Journal: Nature communications

Article Title: Primate-Specific miR-576-3p Sets Host Defense Signaling Threshold

doi: 10.1038/ncomms5963

Figure Lengend Snippet: a , Schematic showing the region of the 3’ UTR of STING that is recognized by the miR-576-3p seed sequence. b , HBEC were transfected with control or miR-576-3p (M-576-3p) mimic for 72 h. Cells were then mock infected or infected with VSV-GFP at an MOI 3 for 3 h. Total RNA was harvested from cells and STING mRNA levels were determined by qPCR and normalized to levels of β-actin. c , HBEC were transfected with miRNA mimic (M-576-3p) or inhibitor (I-576-3p) as in b and mock-infected or infected with VSV-GFP at MOI 0.1 for 18 h. Cell lysates were harvested and subjected to western blot analysis with anti-STING antibodies. β-actin serves as loading control. d,e , HBEC were transfected with 1 µg/ml of poly (I:C) for 6 h ( d ) or treated with 100 U/ml of IFNβ for 18 h ( e ) and levels of pri-mir-576 and SEC24B mRNA were analyzed by qPCR. f,g , HBEC were transfected with poly (I:C) as in d after siRNA knockdown of NFκB (P65) or IRF3. Relative pri-mir-576 ( f ) or SEC24B mRNA ( g ) levels were measured by qPCR. h , HBEC expressing control or miR-576-3p mimic (M-576-3p) or an siRNA targeting STING were transfected with poly (I:C) and levels of IFNβ mRNA were measured by qPCR. i , HBEC were transfected with control or miR-576-3p mimic (M-576-3p) and pre-treated with 100 U/ml of IFNβ prior to VSV infection. Cell viability was determined by measuring ATP levels of mock or infected cells. j , HBEC expressing control or miR-576-3p inhibitor (I-576-3p) were transfected with poly (I:C) and levels of IFNβ were measured by qPCR. Data are representative of three independent experiments. Unpaired two tailed t-test was used and error bars represent SD. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Recombinant human IFNβ was obtained from R&D systems.

Techniques: Sequencing, Transfection, Control, Infection, Western Blot, Knockdown, Expressing, Two Tailed Test

Journal: Nature communications

Article Title: Primate-Specific miR-576-3p Sets Host Defense Signaling Threshold

doi: 10.1038/ncomms5963

Figure Lengend Snippet: a , HBEC were transfected with 1 µg/ml of poly (I:C) for 6 h or treated with 100 U/ml of IFNβ for 18 h and levels of mature miR-576-3p were determined by qPCR. b , HBEC were mock infected or infected with HSV-1 at MOI 10 for 3 h and levels of miR-576-3p were measured by qPCR. Unpaired two tailed t-test was used and error bars represent SD. *p<0.05, **p<0.01. ns, non significant with respect to control.

Article Snippet: Recombinant human IFNβ was obtained from R&D systems.

Techniques: Transfection, Infection, Two Tailed Test, Control

Journal: Nature communications

Article Title: Primate-Specific miR-576-3p Sets Host Defense Signaling Threshold

doi: 10.1038/ncomms5963

Figure Lengend Snippet: a , Model illustrates regulation of IFN expression by miR-576-3p as a feedback mechanism. Targets of miR-576-3p are indicated by inhibitory red lines. b , Public available microRNA array datasets (GSE37425 and GSE37426) of synovial or renal tissues from RA or SLE patients, respectively, along with controls were obtained from the GEO database . Normalized expression values of miR-576-3p from each sample were used to calculate the relative levels and p-values of miR-576-3p for SLE or RA patients as compared to controls. Unpaired two tailed t-test was used and error bars represent SD. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Recombinant human IFNβ was obtained from R&D systems.

Techniques: Expressing, Two Tailed Test

Journal: Chemical science

Article Title: Dual-targeting biomimetic delivery for anti-glioma activity via remodeling the tumor microenvironment and directing macrophage-mediated immunotherapy.

doi: 10.1039/c7sc04853j

Figure Lengend Snippet: Fig. 4 (A) The cytotoxicity test (IC50) in the U87 and GL261 cells. (B) The cytotoxicity test in the U87 cells cultured with M1-CM or M2-CM. (C) The expression of MRs in TAM1 and TAM2 and the polarization modulation of the drugs. (D) The re-education of TAM2 treated with drugs and the expression of MRs and B7-H4. The ELISA analysis of the expression of TNF-a (E) and TGF-b1 (F) in TAM1 and TAM2 after drug treatment. The ELISA analysis of the expression of TNF-a (G) and TGF-b1 (H) in TAM1 and TAM2 cocultured with U87 cells after treatment.

Article Snippet: The mouse IFN-g Elisa Kit was purchased from R&D Systems (USA).

Techniques: Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Clinical Epigenetics

Article Title: The immunomodulatory anticancer agent, RRx-001, induces an interferon response through epigenetic induction of viral mimicry

doi: 10.1186/s13148-017-0312-z

Figure Lengend Snippet: RRx-001 induced IFN response through upregulation of type I and III IFN expression and JAK/STAT pathway. Cells were transiently (24 h) treated with 0.5 μM RRx-001 or 0.5 μM 5-AZA and subsequently maintained in drug-free medium for an additional 7 days. RRx-001 induced a significant increase in type I IFN (IFN-β) ( a ) and type III IFN (IL-29/IL-28B) ( b ) secretion into culture medium by HCT 116 cells as measured by ELISA. Transcript levels of IL29 / IL28A were also increased as determined by qPCR ( c ). ISGs ( IFI27 , IFi44 , IFI44L , and IFI6 ) were upregulated by 5-AZA and RRx-001 but blocked by the JAK/STAT inhibitor ruxolitinib (rux) at 2 μM concentration ( d ). Expression of ISGs ( IRF7 , ISG15 , DDX58 , and OASL ) was also upregulated in HCT 116 cells cultured in conditioned medium containing secreted IFNs induced by 5-AZA and RRx-001 as determined by qPCR ( e )

Article Snippet: Levels of type I IFN (IFN-β) and type III IFN (IL-29/IL-28B) were determined using a VeriKine Human IFN-beta ELISA Kit (PBL Assay Science, Piscataway Township, NJ, USA) and a Human IL-29/IL-28B (IFN-lambda 1/3) DuoSet ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to manufactures’ instructions, respectively.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture

Journal: Journal of neuroinflammation

Article Title: Zika virus infection of mature neurons from immunocompetent mice generates a disease-associated microglia and a tauopathy-like phenotype in link with a delayed interferon beta response.

doi: 10.1186/s12974-022-02668-8

Figure Lengend Snippet: Fig. 2 A delayed induction of IFNB response hampers the capacity of PCNs to stop ZIKV replication. PCNs from B6 embryos, non-infected (NI), non-treated (NT) or treated with anti-IFNAR antibodies or recIFNB before infection, were mock- or ZIKV-infected at a MOI of 5. a–d Neutralization of type-I IFN response with anti-IFNAR antibodies has no significant effect on ZIKV replication in PCNs until 72 h p.i. while treatment with recIFNB inhibits ZIKV replication from the onset of infection as shown by the relative expression of RNA levels analyzed by RT-qPCR with respect to Rplp0 used as reference gene. Symbols represent values obtained from n = 5 independent experiments with varying conditions analyzed in each experiment with a minimum of two different times p.i. analyzed per condition per experiment. a–c Bars represent means without s.d. with significance assessed in a by two-way ANOVA and Tukey’s multiple comparisons test. d Means are represented in graph. e PCNs from B6 embryos ZIKV-infected at a MOI of 5, either non-treated (NT) or treated with recIFNB added before infection (early recIFNB) or 48 h p.i. (late recIFNB), were collected 64 h p.i.. Results correspond to those obtained from n = 3 independent PCNs shown in different colors. Means are represented in graph with significance assessed by paired t-test. Only early recIFNB significantly inhibited ZIKV RNA production measured by RT-qPCR with respect to Rplp0 as reference gene. f MEFs from three different B6 embryos, either non-treated (NT) or treated with anti-IFNAR antibodies, were mock- or ZIKV-infected at a MOI of 5. Neutralization of type-I IFN response with anti-IFNAR antibodies significantly affected ZIKV starting at 48 h p.i. as shown by the relative expression of ZIKV RNA levels analyzed by RT-qPCR with respect to Rplp0 used as reference gene. Symbols represent values obtained from each of the n = 3 different embryos. Bars represent means without s.d. with significance assessed in by two-way ANOVA and Tukey’s multiple comparisons test. P-value < 0.001 (***), < 0.01 (**), < 0.05 (*) and ns not significant

Article Snippet: When indicated, anti-IFNAR antibody or recombinant IFNB (8234-MB, R&D Systems) were added in the culture medium overnight before infection and 48 h p.i. at a final concentration of 6.5 μg/ml and 50 U/well respectively.

Techniques: Infection, Neutralization, Expressing, Quantitative RT-PCR

Journal: Journal of neuroinflammation

Article Title: Zika virus infection of mature neurons from immunocompetent mice generates a disease-associated microglia and a tauopathy-like phenotype in link with a delayed interferon beta response.

doi: 10.1186/s12974-022-02668-8

Figure Lengend Snippet: Fig. 3 Susceptibility to ZIKV infection is correlated with the inability of PCNs to stop viral replication. PCNs from non-susceptible CC001 and susceptible CC071 immunocompetent mice were either non-infected (NI) or ZIKV-infected at a MOI of 5. Levels of ZIKV RNA and of RNAs coding for IFNB and ISGs were analyzed by RT-qPCR with respect to Rplp0 used as reference gene. Symbols represent values obtained from n = 3 and 2 independent experiments for CC001 and CC071, respectively. Bars represent means

Article Snippet: When indicated, anti-IFNAR antibody or recombinant IFNB (8234-MB, R&D Systems) were added in the culture medium overnight before infection and 48 h p.i. at a final concentration of 6.5 μg/ml and 50 U/well respectively.

Techniques: Infection, Quantitative RT-PCR

Journal: Journal of neuroinflammation

Article Title: Zika virus infection of mature neurons from immunocompetent mice generates a disease-associated microglia and a tauopathy-like phenotype in link with a delayed interferon beta response.

doi: 10.1186/s12974-022-02668-8

Figure Lengend Snippet: Fig. 9 ZIKV-infection of neurons signals for IFNB-dependent microglia activation while inducing Tau phosphorylation. ZIKV-infected mature neurons from immunocompetent mice display a delayed IFNB response unable to stop viral replication leading to the accumulation of viral RNA in association with the induction of pathological phosphorylated Tau protein (P-Tau). ZIKV-infection leads to the activation of the expression of the gene coding for the RNA-dependent kinase PKR in PCNs and mouse brain. A role for PKR in the enhancement of P-Tau is proposed in relation with the capacity of PKR to be activated by viral cytoplasmic RNA and in turn activate GSK3B, one of the main kinases responsible of pTau. Following ZIKV infection of neurons, neuronal IFNs-I directly signal microglia for activation and the establishment of a DAM-like phenotype. The diagram was created using BioRender.com

Article Snippet: When indicated, anti-IFNAR antibody or recombinant IFNB (8234-MB, R&D Systems) were added in the culture medium overnight before infection and 48 h p.i. at a final concentration of 6.5 μg/ml and 50 U/well respectively.

Techniques: Infection, Activation Assay, Phospho-proteomics, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Toxoplasma Effector GRA4 Hijacks Host TBK1 to Oppositely Regulate Anti-T. Gondii Immunity and Tumor Immunotherapy.

doi: 10.1002/advs.202400952

Figure Lengend Snippet: Figure 5. Mice vaccinated with ME49Δompdc/gra4 activates stronger IFN-I responses and completely inhibit tumor growth. A) Body weight change of WT and Ifnar−/- mice vaccinated with ME49Δompdc or ME49Δompdc/gra4. B) qPCR analysis of ITS-1 gene expression in splenocytes from mice with or without ME49wt, ME49Δompdc, and ME49Δompdc/gra4 infection. C) qPCR analysis of Ifnb and Isg15 gene expression in splenocytes from mice with or without ME49Δompdc and ME49Δompdc/gra4 immunization. D) ELISA of IFN-𝛽production in serum from mice with or without ME49Δompdc and ME49Δompdc/gra4 immunization. E) Tumor growth (left) and survival curve (right) of WT mice vaccinated with ME49Δompdc, ME49Δompdc/gra4 or PBS, followed by implanted B16-F10 tumor cells. F) The size and location of the tumors detected in mice (top), and changes in the tumor or spleen (bot- tom) volume dissected from mice vaccinated with ME49Δompdc/gra4, ME49Δompdc, or PBS, followed by implanted B16-F10 tumor cells. G) Represen- tative flow plots (left) and histogram (right) of CD4+ and CD8+ T cells within splenocytes from mice vaccinated with ME49Δompdc, ME49Δompdc/gra4, or PBS, followed by implanted B16-F10 tumor cells. H) Representative quantification of PD-1 expression in CD4+ (top) and CD8+ (bottom) T cells from mice vaccinated with ME49Δompdc, ME49Δompdc/gra4, or PBS, followed by implanted B16-F10 tumor cells. I) Representative plots (left) and histogram (right) of IFN-𝛾of CD4+ T and CD8+ T cells within splenocytes from mice vaccinated with ME49Δompdc, ME49Δompdc/gra4, or PBS, followed by implanted B16-F10 tumor cells. J) Macroscopic evaluation of B16-F10 tumors metastasis in the lungs of mice treated with PBS (isotype con-

Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA): IFN-β in cell supernatants and mice serum was measured with the Mouse IFN-beta DuoSet ELISA kit (R&D SYSTEMS, Cat# DY8234-05) following the assay procedure.

Techniques: Gene Expression, Infection, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Schematic of the urea cycle. b , Related metabolites in BMDMs treated with VSV for 6 h or not (NT), assayed by LC–MS/MS. log 2 fold changes are relative to NT. Citrulline, argininosuccinate, fumarate, aspartic acid, putrescine and glutamine increased significantly ( P < 0.05, two-tailed Student’s t -test) ( n = 3). c , Heatmap of gene expression in BMDMs treated with VSV for 6 h or not (NT), assessed by qPCR. log 2 fold changes are relative to NT. SMS , SAT1 , SMOX , ASS1 , IDH3b and CS increased significantly ( P < 0.05, two-tailed unpaired Student’s t -test) ( n = 3). d , IFNβ expression in BMDMs pretreated with indicated metabolites overnight and infected with VSV for 6 h or not (NT), assessed by qPCR ( n = 3). e , IFNβ expression in BMDMs pretreated with DMF (5 µM), MMF (20 µM), FHINI (20 µM) or DMSO (Ctrl) overnight, followed by VSV infection for 6 h or not (NT), determined by qPCR ( n = 3). f , Ki67 + BMDM percentage (left) and survival rate (right) after culture in fumarate (2 mM), DMF (5 µM) or MMF (20 µM) overnight, measured by FACS ( n = 3). g , IFNβ levels in BMDM supernatants pretreated overnight with DMF (5 µM) or DMSO followed by VSV infection for 6 h or not (NT), determined by ELISA ( n = 3). h , VSV transcript in BMDMs pretreated overnight with DMF (5 µM) or DMSO (−) followed by VSV (+) infection for 6 h, analysed by qPCR ( n = 3). i – n , Mice treated with DMF or DMSO for 7 days before the VSV challenge. Serum fumarate levels ( i ; n = 3) and IFNβ mRNA ( j ), serum IFNβ protein ( k ), VSV mRNA ( l ), lung histology ( m ), and ASS1 and IFNβ expression in peritoneal macrophages ( n ) were assessed ( j – n ; n = 5). The scale bar (200 µm) applies to all images in m . Statistical analysis: two-tailed Student’s t -test ( b , c , f , h and i ) or two-way ANOVA ( d , e , g , j , k , l and n ). Data are the mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results. CPS1, carbamoyl phosphate synthetase 1; OTC, ornithine transcarbamylase; ASL, argininosuccinate lyase; ARG1, arginase 1; ODC, ornithine decarboxylase; ASS1, argininosuccinate synthase.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Gene Expression, Expressing, Infection, Enzyme-linked Immunosorbent Assay

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Heatmap of gene expression in VSV-infected BMDMs. Log2 fold changes are relative to uninfected BMDMs (0 h) (n = 3). b - c , IFN-β transcripts in iBMDMs pretreated with metabolites, and then infected with VSV for 6 h or not (NT) (n = 3). d , VSV plaque morphology in Vero cells after DMF (20 μM) or DMSO treatment for 6 h (n = 4). e - g , iBMDMs pretreated with DMF (5 µM) or DMSO, followed by VSV infection for 6 h or not (NT). IFN-β and ASS1 transcripts ( e ), VSV expression ( f ), and IFN-β protein ( g ) (n = 3). h , ASS1 enzymatic activity in BMDMs infected with VSV for 6 h or not (−) (n = 3). i , Enzymatic activity of ASS1, purified from HEK293 cells infected with VSV or not, was analyzed (n = 3). j , Expression of OTC, ASS1, ASL and ARG1 in BMDMs treated with VSV or not (−), measured by Western blot. k , NO levels in ASS1 +/+ and ASS1 −/− BMDMs infected with VSV or not (−) (n = 3). l-n , ASS1 transcripts in BMDMs treated with IFN-β (10 pg/ml) for different time points ( l ), treated with increasing concentrations of IFN-β for 6 h ( m ), and infected with VSV or not in the presence of IFN-β ( n ) (n = 3). o , IFN-β protein levels in supernatants of BMDMs infected with VSV or not (−), determined by ELISA (n = 3). p , ASS1 expression in BMDMs infected with VSV or not (−) in the presence of IFN-β (100 pg/ml) or not (−) for 6 h, measured by qPCR (n = 3). Statistical analysis: two-tailed Student’s t -test ( a , d , f , h , i, l , m , o and p ) or two-way ANOVA ( b , c, e, g and k ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Gene Expression, Infection, Expressing, Activity Assay, Purification, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Fumarate, malate and aspartate levels in BMDMs derived from macrophage-specific ASS1 knockout ( ASS1 −/− ) mice and corresponding wild-type ( ASS1 +/+ ) mice pretreated with AOAA (5 mM) or not (−), followed by VSV infection for 6 h or not (−), measured by LC–MS/MS/MS ( n = 3). b , Atom-transition map showing that the isotope carbon-13 ( 13 C) transfers from [U- 13 C 6 ]glutamine through the AAS shunt. The open circles represent carbon-12 ( 12 C); the green circles indicate 13 C from [U- 13 C 6 ]glutamine. c – e , Incorporation of 13 C atoms into fumarate ( c ), aspartate ( d ) and malate ( e ) in BMDMs from ASS1 +/+ and ASS1 −/− mice cultured with [U- 13 C 6 ]glutamine and treated with VSV or not (−) for 6 h, determined by LC–MS/MS ( n = 3). f , g , Expression of ASS1, IFNβ and VSV ( f ) in BMDMs pretreated overnight with DMF (5 µM) or DMSO (−) followed by VSV infection for 6 h or not (−), determined by qPCR and IFNβ protein levels by ELISA ( g ) ( n = 3). h , OCR analysis of BMDMs from ASS1 +/+ and ASS1 −/− mice infected with VSV for 6 h or not (−). Basal respiration, maximal respiration, proton leak and reserve respiratory capacity were analysed ( n = 4). i , Schematic illustration of the respiratory chain. j , Expression of respiratory chain genes in BMDMs infected with VSV for 6 h or not (NT), analysed by qPCR. log 2 fold changes are relative to BMDMs not infected with VSV (NT). Significant decreases in NDUFA1 , NDUFA3 , NDUFA4 and others ( n = 3). k , OCR analysis of ASS1 +/+ ( n = 4) and ASS1 −/− ( n = 3) BMDMs treated with VSV infection for 6 h or not (−) in the presence or absence of 1 mM succinate. Statistical analysis: two-tailed Student’s t -test ( a and j ) or two-way ANOVA ( c – g ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results. CoQ, coenzyme Q; Cyt c, cytochrome complex; Suc, Succinate. Panel i created with BioRender.com .

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Knock-Out, Infection, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a - e , The incorporation of 13 C atoms into α-ketoglutarate ( a ), succinate ( b ), isocitrate ( c ), aconitate ( d ) and citrate ( e ) in BMDMs from ASS1 +/+ and ASS1 −/− mice cultured in medium containing 2 mM [U- 13 C 6 ]-glutamine and treated with VSV or not (−) for 6 h was determined by LC-MS/MS. The isotopic labeling of each metabolite is denoted as m + n, where n is the number of 13 C atoms (n = 3). f , Luciferase assay analysis of IFN-β promoter activity in HEK293 cells transfected with control siRNA (siCtrl) or ASS1 siRNA (siASS1) and then treated with VSV for a further 6 h (n = 3). Protein expression was analyzed by Western blot. g , HEK293 cells transfected with an increasing amount of Flag-tagged ASS1 plasmids for 24 h. The expression of IFN-β (left) and ASS1 (right) was determined by qPCR (n = 3). h , IFN-β transcript in BMDMs pretreated with DMF (5 µM) overnight, then followed by VSV infection for 6 h in the presence or absence of AOAA (5 mM) was determined by qPCR (n = 3). i , ASS1 +/+ and ASS1 −/− BMDMs treated with VSV for 6 h or not (−) were used for OCR analysis (n = 4). Relative to Fig. . j , IFN-β expression in BMDMs treated with DECA (10 µM) or not (PBS) followed by VSV infection for 6 h was analyzed by qPCR (n = 3). k , ASS1 +/+ (n = 4) and ASS1 −/− (n = 3) BMDMs treated with VSV or not (−) for 6 h in the presence or absence of 1 mM succinate were used for OCR analysis. Relative to Fig. . Statistical analysis: two-tailed Student’s t -test ( f - h ) or two-way ANOVA ( a-e and i-k ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Cell Culture, Liquid Chromatography with Mass Spectroscopy, Isotopic Labeling, Luciferase, Activity Assay, Transfection, Control, Expressing, Western Blot, Infection, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Luciferase reporter assays for IFNβ promoter activity in HEK293 cells transfected with indicated vectors, treated with DMF (10 µM) or DMSO for 4 h, followed by measurement of IFNβ promoter activity and western blot analysis ( n = 3). b , ASS1 +/+ and ASS1 −/− BMDMs pretreated with DMF (5 µM) or DMSO (−) overnight, followed by VSV infection for 6 h or not (−), analysed by Western blot. c , HEK293 cells transfected with GST-MAVS plasmids were treated with VSV for 6 h or not (−), and MAVS succination was assessed by immunoblotting. d , e , Immunoprecipitated Flag-MAVS from HEK293 cells treated with DMF (10 μM) for 6 h was subjected to mass spectrometry for MAVS succination. Cysteine residues at positions 46 ( d ) and 283 ( e ) in MAVS are susceptible to succination by fumarate. f , HEK293 cells transfected with Flag-MAVS or mutants, treated with 10 μM DMF for 6 h and analysed for MAVS succination by immunoblotting. g , MAVS-deficient iBMDMs transduced with mCherry-MAVS or mutant MAVS (2CS) were treated with VSV for 6 h or not (−) and analysed for MAVS succination. h , MAVS-deficient iBMDMs transduced with mCherry-MAVS or mutant MAVS (2CS) treated VSV for 6 h and IFNβ and VSV expression analysed by qPCR (left) ( n = 3). Protein expression measured by western blot (right). i , MAVS-deficient iBMDMs transduced with mCherry-MAVS or mutant MAVS (2CS) treated with DMF (5 μM) or not for 6 h and analysed for MAVS succination by immunoblotting. j – m , MAVS-deficient iBMDMs transduced with mCherry-MAVS or mutant MAVS (2CS) were pretreated with DMF (5 μM) or not (−), followed by VSV infection for 6 h. IFNβ, MAVS and VSV transcripts were determined by qPCR ( j ) ( n = 3). Cell lysates were analysed by SDS–PAGE ( k and m ), and MAVS aggregation was examined by SDD–AGE ( l ). Statistical analysis: two-tailed Student’s t -test ( a and h , right) or two-way ANOVA ( h , left, and j ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results. Luc, luciferase; WB, western blot.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Luciferase, Activity Assay, Transfection, Western Blot, Infection, Immunoprecipitation, Mass Spectrometry, Transduction, Mutagenesis, Expressing, SDS Page, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Protein expression of BMDMs pretreated with DMF (5 μM) overnight or not (−), followed by VSV infection, was determined by Western blot. b , Protein expression in BMDMs pretreated overnight with the indicated metabolites followed by VSV infection or not (−) was determined by Western blot. c , HEK293 cells transfected with GST-MAVS plasmids were treated with MMF (20 μM), DMF (10 μM or 20 μM), fumarate (200 μM) respectively or not (−) for 6 h. MAVS succination was analyzed by immunoblotting (left), and densitometry quantified the succinated-to-total MAVS ratio (right). d , Schematic of RIG-I-like Receptor (RLR) signaling. e , MAVS-deficient iBMDMs transduced with human MAVS were transfected with siRNAs targeting RIG-I and MDA5 or vector control (siCtrl), followed by VSV infection and DMF (5 μM) treatment for 6 h. Whole cell lysates and anti-MAVS immunoprecipitants were analyzed by immunoblotting. f , MAVS-deficient iBMDMs transduced with human MAVS were transfected with both siRNAs targeting RIG-I and MDA5 or vector control (siCtrl), followed by VSV infection or not (−) for 6 h. Whole cell lysates were analyzed by immunoblotting. g , MAVS-deficient iBMDMs transduced with human MAVS or mutant MAVS (2CS) were transfected with siRNAs targeting RIG-I and MDA5, or vector control (siCtrl, -), followed by VSV infection for 6 h. IFN-β expression(left) and protein levels were measured (n = 3). h , Purified Flag-MAVS protein from HEK293 cells was used in in vitro assays to assess succination and aggregation of MAVS in the presence or absence of fumarate (1 mM) for 6 h. i , Purified Flag-MAVS and MAVS (2CS) proteins from HEK293 cells were used in in vitro assays to assess succination and aggregation of MAVS. Statistical analysis: two-tailed Student’s t -test ( c right) or two-way ANOVA ( g ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Expressing, Infection, Western Blot, Transfection, Transduction, Plasmid Preparation, Control, Mutagenesis, Purification, In Vitro, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a - b , ASS1 +/+ and ASS1 −/− BMDMs were infected with SeV or left uninfected (−) for 6 h. Transcripts of ASS1, IFN-β and SeV in were analyzed by qPCR, and fumarate levels were analysed by LC-MS/MS ( a ) (n = 3). Whole cell lysates and anti-MAVS immunoprecipitants were analyzed for MAVS succination by immunoblotting ( b ). c - d , ASS1 +/+ and ASS1 −/− BMDMs were infected with IAV or left uninfected (−) for 6 h. Transcripts of ASS1, IFN-β and IAV in were analyzed by qPCR, and fumarate levels were analyzed by LC-MS/MS ( c ) (n = 3). Whole cell lysates and anti-MAVS immunoprecipitants were analyzed for MAVS succination by immunoblotting ( d ). e - f , ASS1 +/+ and ASS1 −/− BMDMs were infected with HSV-1 or left uninfected (−) for 6 h. Transcripts of ASS1, IFN-β and HSV-1 were analyzed by qPCR, and fumarate levels were analyzed by LC-MS/MS ( e ) (n = 3). Whole cell lysates and anti-MAVS immunoprecipitants were analyzed for MAVS succination by immunoblotting ( f ). g , Lysates from ASS1 +/+ and ASS1 −/− BMDMs infected with SeV (left), IAV (middle), or HSV-1 (right) or left uninfected (−) for 6 h was analyzed by western blot. h , IFN-β expression in MAVS-deficient iBMDMs transduced with human MAVS or mutant MAVS(2CS) infected for 6 h with SeV (left), IAV (middle), or HSV-1 (right) or left uninfected (−) was measured by qPCR (n = 3). i , MAVS-deficient iBMDMs transduced with human MAVS or mutant MAVS(2CS) were infected with SeV, IAV, or HSV-1 or left uninfected (−) for 6 h. Whole cell lysates and anti-MAVS immunoprecipitants were analyzed by immunoblotting. Statistical analysis: two-tailed Student’s t -test (the fourth section of a , c and e ) or two-way ANOVA (the first three part of a , c and e , and h ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Infection, Liquid Chromatography with Mass Spectroscopy, Western Blot, Expressing, Transduction, Mutagenesis, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Expression of IFNβ in hMDMs expressing sgRNA-Ctrl ( ASS1 +/+ hMDMs) and hMDMs expressing sgRNA-ASS1 ( ASS1 −/− hMDMs) not infected or infected with VSV (left) or SeV in the presence or absence of DMF (10 µM) for 6 h was determined by qPCR analysis ( n = 3). b , Expression of VSV in ASS1 +/+ and ASS1 −/− hMDMs infected with VSV or SeV in the presence or absence of 10 µM DMF for 6 h was measured by qPCR analysis ( n = 3). c , Fumarate levels in ASS1 +/+ and ASS1 −/− hMDMs infected for 6 h with VSV or SeV, or uninfected (NT), were measured by LC–MS analysis ( n = 3). d , ASS1 +/+ and ASS1 −/− hMDMs in the presence or absence of 10 µM DMF were infected with VSV or left uninfected (−) for 6 h. Whole-cell lysates and anti-MAVS immunoprecipitants were analysed by immunoblotting. e , Whole lysates and immunoprecipitants with anti-MAVS from ASS1 +/+ and ASS1 −/− hMDMs in the presence or absence of DMF (10 µM) infected with SeV or not (−) were analysed by western blot. Statistical analysis: two-way ANOVA ( a – c ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Expressing, Infection, Liquid Chromatography with Mass Spectroscopy, Western Blot

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , Lysates from ASS1 +/+ and ASS1 −/− hMDMs infected with VSV or SeV or not (−) for 6 h in the presence or absence of DMF (10 µM) were analyzed by Western blot. b , ASS1 enzymatic activity in hMDMs treated with MDLA (10 mM) or not (−) for 6 h (left). Protein expression was also determined by immunoblotting (right) (n = 3). c , Fumarate levels in hMDMs treated with or without MDLA (10 mM) for 6 h was measured by LC-MS/MS (n = 3). d-f , hMDMs treated with MDLA (10 mM) or not (−) were infected with VSV ( d ), SeV ( e ), IAV ( f ), or left uninfected (−) for 6 h. Transcripts of ASS1, IFN-β, VSV, SeV and IAV were measured by qPCR respectively (n = 3). g , Lysates from hMDMs treated with MDLA (10 mM, +) or not (−) were infected with VSV, SeV, IAV, or left uninfected (−) were analyzed by Western blot. Statistical analysis: two-tailed Student’s t -test ( b-c ) or two-way ANOVA ( d-f ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Infection, Western Blot, Activity Assay, Expressing, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , b , In silico bioinformatic analysis of the expression of ASS1 using public databases. Expression of ASS1 was analysed between healthy people (Healthy) and patients infected with EVD using public databases ( GSE83565 ) ( a ). Box plots showing ASS1 expression in primary hMDMs treated with EBOV or MOCK, using data from the GSE84188 database ( b ). The box plot shows the minimum, maximum, median and interquartile range (IQR; 25th to 75th percentiles). Whiskers extend to 1.5 × IQR, and outliers are shown as individual points. c – g , ASS1 +/+ ( n = 5) and ASS1 −/− ( n = 5) mice were treated daily for seven consecutive days with 50 mg kg −1 DMF (dissolved in 10% DMSO) or 10% DMSO (−) by oral gavage, followed by intraperitoneal injection with VSV (2 × 10 7 PFU per mouse, +) or not (−). Mice were killed 6 h post-infection. c , d , mRNA levels of IFNβ ( c ) and VSV ( d ) in the lung and spleen were determined by qPCR analysis. e , Serum IFNβ protein levels were determined by ELISA analysis. f , IFNβ expression in splenic macrophage cells was analysed by qPCR (left, n = 3), and endogenous MAVS succination in splenic macrophage cells was measured by western blot (right). g , H&E staining of lung sections (scale bar, 100 μm). The scale bar applies to all images in g . h , Survival of ASS1 +/+ and ASS1 −/− mice intraperitoneally injected with VSV in the absence (VSV- ASS1 +/+ , n = 9; VSV -ASS1 −/− , n = 9) or presence of DMF dissolved in 10% DMSO (50 mg kg −1 , by oral gavage for seven consecutive days) (VSV, DMF- ASS1 +/+ , n = 10; VSV, DMF- ASS1 −/− , n = 12). i , Schematic illustrating the metabolic remodelling and upregulation of ASS1 to form the AAS cycle induced by viral infection to produce fumarate in the cytosol, promoting the antiviral response. Statistical analysis: two-tailed Student’s t -test ( a and b ), two-way ANOVA ( c – f ) or Kaplan–Meier survival analysis ( h ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results. Arg, arginine; Orn, ornithine; Cit, citrulline; Asp, aspartate; Mal, malate; Suc, succinate. Panel i created with BioRender.com .

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: In Silico, Expressing, Infection, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Two Tailed Test

Journal: Nature Microbiology

Article Title: Metabolic remodelling produces fumarate via the aspartate–argininosuccinate shunt in macrophages as an antiviral defence

doi: 10.1038/s41564-025-01985-x

Figure Lengend Snippet: a , ASS1 +/+ and ASS1 −/− mice were intraperitoneally injected with VSV (2×10 7 pfu per mouse) or not (−). Mice were sacrificed 6 h post-infection. Endogenous MAVS succination was analyzed in the lungs of mice by Western blot. b , ASS1 +/+ and ASS1 −/− mice were treated daily for seven consecutive days with 50 mg/kg DMF (dissolved in 10% DMSO) or 10% DMSO (−) by oral gavage, followed by intraperitoneal injection with VSV (2 × 10 7 pfu per mouse) or not (−). Mice were sacrificed 6 h post-infection. Endogenous MAVS succination was analyzed in the lungs of mice by Western blot. c-j , ASS1 +/+ and ASS1 −/− BMDMs were infected with VSV for different time points as indicated. ASS1 transcripts ( c ), IFN-β protein levels ( d ), VSV ( e ) and IFN-β ( f ) transcripts, fumarate levels ( g ), phosphorylation of TBK1 and IRF3 ( h ), ASS1 activity ( i ), FH transcripts ( j ) were determined respectively (n = 3). k - l , Aspartate and citrulline levels in BMDM infected with VSV at different time points were measured by LC-MS/MS (n = 3). Statistical analysis: two-tailed Student’s t -test ( i, k and l ) or two-way ANOVA ( c-g and j ). Data are mean ± s.d. P values are indicated. Experiments were performed at least three times with similar results.

Article Snippet: Mouse IFNβ proteins in cell culture supernatants and mouse sera were detected using a mouse IFNβ ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Injection, Infection, Western Blot, Phospho-proteomics, Activity Assay, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test