irf3 4302 pirf3 s396  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: Activation of IRF3 in cardiomyocytes impairs mitochondrial oxidative function through PGC-1α inhibition and drives heart failure

doi: 10.1038/s41467-026-69792-4

Figure Lengend Snippet: a Immunoblot showing expression of p-IRF3 and IRF3 levels in the left ventricle tissue of humans with ischemic cardiomyopathy (ICM) compared to non-failing (NF) hearts, NF ( n = 6) and ICM ( n = 6), biological replicates. Vinculin was used as a loading control. b Quantification of the immunoblot shown in Fig. 1a. Statistical significance was calculated by unpaired two-tailed Student’s t test, NF ( n = 6) and ICM ( n = 6), *** P = 5.1 × 10 -7 . c mRNA expression of IRF3 and target genes in the left ventricle tissue of human patients with ICM compared to NF hearts determined by qPCR. Statistical significance was calculated by unpaired two-tailed Student’s t test, samples from NF ( n = 7) and ICM patients ( n = 10) are biological replicates, IRF3 : ** P = 0 0.0230; IFIT2 : * P = 0.0534. d UMAP showing single-cell analysis of genes regulated in the left ventricle tissue of patients with heart failure compared to healthy control hearts. e Gene expression of IRF3 in cardiomyocytes isolated from patients with heart failure. f Immunoblot showing expression of p-IRF3 and IRF3 levels in the left ventricle tissue of mice with LAD induced myocardial infarction (MI) compared to Sham operated αMHC Cre control. Vinculin was used as a loading control. N = 3 per group, biological replicates. g Box plot showing gene expression of Irf3 and target genes regulating type I IFN signaling in the left ventricle tissue of mice with LAD induced MI determined by qPCR. Sham ( n = 7) and LAD ( n = 8), biological replicates. Box and whiskers plot showing all minimum to maximum points. Significance was calculated by unpaired two-tailed Student’s t test, Irf3 : ** P = 0.0047; Ifnβ : ** P = 0.0041; Ifit1 : ** P = 0.0022; Ifit2 : ** P = 0.0099; Ifit3 : ** P = 0.0046; Rsad2 : * P = 0.0438; Ccl2: * P = 0.0311. h Immunoblot showing expression of OXPHOS proteins [NADH: ubiquinone oxidoreductase (NDUFB8), Succinate dehydrogenase (SDHB), Cytochrome c oxidase I (CoI), Ubiquinol Cytochrome c oxidoreductase (UQCRC2), ATP synthase (ATP5A)] in the left ventricle tissue of humans with ICM compared to NF hearts, n = 6 per group, biological replicates. i Quantification of the immunoblot shown in Fig. 1h. Statistical significance was calculated by unpaired two-tailed Student’s t test, n = 6 per group, biological replicates, NDUFB8: *** P = 0.0005; SDHB: * P = 0.0269; UQCRC2: * P = 0.0162; COI: ** P = 0.0024; ATP5A: ** P = 0.0015. j Gene expression of mitochondrial OXPHOS marker genes by qPCR in the left ventricle tissue of mice with LAD induced MI. Box and whiskers plot showing all minimum to maximum points. Sham ( n = 7) and LAD ( n = 8), biological replicates. Significance was calculated by unpaired two-tailed Student’s t test and P values are shown in the box plot. k Nd1 , 16sRNA (mitochondrial) and Sdhb (nuclear) levels in mtDNA isolated from rat left ventricle was quantified by qPCR. Data points represent mtDNA samples ( n = 4), biological replicates. l Immunoblot showing pIRF3 levels in primary cardiomyocytes treated with 1 µg/ml of freshly isolated mtDNA for 6 h. N = 3 independent replicates per group. Data in all panels are represented as mean ± SEM. Source data are provided as a Source file.

Article Snippet: Antibodies were purchased from Cell Signaling (IRF3, 4302; pIRF3 [S396], 29047; Vinculin, 4650; HA-Tag, 3724; VDAC [D73D12], 4661; Histone H3 [D1H2], 4499; OGT [D1D8Q], 24083; p-p38MAPK [Thr180/Tyr182] 9211; p38MAPK, 9212; pAMPKα [Thr172], 2535; AMPKα, 2532; pAKT [S473], 4051; AKT, 9272; O-GlcNAc [CTD110.6], 9875), Abcam (OXPHOS, ab110413), MerckMillipore (PGC-1α, ST1202), and Sigma (Flag M2 [F1804], GAPDH [G8795]).

Techniques: Western Blot, Expressing, Control, Two Tailed Test, Single-cell Analysis, Gene Expression, Isolation, Marker

Journal: Nature Communications

Article Title: Activation of IRF3 in cardiomyocytes impairs mitochondrial oxidative function through PGC-1α inhibition and drives heart failure

doi: 10.1038/s41467-026-69792-4

Figure Lengend Snippet: a Schematic representation of cardiomyocyte-specific IRF3 deletion using mouse with floxed IRF3 allele crossed to αMHC-Cre (designated as Cre in this Figure). b Gene expression of Irf3 mRNA levels in isolated cardiomyocytes from 12wk old αMHC-Cre and CMI3KO mice. n = 6 (αMHC-Cre) and n = 4 (CMI3KO), biological replicates. Significance was calculated by unpaired two-tailed Student’s t test, *** P = 1.8 × 10 -5 . c Immunoblot showing IRF3 protein levels in isolated cardiomyocytes from 12wk old αMHC-Cre and CMI3KO mice, n = 2 per group, biological replicates. Experiment was repeated twice with similar results. d Schematic representation of experimental plan to determine cardiac function in CMI3KO mice upon LAD induced MI. αMHC-Cre served as control. e Representative images of echocardiography performed in 12wk old αMHC-Cre control and CMI3KO mice. f-g Cardiac function in CMI3KO mice compared to αMHC-Cre controls determined by echocardiography, n = 8 (αMHC-Cre), n = 8 (CMI3KO), biological replicates. Data analyzed by one-way analysis of variance (ANOVA) with Šídák’s multiple comparison test, %EF Cre LAD vs Cre Sham: **** P = < 0.0001; %EF CMI3KO KO LAD vs Cre LAD: * *P = 0.0051; %FS Cre LAD vs Cre Sham: **** P = < 0.0001; %EF CMI3KO KO LAD vs Cre LAD: * *P = 0.0072. h Heart weight/body weight ratio in αMHC-Cre control and CMI3KO mice. Sham: n = 7 (αMHC-Cre), n = 8 (CMI3KO); LAD: n = 7 (αMHC-Cre), n = 9 (CMI3KO), biological replicates, data analyzed by one-way analysis of variance (ANOVA) with Šídák’s multiple comparison test, *P = 0.0145. i Lung weight/body weight ratio in αMHC-Cre control and CMI3KO mice. Sham: n = 7 (αMHC-Cre), n = 8 (CMI3KO); LAD: n = 7 (αMHC-Cre), n = 9 (CMI3KO), biological replicates. j Representative images of Masson Trichrome staining in cardiac tissue of αMHC-Cre control and CMI3KO mice. k Quantification of Masson Trichrome staining in Fig. 2j and analysis using one-way analysis ANOVA with Šídák’s multiple comparison test, ****P = < 0.0001. Box and whiskers plot showing all minimum to maximum points. n = 5 (Cre control LAD), n = 5 (CMI3KO LAD), n = 3 (Cre control Sham), n = 4 (CMI3KO Sham), biological replicates. Data in all panels are represented as mean ± SEM. Source data are provided as a Source data file.

Article Snippet: Antibodies were purchased from Cell Signaling (IRF3, 4302; pIRF3 [S396], 29047; Vinculin, 4650; HA-Tag, 3724; VDAC [D73D12], 4661; Histone H3 [D1H2], 4499; OGT [D1D8Q], 24083; p-p38MAPK [Thr180/Tyr182] 9211; p38MAPK, 9212; pAMPKα [Thr172], 2535; AMPKα, 2532; pAKT [S473], 4051; AKT, 9272; O-GlcNAc [CTD110.6], 9875), Abcam (OXPHOS, ab110413), MerckMillipore (PGC-1α, ST1202), and Sigma (Flag M2 [F1804], GAPDH [G8795]).

Techniques: Gene Expression, Isolation, Two Tailed Test, Western Blot, Control, Comparison, Staining

Journal: Nature Communications

Article Title: Activation of IRF3 in cardiomyocytes impairs mitochondrial oxidative function through PGC-1α inhibition and drives heart failure

doi: 10.1038/s41467-026-69792-4

Figure Lengend Snippet: a Representative model showing strategy of CMI3OE transgenic mouse generation. Irf3-2D fl/fl mice were bred with αMHCMerCreMer (αMHCMCM) mice to generate tamoxifen inducible cardiomyocyte-specific CMI3OE ( Irf3-2D fl/αMHCMCM) mice. b Schematic representation of cardiomyocyte-specific expression of murine IRF3-2D by oral gavage of low dose tamoxifen (30 mg/kg) to induce MerCreMer-mediated excision of cardiomyocyte-specific floxed cassette in 12wk old male mice. c Gross morphology of αMHCMCM (Cre) and CMI3OE male mice at 12wks of age. d Kaplan-Meier curve showing survival of CMI3OE compared to αMHCMCM (Cre) mice. e Western blot showing protein levels of IRF3 in the cardiac ventricular tissue, n = 3 per group, biological replicates. f Quantification of the Western blot shown in Fig. 3e, n = 3 per group, biological replicates. Data analyzed by unpaired two-tailed Student’s t test ***P = 2 × 10 -5 . g mRNA expression of Irf3 and its target genes in the cardiac ventricular tissue determined by qPCR, n = 7 per group, biological replicates. Data analyzed by unpaired two-tailed Student’s t test, Irf3 : *** P = 1.8 × 10 -8 ; Ifnβ : * P < 0.0280; Ifit1 : *** P < 1.5 × 10 -9 ; Ifit2 : *** P < 0.0005; Ifit3 : *** P < 0.0002; Rsad2 : *** P < 9.9 × 10 -7 ; Ccl2 : ** P < 0.0030. h-j Plasma cytokine levels in CMI3OE mice determined by ELISA. Samples used are biological replicates. Data analyzed by unpaired two-tailed Student’s t test, CCL2: * P = 0.0430 [ n = 5 per group]; TNFα: * P = 0.0229 [ n = 5 per group]; IL-6: * P = 0.0532 [ n = 6 (Cre), n = 9 (CMI3OE)]. k Body weight measured in αMHCMCM (Cre) and CMI3OE mice, n = 5 per group. l Representative echocardiography images from αMHCMCM (Cre) and CMI3OE mice. m – q Cardiac function in αMHCMCM (Cre) and CMI3OE mice determined by echocardiography, n = 5 per group, biological replicates. Data analyzed by unpaired two-tailed Student’s t test, %EF: *** P = 2.3 × 10 -5 ; %FS: *** P = 5.8 × 10 -5 ; LVPWd: * P = 0.0448; LVmass/BW: * P = 0.0533. Cardiac function was measured in three independent cohorts with similar results. r Heart weight to tibia length determined in αMHCMCM (Cre) and CMI3OE mice, n = 5 per group, data analyzed by unpaired two-tailed Student’s t test, * P = 0.0399. s Lung weight to tibia length determined in αMHCMCM (Cre) and CMI3OE mice, n = 5 per group. Survival curve, gene expression analysis, and echocardiography experiments were performed using independent cohorts. Data in all panels are represented as mean ± SEM. Source data are provided as a Source data file.

Article Snippet: Antibodies were purchased from Cell Signaling (IRF3, 4302; pIRF3 [S396], 29047; Vinculin, 4650; HA-Tag, 3724; VDAC [D73D12], 4661; Histone H3 [D1H2], 4499; OGT [D1D8Q], 24083; p-p38MAPK [Thr180/Tyr182] 9211; p38MAPK, 9212; pAMPKα [Thr172], 2535; AMPKα, 2532; pAKT [S473], 4051; AKT, 9272; O-GlcNAc [CTD110.6], 9875), Abcam (OXPHOS, ab110413), MerckMillipore (PGC-1α, ST1202), and Sigma (Flag M2 [F1804], GAPDH [G8795]).

Techniques: Transgenic Assay, Expressing, Western Blot, Two Tailed Test, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Gene Expression

Journal: Nature Communications

Article Title: Activation of IRF3 in cardiomyocytes impairs mitochondrial oxidative function through PGC-1α inhibition and drives heart failure

doi: 10.1038/s41467-026-69792-4

Figure Lengend Snippet: a Heat map showing differential gene expression and GO biological processes altered in 12wk old male CMI3OE and Cre control mice. b Log2 ratio in the y axis is computed at P value less than 0.05 significance threshold from the bulk RNA-seq differential expression gene dataset showing expression of significantly altered inflammatory, mitochondrial and fibrotic marker genes in CMI3OE compared to Cre control mice. n = 4 per group, biological replicates. c Immunoblot showing cardiac OXPHOS protein levels [NDUFB8 (Complex I), SDHB (Complex II), CoI (Complex IV), UQCRC2 (Complex III), ATP5A (Complex V)] in the CMI3OE mice compared to Cre controls. n = 4 per group, biological replicates. d Quantification of the immunoblots shown in Fig. 4c, n = 4 per group. Data analyzed by unpaired two-tailed Student’s t test, CI: * P = 0.0241; CII: ** P = 0.0102; CIII: *** P = 0.0006; CIV: ** P = 0.0030. e Schematic representation of creatine kinase regulating phosphocreatine/creatine shuttle in cardiac cytosol and mitochondria. f Plasma levels of cardiac creatine kinase (CK-MB) levels in CMI3OE compared to Cre control mice determined using ELlSA kit. Data analyzed by unpaired two-tailed Student’s t test, * P = 0.0504, n = 5 (Cre) n = 7 (CMI3OE) per group, biological replicates. Box and whiskers plot showing all minimum to maximum points. g Relative quantification of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) was performed in mtDNA isolated from 12wk old CMI3OE and Cre control mice by amplification of Nd1 and 16sRNA belonging to the stable part of the mtDNA and normalized to hexokinase 2 (Hk2) gene. n = 6 (Cre), n = 4 (CMI3OE). Box and whiskers plot showing all minimum to maximum points. h Expression of mitochondrial transcription marker genes in the cardiac ventricle tissue of CMI3OE mice compared to Cre Control. Data analyzed by unpaired two-tailed Student’s t test, Tfb2m: * P = 0.0186; Ppargc1a: *** P = 0.0001, n = 5 per group, biological replicates. i Expression of Flag-PGC-1α and HA-IRF3-2D was obtained by transfection in neonatal rat cardiomyocytes. The immunoblot shows co-immunoprecipitation of Flag-PGC-1α with HA-IRF3-2D. j Cardiomyocytes were isolated from CMI3OE and Cre control mice using Langendorff-free method. The immunoblot shows co-immunoprecipitation of IRF3 with PGC-1α in cardiomyocytes. Data in all panels are represented as mean ± SEM. Source data are provided as a Source data file.

Article Snippet: Antibodies were purchased from Cell Signaling (IRF3, 4302; pIRF3 [S396], 29047; Vinculin, 4650; HA-Tag, 3724; VDAC [D73D12], 4661; Histone H3 [D1H2], 4499; OGT [D1D8Q], 24083; p-p38MAPK [Thr180/Tyr182] 9211; p38MAPK, 9212; pAMPKα [Thr172], 2535; AMPKα, 2532; pAKT [S473], 4051; AKT, 9272; O-GlcNAc [CTD110.6], 9875), Abcam (OXPHOS, ab110413), MerckMillipore (PGC-1α, ST1202), and Sigma (Flag M2 [F1804], GAPDH [G8795]).

Techniques: Gene Expression, Control, RNA Sequencing, Quantitative Proteomics, Expressing, Marker, Western Blot, Two Tailed Test, Clinical Proteomics, Isolation, Amplification, Transfection, Immunoprecipitation

Journal: Nature Communications

Article Title: Activation of IRF3 in cardiomyocytes impairs mitochondrial oxidative function through PGC-1α inhibition and drives heart failure

doi: 10.1038/s41467-026-69792-4

Figure Lengend Snippet: a Effect on Ppargc1α mRNA levels was determined by qPCR in neonatal rat cardiomyocytes transduced with murine PGC-1α and murine IRF3-2D. n = 6 replicates per group. Data analyzed by unpaired two-tailed Student’s t test, IRF3-2D: *** P = 3.9 × 10 -5 ; PGC-1α: *** P = 2.2 × 10 -7 . b Effect on Irf3 mRNA levels was determined by qPCR in neonatal rat cardiomyocytes transduced with murine PGC-1α and murine IRF3-2D. n = 6 replicates per group. Data analyzed by unpaired two-tailed Student’s t test, IRF3-2D: *** P = 2.2 × 10 -8 ; PGC-1α: *** P = 0.0003. c Effect on IRF3 target genes upon Ppargc1α expression in neonatal rat cardiomyocytes was determined by qPCR, n = 6 replicates per group. Data analyzed by unpaired two-tailed Student’s t test, Ifit2 : * P = 0.0163; Ifit3 : *** P = 1.9×10 -5 ; Isg15 : *** P = 8.5 × 10 -5 ; Rsad2 : *** P = 7.3 × 10 -9 . d Effect on mitochondrial marker genes upon Ppargc1α expression in neonatal rat cardiomyocytes was determined by qPCR. n = 6 replicates per group. Data analyzed by unpaired two-tailed Student’s t test, Atp6 *** P = 0.0004; Atp8 : *** P = 0005; Cytb : *** P = 2.1 × 10 -5 ; Cox1 : *** P = 0.0006; Cox2: *** P = 0.0002; Cox3 : *** P = 5.1 × 10 -5 ; Nd1 : *** P = 8.3 × 10 -5 . e Effect on Ppargc1α mRNA levels upon siRNA mediated Irf3 knockdown in neonatal rat cardiomyocytes was determined by qPCR. n = 6 replicates per group. Data analyzed by unpaired two-tailed Student’s t test, * P = 0.0004. f Luciferase reporter assay was performed in cardiomyocytes isolated from neonatal rats upon transient transfection with PPRE-X3-TK-luc luciferase reporter plasmid. Co-transfection of PPARα, RXRα, Renilla, PGC-1α, and IRF3 expression vector was performed as indicated in the experimental section. Luciferase activity was normalized to Renilla (internal control), n = 6 replicates per group. Box and whiskers plot showing all minimum to maximum points. Data analyzed by unpaired two-tailed Student’s t test, P values are included in the box plot. g Luciferase reporter assay was performed in cardiomyocytes isolated from neonatal rats upon transient transfection with siRNA targeting rat IRF3. Co-transfection of PPRE-X3-TK-luc luciferase reporter, PPARα, RXRα, Renilla, and PGC-1α expression vectors was performed as indicated in the experimental section. Luciferase activity was normalized to Renilla (internal control), n = 8 replicates per group. Box and whiskers plot showing all minimum to maximum points. Data analyzed by unpaired two-tailed Student’s t test, P values are included in the box plot. h Relative mRNA levels of Foxo1 , Mef2a , and Esrra in the left ventricle of CMI3OE mice. n = 4 per group, biological replicates. Data analyzed by unpaired two-tailed Student’s t test, Foxo1 : *** P = 0.0007, Mef2a : *** P = 0.0001; Foxo1: * P = 0.0194. i Immunoblot showing protein levels of PGC-1α, OGT, p-p38MAPK, p38MAPK, pAMPKα, AMPKα, pAKT, and AKT in CMI3OE mice compared to Cre control. n = 4 per group, biological replicates. j Immunoblot showing O-GlcNAcylation of proteins in the left ventricle of CMI3OE mice compared to Cre control. n = 4 per group, biological replicates. k Relative mRNA levels of Ogt and Mapk14 in the left ventricle of CMI3OE mice. n = 4 per group, biological replicates. Data analyzed by unpaired two-tailed Student’s t test, Ogt : * P = 0.0398, Mapk14 : *** P = 0.0011. Experiments in NRCMs were repeated three times with similar results. Data in all panels are represented as mean ± SEM. Source data are provided as a Source data file.

Article Snippet: Antibodies were purchased from Cell Signaling (IRF3, 4302; pIRF3 [S396], 29047; Vinculin, 4650; HA-Tag, 3724; VDAC [D73D12], 4661; Histone H3 [D1H2], 4499; OGT [D1D8Q], 24083; p-p38MAPK [Thr180/Tyr182] 9211; p38MAPK, 9212; pAMPKα [Thr172], 2535; AMPKα, 2532; pAKT [S473], 4051; AKT, 9272; O-GlcNAc [CTD110.6], 9875), Abcam (OXPHOS, ab110413), MerckMillipore (PGC-1α, ST1202), and Sigma (Flag M2 [F1804], GAPDH [G8795]).

Techniques: Transduction, Two Tailed Test, Expressing, Marker, Knockdown, Luciferase, Reporter Assay, Isolation, Transfection, Plasmid Preparation, Cotransfection, Activity Assay, Control, Western Blot

Journal: Nature Communications

Article Title: Activation of IRF3 in cardiomyocytes impairs mitochondrial oxidative function through PGC-1α inhibition and drives heart failure

doi: 10.1038/s41467-026-69792-4

Figure Lengend Snippet: a Schematic representation of the stable isotope labeling using U- 13 C 6 -glucose into glycolysis, PPP and TCA cycle. b – j Cardiomyocytes isolated from each CMI3OE and Cre control was plated in 6 cm dish at a density of ~1 × 10 6 cells/dish for 3 h. Cardiomyocytes were washed with PBS and cultured in medium containing 5.5 mM U- 13 C 6 -glucose for 10 min and 120 min. Metabolites were extracted and analyzed by LC-MS. Graphs show relative peak area of the isotopologue from glycolysis, PPP and TCA cycle pathways. Samples group, 0 min: Cre ( n = 4), CMI3OE ( n = 4); 10 min: Cre ( n = 5), CMI3OE ( n = 6); 120 min: Cre ( n = 5), CMI3OE ( n = 4), independent biological replicates. k Schematic representation of the fuel adaptation in the left ventricle tissue of CMI3OE mice. Altered metabolite levels indicate an increase in glycolysis and ketone bodies oxidation whereas β-oxidation, carnitine and NAD metabolism is downregulated upon IRF3 activation in CMI3OE mice. Furthermore, this was associated with overall impaired ETC machinery in CMI3OE mice. Data in all panels are represented as mean ± SEM. Source data are provided as a Source data file.

Article Snippet: Antibodies were purchased from Cell Signaling (IRF3, 4302; pIRF3 [S396], 29047; Vinculin, 4650; HA-Tag, 3724; VDAC [D73D12], 4661; Histone H3 [D1H2], 4499; OGT [D1D8Q], 24083; p-p38MAPK [Thr180/Tyr182] 9211; p38MAPK, 9212; pAMPKα [Thr172], 2535; AMPKα, 2532; pAKT [S473], 4051; AKT, 9272; O-GlcNAc [CTD110.6], 9875), Abcam (OXPHOS, ab110413), MerckMillipore (PGC-1α, ST1202), and Sigma (Flag M2 [F1804], GAPDH [G8795]).

Techniques: Quantitative Proteomics, Isolation, Control, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Activation Assay

Journal: Nature Communications

Article Title: Activation of IRF3 in cardiomyocytes impairs mitochondrial oxidative function through PGC-1α inhibition and drives heart failure

doi: 10.1038/s41467-026-69792-4

Figure Lengend Snippet: a Schematic representation of cardiomyocyte-specific expression of PGC-1α and EGFP using AAV9-TnT-PGC-1α and AAV9-TnT-EGFP and IRF3 activation with low dose tamoxifen gavage in CMI3OE mice. b Gene expression of Ppargc1α and Ppargc1β determined in the left ventricle of CMI3OE mice. n = 5 per group, biological replicates. Box and whiskers plot showing all minimum to maximum points. Statistical significance was assessed by unpaired two tailed Student’s t test, ** P = 0.0035. c Representative echocardiography images from CMI3OE mice treated with AAV9-TnT-PGC-1α and AAV9-TnT-EGFP compared to respective controls. d , e Cardiac function in CMI3OE mice treated with AAV9-TnT-PGC-1α and AAV9-TnT-EGFP was determined by echocardiography, n = 6 (Ctrl-AAV-EGFP), n = 5 (Ctrl-AAV-PGC-1α), n = 6 (CMI3OE-AAV-EGFP), n = 5 (CMI3OE-AAV-PGC-1α), biological replicates. Data analyzed by one-way ANOVA with Šídák’s multiple comparison test, P values are shown in the graph. f Heart weight to body weight ratio determined in CMI3OE mice treated with AAV9-TnT-PGC-1α and AAV9-TnT-EGFP, n = 6 (Ctrl-AAV-EGFP), n = 5 (Ctrl-AAV-PGC-1α), n = 6 (CMI3OE-AAV-EGFP), n = 5 (CMI3OE-AAV-PGC-1α), biological replicates. Statistical significance was assessed by unpaired two tailed Student’s t test. Data in all panels are represented as mean ± SEM. Source data are provided as a Source data file.

Article Snippet: Antibodies were purchased from Cell Signaling (IRF3, 4302; pIRF3 [S396], 29047; Vinculin, 4650; HA-Tag, 3724; VDAC [D73D12], 4661; Histone H3 [D1H2], 4499; OGT [D1D8Q], 24083; p-p38MAPK [Thr180/Tyr182] 9211; p38MAPK, 9212; pAMPKα [Thr172], 2535; AMPKα, 2532; pAKT [S473], 4051; AKT, 9272; O-GlcNAc [CTD110.6], 9875), Abcam (OXPHOS, ab110413), MerckMillipore (PGC-1α, ST1202), and Sigma (Flag M2 [F1804], GAPDH [G8795]).

Techniques: Expressing, Activation Assay, Gene Expression, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Activation of IRF3 in cardiomyocytes impairs mitochondrial oxidative function through PGC-1α inhibition and drives heart failure

doi: 10.1038/s41467-026-69792-4

Figure Lengend Snippet: a Heatmap showing differential gene expression between CMI3OE expressing EGFP or PGC-1α using CMI3OE-AAV9-TnT-EGFP or CMI3OE-AAV9-TnT-PGC-1α, respectively. b Log2 ratio in the y axis is computed at P value less than 0.05 significance threshold from the bulk RNA-seq differential expression gene dataset showing gene expression of inflammatory and fibrotic marker genes in CMI3OE-AAV-PGC-1α mice compared to CMI3OE-AAV-EGFP. N = 4 per group, biological replicates. c Mitochondrial transcription marker genes expression by qPCR in the cardiac ventricle tissue of CMI3OE-AAV-PGC-1α mice compared to CMI3OE-AAV-EGFP. n = 4 per group, biological replicates. Statistical significance was assessed by unpaired two tailed Student’s t test, mt-CoI : * P = 0.0160; mt-Co2 : ** P = 0.0100; mt-Co3 : ** P = 0.0027; mt-Nd1 : * P = 0.0212; mt-Nd2 : * P = 0.0189; mt-Nd3 : * P = 0.0455; mt-Nd4 : ** P = 0.0094; mt-Nd4l : * P = 0.0372; mt-Nd5 : ** P = 0.0091; mt-Nd6 : * P = 0.0154; mt-Cytb : ** P = 0.0095. d Immunoblot showing cardiac OXPHOS protein levels of NADH: ubiquinone oxidoreductase (NDUFB8), Succinate dehydrogenase (SDHB), Cytochrome c oxidase I (CoI), Ubiquinol Cytochrome c oxidoreductase (UQCRC2), ATP Synthase (ATP5A) determined in the CMI3OE-AAV-PGC-1α mice compared to CMI3OE-AAV-EGFP. n = 5 per group, biological replicates. e Quantification of the immunoblot shown in Fig. 9d, n = 5 per group, biological replicates. Statistical significance was assessed by unpaired two tailed Student’s t test, NDUFB8: * P = 0.0199; SDHB: ** P = 0.0040; UQCRC2: ** P = 0.0050; COI: * P = 0.0207; ATP5A: * P = 0.0476. f Relative expression of genes regulating NAD metabolism in the left ventricle of CMI3OE-AAV-PGC-1α compared to CMI3OE-AAV-EGFP mice by Reactome analysis. N = 4 per group, biological replicates. Box and whiskers plot showing all minimum to maximum points. Significance was assessed by unpaired two tailed Student’s t test, Idh2 : * P = 0.0289; Nadk2 : * P = 0.0248; Naprt : * P = 0.0246; Naxe : ** P = 0.0047; Nmnat1 : ** P = 0.0106; Nmnat3 : * P = 0.0293. g – i Determination of intracellular NAD + , NADH, NAD + /NADH ratio in neonatal rat cardiomyocytes upon expression of LacZ, IRF3-2D and PGC-1α by adenovirus mediated transduction using NAD + /NADH Quantification kit. n = 4 per replicates group. Data analyzed by one-way ANOVA with Tukey’s multiple comparison test, P values are shown in the graph. Data in all panels are represented as mean ± SEM. Source data are provided as a Source data file.

Article Snippet: Antibodies were purchased from Cell Signaling (IRF3, 4302; pIRF3 [S396], 29047; Vinculin, 4650; HA-Tag, 3724; VDAC [D73D12], 4661; Histone H3 [D1H2], 4499; OGT [D1D8Q], 24083; p-p38MAPK [Thr180/Tyr182] 9211; p38MAPK, 9212; pAMPKα [Thr172], 2535; AMPKα, 2532; pAKT [S473], 4051; AKT, 9272; O-GlcNAc [CTD110.6], 9875), Abcam (OXPHOS, ab110413), MerckMillipore (PGC-1α, ST1202), and Sigma (Flag M2 [F1804], GAPDH [G8795]).

Techniques: Gene Expression, Expressing, RNA Sequencing, Quantitative Proteomics, Marker, Two Tailed Test, Western Blot, Transduction, Comparison

Journal: Nature Communications

Article Title: Activation of IRF3 in cardiomyocytes impairs mitochondrial oxidative function through PGC-1α inhibition and drives heart failure

doi: 10.1038/s41467-026-69792-4

Figure Lengend Snippet: a Upregulated pathways in the left ventricle of CMI3OE mice expressing PGC-1α or EGFP (CMI3OE-AAV-PGC-1α vs CMI3OE-AAV-EGFP). b-c Heatmap showing relative expression of genes regulating fatty acid oxidation, fatty acid metabolism and TCA cycle identified by Reactome enrichment analysis in the left ventricle of CMI3OE-AAV-PGC-1α compared to CMI3OE-AAV-EGFP mice. d , e Fuel flex assay using Seahorse analyzer to determine glucose and fatty acid dependency in NRCMs expressing IRF3-2D and PGC-1α compared to LacZ control. For glucose dependency: LacZ ( n = 7), PGC-1α ( n = 6), IRF3-2D ( n = 4), IRF3-2D + PGC-1α ( n = 7). For FA dependency: LacZ ( n = 6), PGC-1α ( n = 6), IRF3-2D ( n = 5), IRF3-2D + PGC-1α ( n = 5). Box and whiskers plot showing all minimum to maximum points. Data analyzed by one-way ANOVA with Tukey’s multiple comparison test, P values are shown in the graph. f Schematic representation of the effect of IRF3 activation in cardiomyocytes leading to cardiac dysfunction by downregulation of PGC-1α and mitochondrial OXPHOS function. The figure also shows p-IRF3 and PGC-1α levels exist in inverse correlation within cardiomyocytes. Data in all panels are represented as mean ± SEM. Source data are provided as a Source data file.

Article Snippet: Antibodies were purchased from Cell Signaling (IRF3, 4302; pIRF3 [S396], 29047; Vinculin, 4650; HA-Tag, 3724; VDAC [D73D12], 4661; Histone H3 [D1H2], 4499; OGT [D1D8Q], 24083; p-p38MAPK [Thr180/Tyr182] 9211; p38MAPK, 9212; pAMPKα [Thr172], 2535; AMPKα, 2532; pAKT [S473], 4051; AKT, 9272; O-GlcNAc [CTD110.6], 9875), Abcam (OXPHOS, ab110413), MerckMillipore (PGC-1α, ST1202), and Sigma (Flag M2 [F1804], GAPDH [G8795]).

Techniques: Expressing, Control, Comparison, Activation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of STING-induced mitochondrial Drp1/N-GSDMD-mediated MtDNA release alleviates Sepsis-induced lung injury

doi: 10.1007/s00018-025-05774-x

Figure Lengend Snippet: GSDMD signal was correlatively activated with STING during ALI. A UMAP projections illustrating various cell types from samples obtained from COVID-19 patients. B Hierarchical clustering of gene expression data related to STING (TMEM173 for STING) and pyroptosis. This expression data were sourced from the GEO database ( GSE145926 ). C Correlation heatmaps depicting the expression of STING-related and pyroptosis-related genes. D - F Representative results of H&E staining after a time course of LPS challenge ( n = 6), with quantifications of the lung injury score and the ratio of lung wet weight to dry weight provided on the right. Images are displayed at 200× magnification, with a scale bar of 100 μm. G - H Relative quantification of mitochondrial DNA (D-loop and COXI) in the BALF of mice following a time course of LPS challenge ( n = 6). I Western blot images showing the STING/TBK1/IRF3 pathway in lung tissues after a time course of LPS challenge ( n = 6). J Western blot images for the NLRP3/Caspase1/GSDMD/IL-1β pathway in lung tissues after a time course of LPS challenge ( n = 6). K - N Relative mRNA levels of IL-1β, IL-6, TNF-α, and IFN-β in lung tissues following a time course of LPS challenge ( n = 6). O - S Representative results from co-immunostaining of F4/80, STING, and N-GSDMD in lung tissues after a time course of LPS challenge ( n = 6), with quantifications shown below

Article Snippet: Phosphorylated IRF3 (pIRF3) , Abmart , TA2436 , 1:1000 , WB.

Techniques: Gene Expression, Expressing, Staining, Quantitative Proteomics, Western Blot, Immunostaining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of STING-induced mitochondrial Drp1/N-GSDMD-mediated MtDNA release alleviates Sepsis-induced lung injury

doi: 10.1007/s00018-025-05774-x

Figure Lengend Snippet: N-GSDMD translocation to mitochondria was attenuated by disulfiram during LPS challenge. A Analysis of total and mitochondrial N-GSDMD in THP-1 cells stimulated with LPS at various time points, using COXIV as a control for mitochondrial membrane enrichment ( n = 5). B Measurement of total and mitochondrial N-GSDMD in THP-1 cells exposed to different concentrations of LPS, with COXIV serving as a control for mitochondrial membrane enrichment ( n = 5). C Representative images from immunofluorescent staining showing N-GSDMD and TOMM20 following LPS or LPS plus ATP stimulation. D Measurement of N-GSDMD in Mitochondria and Plasma Membrane in THP-1 cells following LPS or LPS plus ATP stimulation, with COXIV and ATP5B serving as a control for Mitochondria and Plasma membrane enrichment. E - F Flow cytometry histograms illustrating anti-N-GSDMD staining in mitochondria, with quantification of the percentage of N-GSDMD-positive mitochondria displayed below. G - H Evaluation of mitochondrial N-GSDMD, STING, and p-STING levels in THP-1 cells after LPS stimulation and pretreatment with varying concentrations of DSF, with COXIV as a control for mitochondrial membrane enrichment ( n = 5). Quantifications are provided on the right. I Relative quantification of cytosolic mtDNA levels (ND-1, D-loop, COXIII, and Cytochrome B) in THP-1 cells stimulated with 1 µg/mL LPS for 4 h, following a 2-hour pretreatment with different concentrations of DSF ( n = 4). J Representative images showing PicoGreen and MitoTracker staining in THP-1 cells after LPS stimulation, with a pretreatment of 5 µM DSF. Scale bar = 5 μm. K - L Western blot results depicting the STING/TBK1/IRF3 pathway and GSDMD in THP-1 cells following LPS stimulation and pretreatment with various concentrations of DSF ( n = 5). Quantifications are shown on the right. M Relative mRNA levels of IL-1β, IL-6, and IFN-β in response to LPS stimulation after pretreatment with different concentrations of DSF ( n = 5)

Article Snippet: Phosphorylated IRF3 (pIRF3) , Abmart , TA2436 , 1:1000 , WB.

Techniques: Translocation Assay, Control, Membrane, Staining, Clinical Proteomics, Flow Cytometry, Quantitative Proteomics, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of STING-induced mitochondrial Drp1/N-GSDMD-mediated MtDNA release alleviates Sepsis-induced lung injury

doi: 10.1007/s00018-025-05774-x

Figure Lengend Snippet: Inhibition of STING abrogated mitochondrial translocation of N-GSDMD. A - B Western blot analyses displaying the STING/TBK1/IRF3 pathway in THP-1 cells subjected to varying durations of LPS stimulation ( n = 5). Quantifications are shown on the right. C - D Western blot results illustrating the STING/TBK1/IRF3 pathway in both shCtrl and shSTING THP-1 cells following LPS stimulation ( n = 5). Quantifications are provided on the right. E Relative mRNA expression levels of IL-1β, IL-6, and IFN-β in shCtrl and shSTING THP-1 cells upon LPS stimulation ( n = 5). F Measurement of mitochondrial membrane potential using flow cytometry with JC-1, where JC-1 aggregation indicates normal mitochondrial membrane potential on the y-axis, and JC-1 monomers reflect low membrane potential on the x-axis. G Relative quantification of cytosolic mtDNA levels (ND-1, D-loop, COXIII, and Cytochrome B) in shCtrl and shSTING THP-1 cells following LPS stimulation ( n = 4). H - K Analysis of total and mitochondrial N-GSDMD, STING and p-TBK1 in both shCtrl and shSTING THP-1 cells after LPS stimulation, with COXIV serving as a control for mitochondrial membrane enrichment ( n = 5). Quantifications are presented below. L Representative images from immunofluorescent staining for N-GSDMD and TOMM20 in shCtrl and shSTING THP-1 cells following LPS stimulation. Scale bar is set at 5 μm. Quantifications are displayed below

Article Snippet: Phosphorylated IRF3 (pIRF3) , Abmart , TA2436 , 1:1000 , WB.

Techniques: Inhibition, Translocation Assay, Western Blot, Expressing, Membrane, Flow Cytometry, Quantitative Proteomics, Control, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of STING-induced mitochondrial Drp1/N-GSDMD-mediated MtDNA release alleviates Sepsis-induced lung injury

doi: 10.1007/s00018-025-05774-x

Figure Lengend Snippet: Pharmacological inhibition of STING alleviated ALI via reducing mitochondrial fission. A - E Representative findings from H&E staining and immunofluorescent staining for STING and ROS in lung tissues subjected to LPS challenge, with or without H-151 pretreatment. Scale bar is set at 100 μm. Quantifications of the lung injury score, the ratio of lung wet weight to dry weight and STING and ROS fluorescent intensity are provided on the right. F - G Relative quantifications of mtDNA (D-loop and COXI) in the BALF of mice after LPS challenge, with or without H-151 pretreatment ( n = 6). H - K Relative mRNA expression levels of IL-1β, IL-6, TNF-α, and IFN-β in lung tissues after LPS challenge, with or without H-151 pretreatment ( n = 6). L Western blot analyses depicting the STING/TBK1/IRF3 pathway in lung tissues following LPS challenge, with or without pretreatment using H-151 ( n = 6). M Western blot images showing levels of GSDMD, N-GSDMD, MFN1, MFN2, and Drp1 in lung tissues following LPS challenge, with or without H-151 pretreatment ( n = 6). N Representative images illustrating co-immunostaining for Drp1 and N-GSDMD in lung tissues after LPS challenge, with or without H-151 pretreatment ( n = 6). Scale bar is set at 100 μm. Representative images depicting COXIV in lung tissues following LPS challenge, with or without H-151 pretreatment. Scale bar is set at 5 μm

Article Snippet: Phosphorylated IRF3 (pIRF3) , Abmart , TA2436 , 1:1000 , WB.

Techniques: Inhibition, Staining, Expressing, Western Blot, Immunostaining

Journal: Nature Communications

Article Title: Cascaded immunotherapy with implantable dual-drug depots sequentially releasing STING agonists and apoptosis inducers

doi: 10.1038/s41467-025-56407-7

Figure Lengend Snippet: a Schematic diagram of the experimental design for assessing the effects of drug depots on in-vitro activation of STING-IFNβ pathway in cancer cells and induction of immunogenic apoptosis. Representative calcein AM (live)/PI (dead)-stained fluorescence images ( b ) and cell viabilities ( c ) of B16F10 cells treated with indicated composites for 24 h ( n = 3 independent experiments). Representative flow cytometry pseudo-color plots ( d ) and representative immunofluorescence images with quantified 3D surface plots ( e ) reflecting the fluorescence intensities and distributions of pIRF3 in B16F10 cells treated with indicated composites for 6 h ( n = 3 independent experiments). f Representative western blots of crucial proteins separately involved in general apoptosis (BAX), ERS-induced apoptosis (Casp12 and C-Casp12), and STING-IFNβ pathway (pIRF3) in B16F10 cells treated with indicated composites for 6 h ( n = 4 independent experiments). Representative immunofluorescence images with quantified 3D surface plots showing the fluorescence intensities and distributions of CRT on cell membranes ( g ), the release of HMGB1 ( h ), and the release of ATP ( i ) of B16F10 cells treated with indicated composites for 12 h ( n = 3 independent experiments). j Relative mRNA level of ifnb1 in B16F10 cells treated with indicated composites for 6 h ( n = 3 independent experiments). Data are shown as mean ± SD. Statistical significances were evaluated by one-way ANOVA with Tukey’s multiple comparisons post hoc test. Source data are provided as a Source Data file.

Article Snippet: Rabbit-derived anti-phospho-IRF3 (pIRF3) polyclonal antibody (#AF2436) was purchased from Affinity Biosciences (Changzhou, China).

Techniques: In Vitro, Activation Assay, Staining, Fluorescence, Flow Cytometry, Immunofluorescence, Western Blot