n = 5; LPS 24 h). Data are mean or mean ± s.e.m. n number represents independent biological replicates (mice) from a minimum of three independent experiments. P- values calculated using two-tailed Student’s t test for two group comparisons or multiple two-tailed unpaired t tests corrected for multiple comparisons using Benjamini, Krieger and Yekutieli method. *** P < 0.001 ** P < 0.01 * P < 0.05.

Article Snippet: Where stated, pre-treatment of BMDMs with Ruxolitinib (HY-50856; MedChemExpress; 1 μM) or an anti-IFNAR monoclonal blocking antibody (MAR1-5A3; ThermoFisher Scientific; 20 μg/mL) prior to LPS stimulation were used to inhibit IFNAR signalling.

Techniques: Two Tailed Test

Journal: Nature Communications

Article Title: An inherited mitochondrial DNA mutation remodels inflammatory cytokine responses in macrophages and in vivo in mice

doi: 10.1038/s41467-025-65023-4

Figure Lengend Snippet: a Schematic of U- 13 C-glutamine tracing into the aspartate-argininosuccinate shunt (AAS), indicating oxidative versus reductive labelling patterns and NO production. b m + 3 labelling from U- 13 C-glutamine in aspartate, argininosuccinate ( P = 0.000026) and fumarate ( P = 0.000005) in non-stimulated (non-stim) and lipopolysaccharide (LPS)-stimulated wildtype (WT) and m.5019A > G BMDMs ( n = 5; LPS 6 h). c Schematic of U- 13 C-glucose tracing into the AAS indicating oxidative versus reductive labelling patterns and nitric oxide (NO) production. d m + 3 labelling from U- 13 C-glucose in aspartate ( P = 0.000070; P = 0.000002), argininosuccinate ( P = 0.000015) and fumarate ( P = 0.000039) in non-stim and LPS-stimulated WT and m.5019A > G BMDMs ( n = 3; LPS 24 h). e Nitrite levels in cell culture medium (CCM) in non-stim and LPS-stimulated WT and m.5019A > G BMDMs in the presence or absence of glutamine (Gln) ( n = 3; LPS 24 h) ( P = 0.00000007). f , g Nos2 expression and inducible nitric oxide synthase (iNOS) protein levels from LPS time course analysis in WT and m.5019A > G BMDMs ( n = 3; LPS 0, 1, 2, 6 & 24 h). Representative blot shown. Data are mean ± s.e.m. n number represents independent biological replicates (mice) from a minimum of three independent experiments. P- values calculated using multiple two-tailed unpaired t tests corrected for multiple comparisons using the Benjamini, Krieger and Yekutieli method or one-way ANOVA corrected for multiple comparisons using the Tukey method.

Article Snippet: Where stated, pre-treatment of BMDMs with Ruxolitinib (HY-50856; MedChemExpress; 1 μM) or an anti-IFNAR monoclonal blocking antibody (MAR1-5A3; ThermoFisher Scientific; 20 μg/mL) prior to LPS stimulation were used to inhibit IFNAR signalling.

Techniques: Cell Culture, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: An inherited mitochondrial DNA mutation remodels inflammatory cytokine responses in macrophages and in vivo in mice

doi: 10.1038/s41467-025-65023-4

Figure Lengend Snippet: a Volcano plot of differentially expressed genes from RNA sequencing in lipopolysaccharide (LPS)-stimulated m.5019A > G vs wildtype (WT) BMDMs ( n = 3; LPS 1 h). b , c Ifnb1 expression and interferon-β (IFN-β) release from LPS time course analysis in WT and m.5019A > G BMDMs ( n = 3; LPS 0, 1, 2, 6 & 24 h) ( P = 0.000052). d Phospho-interferon regulatory factor 3 (IRF3) (ser396), IRF3, IRF7, and interferon-stimulated gene 15 (ISG15) protein levels from LPS time course analysis in WT and m.5019A > G BMDMs ( n = 3; LPS 0, 1, 2, 6 & 24 h). Representative blot shown. e Oxylipin profiling of cell culture medium (CCM) in non-stimulated (non-stim) and LPS-stimulated WT and m.5019A > G BMDMs ( n = 3; LPS 6 h) ( P = 0.00001417). f Olink target T48 mouse cytokine and chemokine profiling of CCM in LPS-stimulated m.5019 A > G vs WT BMDMs ( n = 3; LPS 6 h). g Cyclooxygenase 2 (COX2) and pro-interleukin-1β (pro-IL-1β) protein levels from LPS time course analysis in WT and m.5019A > G BMDMs ( n = 3; LPS 0, 1, 2, 6 & 24 h). Representative blot shown. h COX2 and pro-IL-1β protein levels in non-stim and LPS-stimulated WT and m.5019A > G BMDMs treated with an anti-interferon-α/β receptor (IFNAR) monoclonal antibody (Ab) or isotype control Ab ( n = 3; LPS 6 h). Representative blot shown. i COX2 and pro-IL-1β protein levels in non-stim and LPS-stimulated WT and m.5019A > G BMDMs treated with Ruxolitinib or vehicle control Ab ( n = 3; LPS 6 h). Representative blot shown. j Inducible nitric oxide synthase (iNOS) protein levels in non-stim and LPS-stimulated WT and m.5019A > G BMDMs treated with Ruxolitinib or vehicle control Ab ( n = 3; LPS 24 h). Representative blot shown. Data are log 2 FC or mean ± s.e.m. n number represents independent biological replicates (mice) from a minimum of three independent experiments. P- values calculated using two-tailed Student’s t test for two group comparisons, multiple two-tailed unpaired t tests corrected for multiple comparisons using Benjamini, Krieger and Yekutieli method or one-way ANOVA corrected for multiple comparisons using Tukey method.

Article Snippet: Where stated, pre-treatment of BMDMs with Ruxolitinib (HY-50856; MedChemExpress; 1 μM) or an anti-IFNAR monoclonal blocking antibody (MAR1-5A3; ThermoFisher Scientific; 20 μg/mL) prior to LPS stimulation were used to inhibit IFNAR signalling.

Techniques: RNA Sequencing, Expressing, Cell Culture, Control, Two Tailed Test

Journal: Nature Communications

Article Title: An inherited mitochondrial DNA mutation remodels inflammatory cytokine responses in macrophages and in vivo in mice

doi: 10.1038/s41467-025-65023-4

Figure Lengend Snippet: a , b Representative immunofluorescence staining of cytochrome c (Cyt c) and translocase of the outer membrane 20 (TOM20) coupled to confocal microscopy in non-stimulated (non-stim) wildtype (WT) and m.5019A > G BMDMs ( a ) and mitochondrial morphology analysis in non-stim WT ( n = 4), non-stim m.5019A > G ( n = 6), lipopolysaccharide (LPS)-stimulated WT ( n = 3) and LPS-stimulated m.5019A > G ( n = 4) BMDMs ( b ) (LPS 6 h; minimum of 20 cells analysed per condition per biological replicate) ( P = 0.000038). Scale bars: 5 μm. c , d Representative immunofluorescence staining of TOM20 and ATP synthase coupled to super-resolution microscopy in non-stim WT and m.5019A > G BMDMs ( c ) and mitochondrial morphology analysis of non-stim WT ( n = 3), non-stim m.5019A > G ( n = 3), LPS-stimulated WT ( n = 2) and LPS-stimulated m.5019A > G ( n = 3) BMDMs ( d ) (LPS 6 h; minimum of 33 cells analysed from independent biological replicates) ( P = 0.000000183; P = 0.0000000003). Scale bars: 5 μm. e , f Representative immunofluorescence staining of dynamin-related protein 1 (DRP1) and TOM20 coupled to confocal microscopy ( e ) and Pearson r correlation analysis ( f ) in non-stim and LPS-stimulated WT and m.5019A > G BMDMs ( n = 3; LPS 6 h; minimum of 20 cells analysed per condition per biological replicate) ( P = 0.000059). Scale bars: 5 μm. Data are mean ± s.e.m or ± s.d. n number represents independent biological replicates (mice) from a minimum of two independent experiments. P- values calculated using multiple two-tailed unpaired tests corrected for multiple comparisons using the Holm-Sidak method or one-way ANOVA corrected for multiple comparisons using the Kruskal-Wallis method.

Article Snippet: Where stated, pre-treatment of BMDMs with Ruxolitinib (HY-50856; MedChemExpress; 1 μM) or an anti-IFNAR monoclonal blocking antibody (MAR1-5A3; ThermoFisher Scientific; 20 μg/mL) prior to LPS stimulation were used to inhibit IFNAR signalling.

Techniques: Immunofluorescence, Staining, Membrane, Confocal Microscopy, Super-Resolution Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: An inherited mitochondrial DNA mutation remodels inflammatory cytokine responses in macrophages and in vivo in mice

doi: 10.1038/s41467-025-65023-4

Figure Lengend Snippet: a , b Representative transmission electron microscopy (TEM) images ( a ) and cristae and mitochondrial aspect ratio (length/width) analysis ( b ) of non-stimulated (non-stim) and lipopolysaccharide (LPS)-stimulated wildtype (WT) and m.5019A > G BMDMs ( n = 3; LPS 6 h; mitochondria from a minimum of 9 cells were analysed per condition per biological replicate). Scale bars: 0.5 μm. Black arrows indicate mitochondria. c Mitochondrial (mt)DNA and mtRNA levels in cytosolic fraction of non-stim WT and m.5019A > G BMDMs ( n = 6). d , e Representative immunofluorescence staining of DNA and translocase of the outer membrane 20 (TOM20) coupled to confocal microscopy in LPS-stimulated WT and m.5019A > G BMDMs ( d ) and cytosolic DNA foci quantification in non-stim and LPS-stimulated WT and m.5019 A > G BMDMs ( e ) ( n = 3; LPS 24 h; minimum of 20 cells analysed per condition per biological replicate). White arrows indicate cytosolic DNA foci. f Ifnb1 expression (LPS 24 h) and interferon-β (IFN-β) release (LPS 6 h) in LPS-stimulated WT and m.5019 A > G macrophages pre-treated with cyclic GMP-AMP synthase (cGAS) inhibitor RU.521 or vehicle control (DMSO) for 1 h ( n = 3) ( P = 0.000262). g mt-Nd1 ( P = 0.000000008; P = 0.000946773), mt-Co3 ( P = 0.000000005; P = 0.000236469), Ifnb1 ( P = 0.000402; P = 0.000402) expression and IFN-β release in LPS-stimulated WT and m.5019A > G macrophages pre-treated with inhibitor of mitochondrial transcription 1 (IMT1) or vehicle control (DMSO) for 24 h ( n = 3; LPS 24 h). Data are mean ± s.e.m. n number represents independent biological replicates (mice) from a minimum of three independent experiments. P- values calculated using multiple two-tailed unpaired t tests corrected for multiple comparisons using the Benjamini, Krieger and Yekutieli method.

Article Snippet: Where stated, pre-treatment of BMDMs with Ruxolitinib (HY-50856; MedChemExpress; 1 μM) or an anti-IFNAR monoclonal blocking antibody (MAR1-5A3; ThermoFisher Scientific; 20 μg/mL) prior to LPS stimulation were used to inhibit IFNAR signalling.

Techniques: Transmission Assay, Electron Microscopy, Immunofluorescence, Staining, Membrane, Confocal Microscopy, Expressing, Control, Two Tailed Test

Journal: Nature Communications

Article Title: An inherited mitochondrial DNA mutation remodels inflammatory cytokine responses in macrophages and in vivo in mice

doi: 10.1038/s41467-025-65023-4

Figure Lengend Snippet: a Schematic of m.5019A > G mt-Ta mutation model. b Pyrosequencing results of pre-differentiation bone marrow and post-differentiation bone marrow-derived macrophages (BMDMs) from m.5019A > G mice ( n = 6). Heteroplasmy range between 70% and 87%. Colours indicate matched bone marrow and BMDMs. c Mitochondrial DNA (mtDNA) copy number in non-stimulated (non-stim) and lipopolysaccharide (LPS)-stimulated wildtype (WT) and m.5019A > G BMDMs ( n = 3; LPS 6 h). d 35 S-methionine labelling and quantification of mitochondrial proteins in non-stim WT and m.5019A > G BMDMs ( n = 3). e Seahorse XFe24 oxygen consumption rate (OCR) trace in non-stim WT ( n = 3) and m.5019A > G BMDMs ( n = 4). f Coenzyme Q (CoQ) redox measurements with or without antimycin A (Ant A) in non-stim WT and m.5019A > G BMDMs ( n = 3). g Heatmap of all identified complex I (CI), CIII and CIV subunits and assembly factors in non-stim WT ( n = 6) and m.5019A > G ( n = 7) BMDMs. h CV-ATP5A, CIII-UQCRC2, CIV-MT-COI, CII-SDHB and CI-NDUFB8 protein levels in non-stim and LPS-stimulated WT and m.5019A > G BMDMs ( n = 3; LPS 6 h & 24 h). Representative blot shown. i Comparison of log 2 FC values of CI structural subunits from proteomics ( n = 6; WT and n = 7; m.5019A > G ) and RNA sequencing ( n = 3) data with Pearson r correlation and two-tailed statistical analysis applied. j Mitochondrial mass ( P = 0.0000000322) and ( k ) normalised mitochondrial membrane potential (MMP) measurements in non-stim m.5019A > G vs WT BMDMs using MitoTracker Green (MTG) and tetramethyl rhodamine methyl ester (TMRM) ( n = 8). Data are scaled log 2 intensities, log 2 FC or mean ± s.e.m. n number represents independent biological replicates (mice) from a minimum of two independent experiments. P- values calculated using two-tailed Student’s t test for two group comparisons or multiple two-tailed unpaired t tests corrected for multiple comparisons using Benjamini, Krieger and Yekutieli method. a Created in BioRender. Dwane, L. (2025) https://BioRender.com/to6x4hj .

Article Snippet: Pre-treatment of BMDMs with IMT1 (HY-134539; MedChemExpress; 10 μM) for 24 h or RU.521 (HY-114180; MedChemExpress; 10 μM) for 1 h prior to LPS stimulation were used to inhibit mitochondrial transcription and the cGAS-STING pathway, respectively.

Techniques: Mutagenesis, Derivative Assay, Comparison, RNA Sequencing, Two Tailed Test, Membrane

Journal: Nature Communications

Article Title: An inherited mitochondrial DNA mutation remodels inflammatory cytokine responses in macrophages and in vivo in mice

doi: 10.1038/s41467-025-65023-4

Figure Lengend Snippet: a , b Overrepresentation analysis (ORA) using KEGG terms of all differentially expressed genes from RNA sequencing ( n = 3) and differentially abundant proteins ( n = 6; WT and n = 7; m.5019A > G ) increased in non-stimulated (non-stim) m.5019A > G vs wildtype (WT) BMDMs. c , d Proton efflux rate (PER) measurements in non-stim WT and m.5019A > G BMDMs ( n = 3; WT and n = 4; m.5019A > G )( P = 0.000036). e Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements in non-stim and lipopolysaccharide (LPS)-stimulated WT and m.5019A > G BMDMs ( n = 6; WT and n = 7; m.5019A > G ). f Lactate/pyruvate ratio in cell culture medium (CCM) from metabolomics in non-stim and LPS-stimulated WT and m.5019A > G BMDMs ( n = 3; LPS 6 h & 24 h). g Heatmap of hypoxia-inducible factor 1-α (HIF-1α) targets and glycolytic enzymes from proteomics in LPS-stimulated WT and m.5019A > G BMDMs ( n = 4; WT and n = 5; m.5019A > G ; LPS 6 h). h Schematic of U- 13 C-glucose tracing into lactate and the tricarboxylic acid (TCA) cycle, indicating the first round labelling pattern. i m + 3 labelling in lactate and m + 2 labelling in citrate from U- 13 C-glucose in non-stim and LPS-stimulated WT and m.5019A > G BMDMs ( n = 3; LPS 6 h). Data are scaled log 2 intensities or mean ± s.e.m. n number represents independent biological replicates (mice) from a minimum of two independent experiments. P- values calculated using two-tailed Student’s t test for two group comparisons or multiple two-tailed unpaired t tests corrected for multiple comparisons using Benjamini, Krieger and Yekutieli method.

Article Snippet: Pre-treatment of BMDMs with IMT1 (HY-134539; MedChemExpress; 10 μM) for 24 h or RU.521 (HY-114180; MedChemExpress; 10 μM) for 1 h prior to LPS stimulation were used to inhibit mitochondrial transcription and the cGAS-STING pathway, respectively.

Techniques: RNA Sequencing, Cell Culture, Two Tailed Test

Journal: Nature Communications

Article Title: An inherited mitochondrial DNA mutation remodels inflammatory cytokine responses in macrophages and in vivo in mice

doi: 10.1038/s41467-025-65023-4

Figure Lengend Snippet: a Heatmap comparing metabolite levels in non-stimulated (non-stim) (α-KG, P = 0.001812; Fumarate, P = 0.006172; Malate, P = 0.000451) and lipopolysaccharide (LPS)-stimulated (α-KG, P = 0.005704; Succinyl-CoA, P = 0.025485; Aspartate, P = 0.047568) wildtype (WT) and m.5019A > G BMDMs ( n = 5; LPS 6 h; left), and comparing metabolite levels in non-stim WT and m.5019A > G BMDMs in the presence or absence of glutamine (Gln) ( n = 3; LPS 4 h; right) (Citrate, P = 0.008855; Isocitrate, P = 0.000054; α-KG, P = 0.0000001; Succinate, P = 0.0128505; Fumarate, P = 0.0000541; Malate, P = 0.0000116; Aspartate, P = 0.0297496). b α-Ketoglutarate (α-KG)/succinyl-CoA and α-KG/succinate ratio in non-stim WT and m.5019A > G BMDMs ( n = 5). c Oxoglutarate dehydrogenase complex (OGDHC) and pyruvate dehydrogenase complex (PDHC) E3 subunit (DLD) levels from proteomics in non-stim WT ( n = 4), non-stim m.5019A > G ( n = 5), LPS-stimulated WT ( n = 6; 6 h) and LPS-stimulated m.5019A > G BMDMs ( n = 7; 6 h) ( P = 0.000047). d Schematic of U- 13 C-glutamine tracing into the tricarboxylic acid (TCA) cycle, indicating oxidative versus reductive labelling patterns. e m + 5 labelling from U- 13 C-glutamine in isocitrate and citrate in non-stim and LPS-stimulated WT and m.5019A > G BMDMs ( n = 5; LPS 6 h). f m + 5 labelling in L-2-hydroxyglutarate (L-2-HG) from U- 13 C-glutamine in non-stim and LPS-stimulated WT and m.5019A > G BMDMs ( n = 3; LPS 6 h). g Schematic of U- 13 C-glucose tracing into the TCA cycle, indicating oxidative versus reductive labelling patterns. h m + 3 labelling from U- 13 C-glucose in citrate, malate ( P = 0.000405; P = 0.000405) and succinate in non-stim and LPS-stimulated WT and m.5019A > G BMDMs ( n = 5; LPS 24 h). Data are mean or mean ± s.e.m. n number represents independent biological replicates (mice) from a minimum of three independent experiments. P- values calculated using two-tailed Student’s t test for two group comparisons or multiple two-tailed unpaired t tests corrected for multiple comparisons using Benjamini, Krieger and Yekutieli method. *** P < 0.001 ** P < 0.01 * P < 0.05.

Article Snippet: Pre-treatment of BMDMs with IMT1 (HY-134539; MedChemExpress; 10 μM) for 24 h or RU.521 (HY-114180; MedChemExpress; 10 μM) for 1 h prior to LPS stimulation were used to inhibit mitochondrial transcription and the cGAS-STING pathway, respectively.

Techniques: Two Tailed Test

Journal: Nature Communications

Article Title: An inherited mitochondrial DNA mutation remodels inflammatory cytokine responses in macrophages and in vivo in mice

doi: 10.1038/s41467-025-65023-4

Figure Lengend Snippet: a Schematic of U- 13 C-glutamine tracing into the aspartate-argininosuccinate shunt (AAS), indicating oxidative versus reductive labelling patterns and NO production. b m + 3 labelling from U- 13 C-glutamine in aspartate, argininosuccinate ( P = 0.000026) and fumarate ( P = 0.000005) in non-stimulated (non-stim) and lipopolysaccharide (LPS)-stimulated wildtype (WT) and m.5019A > G BMDMs ( n = 5; LPS 6 h). c Schematic of U- 13 C-glucose tracing into the AAS indicating oxidative versus reductive labelling patterns and nitric oxide (NO) production. d m + 3 labelling from U- 13 C-glucose in aspartate ( P = 0.000070; P = 0.000002), argininosuccinate ( P = 0.000015) and fumarate ( P = 0.000039) in non-stim and LPS-stimulated WT and m.5019A > G BMDMs ( n = 3; LPS 24 h). e Nitrite levels in cell culture medium (CCM) in non-stim and LPS-stimulated WT and m.5019A > G BMDMs in the presence or absence of glutamine (Gln) ( n = 3; LPS 24 h) ( P = 0.00000007). f , g Nos2 expression and inducible nitric oxide synthase (iNOS) protein levels from LPS time course analysis in WT and m.5019A > G BMDMs ( n = 3; LPS 0, 1, 2, 6 & 24 h). Representative blot shown. Data are mean ± s.e.m. n number represents independent biological replicates (mice) from a minimum of three independent experiments. P- values calculated using multiple two-tailed unpaired t tests corrected for multiple comparisons using the Benjamini, Krieger and Yekutieli method or one-way ANOVA corrected for multiple comparisons using the Tukey method.

Article Snippet: Pre-treatment of BMDMs with IMT1 (HY-134539; MedChemExpress; 10 μM) for 24 h or RU.521 (HY-114180; MedChemExpress; 10 μM) for 1 h prior to LPS stimulation were used to inhibit mitochondrial transcription and the cGAS-STING pathway, respectively.

Techniques: Cell Culture, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: An inherited mitochondrial DNA mutation remodels inflammatory cytokine responses in macrophages and in vivo in mice

doi: 10.1038/s41467-025-65023-4

Figure Lengend Snippet: a Volcano plot of differentially expressed genes from RNA sequencing in lipopolysaccharide (LPS)-stimulated m.5019A > G vs wildtype (WT) BMDMs ( n = 3; LPS 1 h). b , c Ifnb1 expression and interferon-β (IFN-β) release from LPS time course analysis in WT and m.5019A > G BMDMs ( n = 3; LPS 0, 1, 2, 6 & 24 h) ( P = 0.000052). d Phospho-interferon regulatory factor 3 (IRF3) (ser396), IRF3, IRF7, and interferon-stimulated gene 15 (ISG15) protein levels from LPS time course analysis in WT and m.5019A > G BMDMs ( n = 3; LPS 0, 1, 2, 6 & 24 h). Representative blot shown. e Oxylipin profiling of cell culture medium (CCM) in non-stimulated (non-stim) and LPS-stimulated WT and m.5019A > G BMDMs ( n = 3; LPS 6 h) ( P = 0.00001417). f Olink target T48 mouse cytokine and chemokine profiling of CCM in LPS-stimulated m.5019 A > G vs WT BMDMs ( n = 3; LPS 6 h). g Cyclooxygenase 2 (COX2) and pro-interleukin-1β (pro-IL-1β) protein levels from LPS time course analysis in WT and m.5019A > G BMDMs ( n = 3; LPS 0, 1, 2, 6 & 24 h). Representative blot shown. h COX2 and pro-IL-1β protein levels in non-stim and LPS-stimulated WT and m.5019A > G BMDMs treated with an anti-interferon-α/β receptor (IFNAR) monoclonal antibody (Ab) or isotype control Ab ( n = 3; LPS 6 h). Representative blot shown. i COX2 and pro-IL-1β protein levels in non-stim and LPS-stimulated WT and m.5019A > G BMDMs treated with Ruxolitinib or vehicle control Ab ( n = 3; LPS 6 h). Representative blot shown. j Inducible nitric oxide synthase (iNOS) protein levels in non-stim and LPS-stimulated WT and m.5019A > G BMDMs treated with Ruxolitinib or vehicle control Ab ( n = 3; LPS 24 h). Representative blot shown. Data are log 2 FC or mean ± s.e.m. n number represents independent biological replicates (mice) from a minimum of three independent experiments. P- values calculated using two-tailed Student’s t test for two group comparisons, multiple two-tailed unpaired t tests corrected for multiple comparisons using Benjamini, Krieger and Yekutieli method or one-way ANOVA corrected for multiple comparisons using Tukey method.

Article Snippet: Pre-treatment of BMDMs with IMT1 (HY-134539; MedChemExpress; 10 μM) for 24 h or RU.521 (HY-114180; MedChemExpress; 10 μM) for 1 h prior to LPS stimulation were used to inhibit mitochondrial transcription and the cGAS-STING pathway, respectively.

Techniques: RNA Sequencing, Expressing, Cell Culture, Control, Two Tailed Test

Journal: Nature Communications

Article Title: An inherited mitochondrial DNA mutation remodels inflammatory cytokine responses in macrophages and in vivo in mice

doi: 10.1038/s41467-025-65023-4

Figure Lengend Snippet: a , b Representative immunofluorescence staining of cytochrome c (Cyt c) and translocase of the outer membrane 20 (TOM20) coupled to confocal microscopy in non-stimulated (non-stim) wildtype (WT) and m.5019A > G BMDMs ( a ) and mitochondrial morphology analysis in non-stim WT ( n = 4), non-stim m.5019A > G ( n = 6), lipopolysaccharide (LPS)-stimulated WT ( n = 3) and LPS-stimulated m.5019A > G ( n = 4) BMDMs ( b ) (LPS 6 h; minimum of 20 cells analysed per condition per biological replicate) ( P = 0.000038). Scale bars: 5 μm. c , d Representative immunofluorescence staining of TOM20 and ATP synthase coupled to super-resolution microscopy in non-stim WT and m.5019A > G BMDMs ( c ) and mitochondrial morphology analysis of non-stim WT ( n = 3), non-stim m.5019A > G ( n = 3), LPS-stimulated WT ( n = 2) and LPS-stimulated m.5019A > G ( n = 3) BMDMs ( d ) (LPS 6 h; minimum of 33 cells analysed from independent biological replicates) ( P = 0.000000183; P = 0.0000000003). Scale bars: 5 μm. e , f Representative immunofluorescence staining of dynamin-related protein 1 (DRP1) and TOM20 coupled to confocal microscopy ( e ) and Pearson r correlation analysis ( f ) in non-stim and LPS-stimulated WT and m.5019A > G BMDMs ( n = 3; LPS 6 h; minimum of 20 cells analysed per condition per biological replicate) ( P = 0.000059). Scale bars: 5 μm. Data are mean ± s.e.m or ± s.d. n number represents independent biological replicates (mice) from a minimum of two independent experiments. P- values calculated using multiple two-tailed unpaired tests corrected for multiple comparisons using the Holm-Sidak method or one-way ANOVA corrected for multiple comparisons using the Kruskal-Wallis method.

Article Snippet: Pre-treatment of BMDMs with IMT1 (HY-134539; MedChemExpress; 10 μM) for 24 h or RU.521 (HY-114180; MedChemExpress; 10 μM) for 1 h prior to LPS stimulation were used to inhibit mitochondrial transcription and the cGAS-STING pathway, respectively.

Techniques: Immunofluorescence, Staining, Membrane, Confocal Microscopy, Super-Resolution Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: An inherited mitochondrial DNA mutation remodels inflammatory cytokine responses in macrophages and in vivo in mice

doi: 10.1038/s41467-025-65023-4

Figure Lengend Snippet: a , b Representative transmission electron microscopy (TEM) images ( a ) and cristae and mitochondrial aspect ratio (length/width) analysis ( b ) of non-stimulated (non-stim) and lipopolysaccharide (LPS)-stimulated wildtype (WT) and m.5019A > G BMDMs ( n = 3; LPS 6 h; mitochondria from a minimum of 9 cells were analysed per condition per biological replicate). Scale bars: 0.5 μm. Black arrows indicate mitochondria. c Mitochondrial (mt)DNA and mtRNA levels in cytosolic fraction of non-stim WT and m.5019A > G BMDMs ( n = 6). d , e Representative immunofluorescence staining of DNA and translocase of the outer membrane 20 (TOM20) coupled to confocal microscopy in LPS-stimulated WT and m.5019A > G BMDMs ( d ) and cytosolic DNA foci quantification in non-stim and LPS-stimulated WT and m.5019 A > G BMDMs ( e ) ( n = 3; LPS 24 h; minimum of 20 cells analysed per condition per biological replicate). White arrows indicate cytosolic DNA foci. f Ifnb1 expression (LPS 24 h) and interferon-β (IFN-β) release (LPS 6 h) in LPS-stimulated WT and m.5019 A > G macrophages pre-treated with cyclic GMP-AMP synthase (cGAS) inhibitor RU.521 or vehicle control (DMSO) for 1 h ( n = 3) ( P = 0.000262). g mt-Nd1 ( P = 0.000000008; P = 0.000946773), mt-Co3 ( P = 0.000000005; P = 0.000236469), Ifnb1 ( P = 0.000402; P = 0.000402) expression and IFN-β release in LPS-stimulated WT and m.5019A > G macrophages pre-treated with inhibitor of mitochondrial transcription 1 (IMT1) or vehicle control (DMSO) for 24 h ( n = 3; LPS 24 h). Data are mean ± s.e.m. n number represents independent biological replicates (mice) from a minimum of three independent experiments. P- values calculated using multiple two-tailed unpaired t tests corrected for multiple comparisons using the Benjamini, Krieger and Yekutieli method.

Article Snippet: Pre-treatment of BMDMs with IMT1 (HY-134539; MedChemExpress; 10 μM) for 24 h or RU.521 (HY-114180; MedChemExpress; 10 μM) for 1 h prior to LPS stimulation were used to inhibit mitochondrial transcription and the cGAS-STING pathway, respectively.

Techniques: Transmission Assay, Electron Microscopy, Immunofluorescence, Staining, Membrane, Confocal Microscopy, Expressing, Control, Two Tailed Test