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

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: PLoS ONE

Article Title: Role of Lactobacillus pentosus Strain b240 and the Toll-Like Receptor 2 Axis in Peyer's Patch Dendritic Cell-Mediated Immunoglobulin A Enhancement

doi: 10.1371/journal.pone.0091857

Figure Lengend Snippet: (A) PP cells (1.5×10 6 cells) were cultured with saline (open circles), 1.2×10 6 counts of heat-killed b240 (closed squares), or 1.2 × 10 7 counts of heat-killed b240 (closed circles) for 1, 3, 5, and 7 days. (B, C) In the presence or absence of heat-killed b240 (4.7×10 6 counts), PP cells (5.8×10 5 cells) were cultured with (B) anti-IL-6 mAb (10 μg/ml), anti-IFN-γ mAb (10 μg/ml), anti-TNF mAb (10 μg/ml), rat IgG1 k isotype control (10 μg/ml), (C) LE540 (1 μM), BCMA-Ig+ TACI-Ig (5 μg/ml each), dimethyl sulfoxide, or human IgG1 Fc antibody (10 μg/ml) for 4 days. The stimulation index of each sample was calculated (for example, (b240-treatment and anti-IL-6 Ab treatment)/(saline-treatment and anti-IL-6 Ab treatment) is the stimulation index for anti-IL-6 Ab treatment). (D) PP cells (5.8×10 5 cells) were cultured with a low dose (light gray), medium dose (dark gray), and high dose (black) of rIL-6 (0.4, 2, or 10 ng/ml), rIFN-γ (0.6, 3, or 15 ng/ml), rTNF (0.08, 0.4, or 2 ng/ml), or heat-killed b240 (4.7×10 6 counts) for 4 days. IgA or cytokine in the culture supernatants was determined by ELISA or CBA. Data are expressed as mean ± SEM (n = 3). (A, B) * P <0.05 versus control group by Dunnett's test. (C) Student's t -test was conducted. (D) Statistical analysis was not conducted. Data are representative of 2 independent experiments producing similar results.

Article Snippet: IFN-α and IFN-β were measured using Mouse Interferon Alpha and Mouse Interferon Beta ELISA kit (PBL Biomedical Laboratories, Piscataway, NJ, USA), respectively.

Techniques: Cell Culture, Saline, Control, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Role of Lactobacillus pentosus Strain b240 and the Toll-Like Receptor 2 Axis in Peyer's Patch Dendritic Cell-Mediated Immunoglobulin A Enhancement

doi: 10.1371/journal.pone.0091857

Figure Lengend Snippet: (A, C) In the presence (black) or absence (white) of heat-killed b240 (1.6×10 6 counts), (A) purified WT PP IgD + cells (2×10 5 cells/well) were cultured with or without purified WT PP CD11c + B220 − DCs (1×10 5 cells/well), (B) purified WT PP CD11c + B220 − DCs and purified WT PP IgD + B cells were co-cultured with (black) or without (white) anti-IL-6 mAb (10 μg/ml) in the presence or absence of heat-killed b240 (1.6×10 6 counts), and (C) purified WT or TLR2 −/− PP CD11c + B220 − DCs and purified WT or TLR2 −/− PP IgD + B cells were co-cultured for 7 days. IgA and IL-6 in the culture supernatants were determined by ELISA and CBA, respectively. Data are expressed as mean ± SEM (n = 1–3). ND, not detected. * P <0.05 versus (A) saline group by Welch's t -test, (B) isotype Ig group by Student's t- t est, (C) WT DCs + WT B cells + b240 co-culture by Dunnett's test. Data are representative of 2 independent experiments producing similar results.

Article Snippet: IFN-α and IFN-β were measured using Mouse Interferon Alpha and Mouse Interferon Beta ELISA kit (PBL Biomedical Laboratories, Piscataway, NJ, USA), respectively.

Techniques: Purification, Cell Culture, Enzyme-linked Immunosorbent Assay, Saline, Co-Culture Assay

Journal: Brain, behavior, and immunity

Article Title: STING recognition of viral dsDNA by nociceptors mediates pain in mice.

doi: 10.1016/j.bbi.2024.07.013

Figure Lengend Snippet: Fig. 4. Virus-derived dsDNA-induced pain behaviors rely on TRPV1 expression. (A) Illustration depicting the canonical STING signaling and the hypothesized dependency on TRP channels. (B) Nocifensive behaviors and mechanical sensitivity induced by intraplantar (i.pl.) injection of HSV-60 (5 µg/20 µl) after intra- peritoneal (i.p.) injection of STING agonists or a vehicle control (n = 6 male mice/group). (C-E) Nocifensive behaviors i.pl. injection of HSV-60 (5 µg/20 µl) in mice treated with TBK1 antagonist GSK8612 (5 mg/kg, i.p., C), IFNß antibody (10 µg, i.pl.), TRPA1 inhibitor HC-030031 (35 mg/kg, i.p., E) or TRPM8 inhibitor AMG2850 (30 mg/kg, i.p., E), compared to their respective controls (n = 4–5 male mice/group). (F) Nocifensive behaviors and mechanical sensitivity induced by i.pl. injection of HSV-60 (5 µg/20 µl) after i.p. injection of TRPV1 inhibitor AMG9810 (100 mg/100 ml) or vehicle control (n = 5 male mice/group). Statistical analysis: two-tailed unpaired Student’s t-test (C-E, F; nocifensive behaviors), one-way ANOVA followed by Dunnett’s multiple comparisons test (B; nocifensive behaviors), two-way ANOVA followed by Dunnett’s multiple comparisons test (B; mechanical sensitivity) or ˇSíd´ak’s multiple comparisons test (F; mechanical sensitivity). Error bars indicate mean ± s.e.m. and *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: An ELISA kit for IFNß (# 424101) and IFNα (#421151) were purchased from PBL Assay Science.

Techniques: Virus, Derivative Assay, Expressing, Injection, Control, Two Tailed Test

Journal: Cell

Article Title: Self-Recognition of an Inducible Host lncRNA by RIG-I Feedback Restricts Innate Immune Response.

doi: 10.1016/j.cell.2018.03.064

Figure Lengend Snippet: Figure 2. lnc-Lsm3b Inhibits RIG-I-Mediated Signaling in RNA Virus-Infected Macrophages (A–C) ELISA of IFN-a (A), IFN-b (B), and IL-6 (C) in the supernatants of peritoneal macrophages transfected with si-ctrl or si-lnc-Lsm3b for 48 hr before VSV, SeV, or HSV-1 infection for 16 hr. (D–F) ELISA of IFN-a (D), IFN-b (E), and IL-6 (F) in supernatants of lnc-Lsm3b+/+ and lnc-Lsm3b/ cells infected with VSV for indicated hours. (G and H) IFN-b (G) and NF-kB (H) promoter activity in HEK293T cells transfected with lnc-Lsm3b and RIG-I vectors upon VSV infection. (I) VSV titers by TCID50 assay in supernatants of lnc-Lsm3b+/+ and lnc-Lsm3b/ cells infected with VSV for 12 hr and 24 hr. (J) Immunoblot analysis of RIG-I signaling pathways in lnc-Lsm3b+/+ and lnc-Lsm3b/ cells infected with VSV for indicated hours. Data are shown as mean ± SD (n = 4) or typical photographs of one representative experiment. Similar results were obtained in three independent experiments. *p < 0.05; **p < 0.01. See also Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER poly(I:C) HMW Biotin InvivoGen Cat #tlrl-picb TRIzol reagent Invitrogen Cat #15596-018 Mouse IFN-a Miltenyi Biotec Cat #130-093-130 Mouse IFN-b R&D Systems Cat #8234-MB-010 RNA FISH probe Biosearch technologies N/A GST-RIG-I This paper N/A His-RIG-I This paper N/A GST-CTD This paper N/A GST-CARD This paper N/A His-deltaCARD This paper N/A Critical Commercial Assays NorthernMax kit Thermo Fisher Scientific Cat #AM1940 RNeasy Mini Kit QIAGEN Cat #74104 miRNeasy Serum/Plasma Spike-In Control QIAGEN Cat #219610 MAXIscript SP6/T7 Kit Ambion Cat #AM1320 Streptavidin beads Thermo Fisher Scientific Cat #11206D Mouse IFN-a ELISA kit PBL Biomedical Laboratories Cat #42120-1 Mouse IFN-b ELISA kit PBL Biomedical Laboratories Cat #42400-1 Mouse IL-6 ELISA kit R&D Systems Cat #VAL604 Dual-Luciferase assay kit Promega Cat #TM040 SMARTer RACE 50/30 kit Clontech Cat #634858 ATPase/GTPase Activity Assay Kit Sigma Cat #MAK113 Deposited Data RIP-seq data This paper GEO: GSE106254 iCLIP data This paper GEO: GSE106254 Experimental Models: Cell Lines HEK293T cells American Type Culture Collection N/A L929 cells American Type Culture Collection N/A RAW264.7 cells American Type Culture Collection N/A lnc-Lsm3b / RAW264.7 cells This paper N/A Experimental Models: Organisms/Strains lnc-Lsm3b / mice This paper N/A IFNabR / mice The Jackson Laboratory JAX stock 000819 Recombinant DNA Plasmid: pSIF vector Provided by Dr. Qian Zhang from Second Military Medical University, Shanghai, China N/A Plasmid: pGEM-3zf vector Provided by Dr. Xiaozhong Peng from Peking Union Medical College, Beijing, China N/A cDNA lnc-Lsm3b This paper N/A cDNA lnc-Lsm3b mutants This paper N/A Software and Algorithms FastQC 0.11.5 Babraham Bioinfomatics http://www.bioinformatics.babraham.ac.uk/ projects/fastqc/ Samtools 0.1.19 John Marshall and Petr Danecek http://www.htslib.org/ HISAT2 2.0.4 The Center for Computational Biology http://ccb.jhu.edu/software/hisat2/index.shtml StringTie v1.3.3b The Center for Computational Biology http://ccb.jhu.edu/software/stringtie/ (Continued on next page) e2 Cell 173, 1–14.e1–e6, May 3, 2018 Please cite this article in press as: Jiang et al., Self-Recognition of an Inducible Host lncRNA by RIG-I Feedback Restricts Innate Immune Response, Cell (2018), https://doi.org/10.1016/j.cell.2018.03.064

Techniques: Virus, Infection, Enzyme-linked Immunosorbent Assay, Transfection, Activity Assay, TCID50 Assay, Western Blot, Protein-Protein interactions

Journal: Cell

Article Title: Self-Recognition of an Inducible Host lncRNA by RIG-I Feedback Restricts Innate Immune Response.

doi: 10.1016/j.cell.2018.03.064

Figure Lengend Snippet: Figure 3. lnc-Lsm3-Deficient Mice Produce More Type I IFNs (A) Survival of 6-week-old lnc-Lsm3b+/+ and lnc-Lsm3b/ mice given VSV (1 3 108 pfu/g body weight) via tail intravenous injection (i.v.) (n = 10). Kaplan-Meier method was used to evaluate survival curves. (B) ELISA of type I IFNs (IFN-a and IFN-b) production in sera from lnc-Lsm3b+/+ and lnc-Lsm3b/ mice (n = 5) at 18 hr and 24 hr after i.v. injection with VSV (5 3 107 pfu/g). (C) VSV loads by TCID50 assay and qPCR assay of VSV RNA copies in organs and peritoneal macrophages from mice in (B) (n = 4). (D) Pathology of lnc-Lsm3b+/+ and lnc-Lsm3b/ mice in response to VSV. H&E staining of lung sections from mice in (B). Scale bar, 80 mm. (E and F) qPCR analysis of Ifn-b (E) and lnc-Lsm3b (F) expression in organs and peritoneal macrophages from mice in (B) (n = 5). Data are shown as mean ± SD or typical photographs of one representative experiment. Similar results were obtained in three independent experiments. *p < 0.05; **p < 0.01. See also Figures S3 and S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER poly(I:C) HMW Biotin InvivoGen Cat #tlrl-picb TRIzol reagent Invitrogen Cat #15596-018 Mouse IFN-a Miltenyi Biotec Cat #130-093-130 Mouse IFN-b R&D Systems Cat #8234-MB-010 RNA FISH probe Biosearch technologies N/A GST-RIG-I This paper N/A His-RIG-I This paper N/A GST-CTD This paper N/A GST-CARD This paper N/A His-deltaCARD This paper N/A Critical Commercial Assays NorthernMax kit Thermo Fisher Scientific Cat #AM1940 RNeasy Mini Kit QIAGEN Cat #74104 miRNeasy Serum/Plasma Spike-In Control QIAGEN Cat #219610 MAXIscript SP6/T7 Kit Ambion Cat #AM1320 Streptavidin beads Thermo Fisher Scientific Cat #11206D Mouse IFN-a ELISA kit PBL Biomedical Laboratories Cat #42120-1 Mouse IFN-b ELISA kit PBL Biomedical Laboratories Cat #42400-1 Mouse IL-6 ELISA kit R&D Systems Cat #VAL604 Dual-Luciferase assay kit Promega Cat #TM040 SMARTer RACE 50/30 kit Clontech Cat #634858 ATPase/GTPase Activity Assay Kit Sigma Cat #MAK113 Deposited Data RIP-seq data This paper GEO: GSE106254 iCLIP data This paper GEO: GSE106254 Experimental Models: Cell Lines HEK293T cells American Type Culture Collection N/A L929 cells American Type Culture Collection N/A RAW264.7 cells American Type Culture Collection N/A lnc-Lsm3b / RAW264.7 cells This paper N/A Experimental Models: Organisms/Strains lnc-Lsm3b / mice This paper N/A IFNabR / mice The Jackson Laboratory JAX stock 000819 Recombinant DNA Plasmid: pSIF vector Provided by Dr. Qian Zhang from Second Military Medical University, Shanghai, China N/A Plasmid: pGEM-3zf vector Provided by Dr. Xiaozhong Peng from Peking Union Medical College, Beijing, China N/A cDNA lnc-Lsm3b This paper N/A cDNA lnc-Lsm3b mutants This paper N/A Software and Algorithms FastQC 0.11.5 Babraham Bioinfomatics http://www.bioinformatics.babraham.ac.uk/ projects/fastqc/ Samtools 0.1.19 John Marshall and Petr Danecek http://www.htslib.org/ HISAT2 2.0.4 The Center for Computational Biology http://ccb.jhu.edu/software/hisat2/index.shtml StringTie v1.3.3b The Center for Computational Biology http://ccb.jhu.edu/software/stringtie/ (Continued on next page) e2 Cell 173, 1–14.e1–e6, May 3, 2018 Please cite this article in press as: Jiang et al., Self-Recognition of an Inducible Host lncRNA by RIG-I Feedback Restricts Innate Immune Response, Cell (2018), https://doi.org/10.1016/j.cell.2018.03.064

Techniques: Injection, Enzyme-linked Immunosorbent Assay, TCID50 Assay, Staining, Expressing