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: 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: 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

Journal: EMBO Reports

Article Title: RNF144B negatively regulates antiviral immunity by targeting MDA5 for autophagic degradation

doi: 10.1038/s44319-024-00256-w

Figure Lengend Snippet: ( A ) Rnf144b +/+ and Rnf144b −/− MEFs were infected with EMCV (MOI = 1) for indicated time points, and then cell lysates were analyzed for EMCV replication level by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student’s t-test. **** P = 0.000039 (4 h), *** P = 0.00050 (8 h), **** P = 0.000040 (12 h). ( B ) Rnf144b +/+ and Rnf144b −/− MEFs were infected with EMCV (MOI = 1) for indicated time points. The cell supernatant was harvested and analyzed by TCID 50 assay. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student’s t-test. ** P = 0.00540 (8 h), ** P = 0.00820 (12 h). ( C ) Western blot analysis of the indicated signaling proteins in MEFs from Rnf144b +/+ or Rnf144b −/− mice infected with EMCV (MOI = 1) for the indicated time periods. ( D ) Rnf144b +/+ and Rnf144b −/− MEFs were infected with EMCV (MOI = 1). After 12 h, cell lysates were analyzed for Ifnb1, TNF-α, IL-6 mRNA level by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student’s t-test. **** P = 0.000009 (Ifnb1), *** P = 0.00090 (TNF-α), *** P = 0.00030 (IL-6). ( E ) Rnf144b +/+ and Rnf144b −/− MEFs were infected with EMCV (MOI = 1). 12 h post-infection, cells were treated by CHX (30 μM) for indicated time points. Protein extracts were used for immunoblot analysis of the endogenous MDA5 protein level. ( F , G ) Rnf144b +/+ and Rnf144b −/ − mice were intraperitoneally injected EMCV (5 × 10 6 PFU) for 48 h ( n = 5 per group). qPCR analysis of EMCV replication level in heart and brain, and mRNA level of Ifnb1, TNF-α, IL-6 in brain ( G ). Statistical significance was determined by two-tailed unpaired Student’s t-test. ** P = 0.00530 (heart), * P = 0.02620 (brain), *** P = 0.00030 (Ifnb1), ** P = 0.00170 (TNF-α), *** P = 0.00030 (IL-6). ( H ) ELISA of IFN-β production in serum of Rnf144b +/+ and Rnf144b −/− mice that were intraperitoneally injected with EMCV (5 × 10 6 PFU) for 12 h ( n = 3 per group). Statistical significance was determined by two-tailed unpaired Student’s t-test. **** P = 0.000020. ( I ) Survival of Rnf144b +/+ and Rnf144b −/− mice ( n = 20 per group) intraperitoneally infected with EMCV (6 × 10 7 PFU). Significance was tested using log-rank (Mantel-Cox) test, *** P = 0.00030. Data information: Data shown are representative of at least three biological replicates, with each data point representing a biological experiment. Error bars are presented as mean ± SD. Statistical significance was determined by Student’s t-test. .

Article Snippet: Mouse Interferon β (IFN-β/IFNB) ELISA Kit , CUSABIO , Cat#E04945m.

Techniques: Infection, Two Tailed Test, Western Blot, Injection, Enzyme-linked Immunosorbent Assay

Journal: EMBO Reports

Article Title: RNF144B negatively regulates antiviral immunity by targeting MDA5 for autophagic degradation

doi: 10.1038/s44319-024-00256-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Mouse Interferon β (IFN-β/IFNB) ELISA Kit , CUSABIO , Cat#E04945m.

Techniques: Recombinant, Sequencing, shRNA, Mutagenesis, RNA Extraction, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Soft‐Drug‐Inspired MnSTF Nano‐Adjuvant for Safe and Synergistic cGAS–STING Activation in Tumor Immunotherapy

doi: 10.1002/advs.202515432

Figure Lengend Snippet: MnSTF augments cGAS‐STING activation. (a) Quantification of extracellular 2′,3′‐cGAMP in RAW264.7 cell supernatants after 24 h treatment, as measured by enzyme‐linked immunosorbent assay (ELISA) (n = 3). ND, not detected. (b) Immunoblot analysis of the cGAS–STING pathway proteins in RAW264.7 cells treated after 6 h treatment. (c) Quantitative real‐time PCR (qRT–PCR) analysis of IFN‐β mRNA levels in RAW264.7 cells treated with MnSTF for the indicated times (n = 3). (d) Relative expression of IFN‐β mRNA in RAW264.7 cells after 6 h treatment (n = 3). (e–g) ELISA quantification of IFN‐β (e), IL‐6 (f), and TNF‐α (g) in culture supernatants of RAW264.7 cells following 24 h treatment (n = 3). (h) Flow cytometric analysis of CD80 surface expression on DC2.4 dendritic cells after 24 h treatment (n = 3). (i) Representative flow cytometric histograms of MHC class II expression on DC2.4 cells treated for 24 h. Replicates were biological and the data were expressed as mean ± s.d. Statistical analysis was determined using one‐way ANOVA, **** p < 0.0001.

Article Snippet: The cells were treated as described above with an exposure time of 24 h. The culture supernatant from RAW264.7 cells or DC2.4 was collected for ELISA assays to quantify 2′,3′‐cGAMP (COIBO BIO, Cat. CB15107‐Mu), IFN‐β (ABclonal, Cat. RK00420), TNF‐α (ABclonal, Cat. RK00027), and IL‐6 (Dakewe, Cat. 1 210 602)].

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

Journal: Advanced Science

Article Title: Soft‐Drug‐Inspired MnSTF Nano‐Adjuvant for Safe and Synergistic cGAS–STING Activation in Tumor Immunotherapy

doi: 10.1002/advs.202515432

Figure Lengend Snippet: MnSTF regulates the tumor microenvironment by enhancing intratumoral immune responses and reshaping immune cell composition. (a) Representative IVIS fluorescence images of B16F10‐OVA tumor‐bearing mice illustrating intratumoral retention of free ICG or ICG–MnSTF following intratumoral injection. (b) Quantification of intratumoral ICG retention over time (n = 4). (c) Treatment timeline for B16F10‐OVA tumor‐bearing mice administered a single intratumoral injection of PBS or MnSTF (0.69 µmol Mn equivalent), initiated upon tumor palpation (typically 50–100 mm 3 ). (d) Heat map of selected genes in tumors on days 1, 3, and 5 post‐treatment, generated from transcriptome sequencing (n = 3). (e) Tumor IFN‐β levels on day 1 post‐treatment, measured by ELISA (n = 4). (f–i) Representative flow cytometry plots and quantification of F4/80 + iNOS + (f, g) and CD11c + (h, i) cells within tumor tissues at day 1 post‐treatment (n = 4). (j, k) Representative flow cytometry plots (j) and quantification (k) of CD80 + CD86 + expression on CD11c + cells in the draining lymph nodes (LNs) on day 1 post‐treatment (n = 4). (l, m) Representative ELISpot images of IFN‐γ–secreting splenocytes (l) and quantification of spot‐forming units (SFU) per 4 × 10 5 splenocytes (m) on day 7 after three PBS, MnCl 2 , STF, or MnSTF doses (every three days, n = 5). Replicates were biological, and the data were expressed as mean ± s.d. Statistical analysis was performed using one‐way ANOVA (e, g, i, k, m). Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The cells were treated as described above with an exposure time of 24 h. The culture supernatant from RAW264.7 cells or DC2.4 was collected for ELISA assays to quantify 2′,3′‐cGAMP (COIBO BIO, Cat. CB15107‐Mu), IFN‐β (ABclonal, Cat. RK00420), TNF‐α (ABclonal, Cat. RK00027), and IL‐6 (Dakewe, Cat. 1 210 602)].

Techniques: Fluorescence, Injection, Generated, Sequencing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Enzyme-linked Immunospot

Journal: Advanced Science

Article Title: Soft‐Drug‐Inspired MnSTF Nano‐Adjuvant for Safe and Synergistic cGAS–STING Activation in Tumor Immunotherapy

doi: 10.1002/advs.202515432

Figure Lengend Snippet: MnSTF synergizes with radiotherapy to enhance immune priming and elicit a robust systemic antigen‐specific T cell response. (a) Schematic illustration of enhanced antitumor immunity induced by RT combined with MnSTF. (b) Experimental timeline for B16F10‐OVA tumor‐bearing mice treated with PBS, RT (6 Gy), MnSTF (0.69 µmol Mn equivalent), or their combination (RT + MnSTF). Treatments were administered every four days for two doses. (c) Tumor growth kinetics following different treatments (n = 9). (d) Tumor IFN‐β levels on day 1 post‐treatments, measured by ELISA (n = 4). (e–h) Representative flow cytometry plots and quantification of CD11c + DCs presenting SIINFEKL–H‐2K b (e, f) and expressing CD80 and CD86 (g, h) in TDLNs on day 1 post‐treatment (n = 4). (i, j) Representative IFN‐γ ELISpot images (i) and quantification of SFU per 5 × 10 5 splenocytes (j) from each group at the end of treatments (n = 5). (k) Representative immunofluorescence images of tumors at treatment end, stained for CD4 + (green) and CD8 + (red) T cells, and DAPI (blue). Scale bar, 120 µm. (n = 3). Replicates were biological and the data were expressed as mean ± s.d. Statistical analysis was performed using two‐way ANOVA (c) or one‐way ANOVA (d, f, h, j). Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The cells were treated as described above with an exposure time of 24 h. The culture supernatant from RAW264.7 cells or DC2.4 was collected for ELISA assays to quantify 2′,3′‐cGAMP (COIBO BIO, Cat. CB15107‐Mu), IFN‐β (ABclonal, Cat. RK00420), TNF‐α (ABclonal, Cat. RK00027), and IL‐6 (Dakewe, Cat. 1 210 602)].

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Enzyme-linked Immunospot, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Soft‐Drug‐Inspired MnSTF Nano‐Adjuvant for Safe and Synergistic cGAS–STING Activation in Tumor Immunotherapy

doi: 10.1002/advs.202515432

Figure Lengend Snippet: mRNA vaccine and MnSTF synergistically potentiate antitumor immune responses. (a) Schematic illustration of enhanced antitumor immunity induced by mRNA vaccine combined with MnSTF. (b) Experimental design schematic in B16F10‐OVA tumor‐bearing mice treated with PBS, MnSTF (0.69 µmol Mn equivalent), LNP‐formulated mRNA OVA (10 µg), or their combination (MnSTF + mRNA OVA ). Treatments were administered every three days for three doses. (c) Tumor growth kinetics after indicated treatments (n = 8). (d, e) Representative flow cytometry plots (d) and quantification (e) of SIINFEKL tetramer + CD8 + T cells in splenocytes collected at the end of treatment (n = 4). (f, h, j, l) Representative IFN‐γ ELISpot images of splenocytes stimulated with SIINFEKL (f), SVYDFFVWL (h), SIYRYYGL (j), or KVPRNQDWL (l) at treatment end. (g,i,k,m) Quantification of IFN‐γ SFU per 2 × 10 5 splenocytes for SIINFEKL (g), or per 10 6 splenocytes for SVYDFFVWL (i), SIYRYYGL (k), and KVPRNQDWL (m), n = 4. Replicates were biological, and the data were expressed as mean ± s.d. Statistical analysis was performed using two‐way ANOVA (c) or one‐way ANOVA (e, g, i, k, m). Significance: * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: The cells were treated as described above with an exposure time of 24 h. The culture supernatant from RAW264.7 cells or DC2.4 was collected for ELISA assays to quantify 2′,3′‐cGAMP (COIBO BIO, Cat. CB15107‐Mu), IFN‐β (ABclonal, Cat. RK00420), TNF‐α (ABclonal, Cat. RK00027), and IL‐6 (Dakewe, Cat. 1 210 602)].

Techniques: Flow Cytometry, Enzyme-linked Immunospot

Journal: Advanced Science

Article Title: Soft‐Drug‐Inspired MnSTF Nano‐Adjuvant for Safe and Synergistic cGAS–STING Activation in Tumor Immunotherapy

doi: 10.1002/advs.202515432

Figure Lengend Snippet: MnSTF acts as a vaccine adjuvant boosting tumor antigen‐specific immunity. (a, b) C57BL/6 mice were subcutaneously immunized with PBS, Alum + OVA (50 µg Alum; 100 µg OVA), or MnSTF + OVA (50 µg Mn; 100 µg OVA) every three days for a total of three doses. Spleens were harvested 7 days after the final immunization to quantify OVA‐specific IFN‐γ–producing splenocytes by ELISpot. Representative IFN‐γ ELISpot images (a) and quantification of SFU per 10 6 splenocytes (b). (c) Schematic of the experimental design in B16F10‐OVA tumor‐bearing mice treated subcutaneously with PBS, MnSTF (0.69 µmol Mn equivalent), OVA (100 µg), or their combination (MnSTF + OVA). Treatments were administered every three days for three doses. (d) Tumor growth kinetics following the indicated treatments (n = 8). (e, f) Representative IFN‐γ ELISpot images (e) and quantification of SFU per 10 6 splenocytes (f), n = 5. (g, h) Representative flow cytometry plots (g) and quantification (h) of effector memory CD44 + CD62L−CD8 + T cells (n = 5). Replicates were biological, and the data were expressed as mean ± s.d. Statistical analysis was performed using two‐way ANOVA (d) or one‐way ANOVA (b, f, h). Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The cells were treated as described above with an exposure time of 24 h. The culture supernatant from RAW264.7 cells or DC2.4 was collected for ELISA assays to quantify 2′,3′‐cGAMP (COIBO BIO, Cat. CB15107‐Mu), IFN‐β (ABclonal, Cat. RK00420), TNF‐α (ABclonal, Cat. RK00027), and IL‐6 (Dakewe, Cat. 1 210 602)].

Techniques: Adjuvant, Enzyme-linked Immunospot, Flow Cytometry

Journal: Nature

Article Title: Loss of ADAR1 in tumours overcomes resistance to immune checkpoint blockade

doi: 10.1038/s41586-018-0768-9

Figure Lengend Snippet: a, T cell-dependent killing of ovalbumin-expressing Adar1 p150/p110-null (red), p150-null (orange) and control (grey) B16 tumour cells by OT-I transgenic T cells specific for an ovalbumin-derived peptide in the context of MHC-I at decreasing E:T ratios (1:20, 1:10 and 1:5). Data shown are representative of two independent experiments with n = 3 replicates for each condition; mean ± s.e.m. b, Relative numbers of control, Adar1 p150/p110-null, and Adar1 p150-null B16 tumour cells stimulated with cytokines indicated compared with unstimulated conditions (n = 3 for each condition; data are representative of three independent experiments). c, Annexin V staining in control, Adar1 p110/ p150-null and Adar1 p150-null tumours following stimulation with IFNβ, IFNγ or IFNβ + IFNγ (n = 3 for each condition; data are representative of three independent experiments). d, GSEA of gene signatures in Adar1-null compared with control B16 tumour cells after in vitro culture with IFNβ stimulation. n = 3 for each condition; FDR calculated using GSEA. e, Enzyme-linked immunosorbent assay (ELISA) for IFNβ in supernatant from control, Adar1 p150/p110-null and Adar1 p150-null B16 tumour cells after in vitro culture in unstimulated, IFNβ or IFNγ conditions (n = 3 for each condition; data are representative of three independent experiments). f, Tumour volume following treatment with anti-PD-1 in vivo in genetic epistasis tumour models that lack ADAR1 and components of IFN sensing pathways (n = 5 mice in each group, data representative of two independent experiments). g, ELISA for IFNβ in supernatant from control and Adar1 p150/p110-null B16 tumour cells following irradiation in vitro (n = 3 for each condition; data representative of two independent experiments). h, Tumour volume of control (grey) and Adar1-null (red) B16 tumours following therapeutic irradiation in vivo. n = 10 mice in each group; data are representative of two independent experiments. b, c, e, g, Bars represent mean; f, mean ± s.e.m. a–c, e, f, h, Two-sided Student’s t-test; *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001; NS, not significant.

Article Snippet: After 72 h of incubation at 37 °C, the supernatant was collected, and the concentration of IFNβ was determined using the VeriKine Mouse Interferon Beta ELISA Kit (PBL Assay Science) or Mouse IFN-beta Quantikine ELISA Kit (R&D Systems).

Techniques: Expressing, Control, Transgenic Assay, Derivative Assay, Staining, In Vitro, Enzyme-linked Immunosorbent Assay, In Vivo, Irradiation