mouse anti ifn β  (MedChemExpress)

Journal: Nature Communications

Article Title: cGAS-IFN-I responses by extracting nuclear DNA from dying cells via nucleocytosis

doi: 10.1038/s41467-026-68839-w

Figure Lengend Snippet: a RNA-seq analysis of mRNA expression in GM-BMCs treated with HCQ (100 µM) or cGAMP (5 µg/mL). The top 50 genes induced by HCQ in 12 h among the genes are shown in the heatmap. The line graph of Ifnb1 and IFN-inducible genes is shown in ( b ). c IFN-β concentration in the culture supernatant of GM-BMCs treated with HCQ (80, 100, 120, and 140 µM), CQ (20, 40, 60, and 80 µM), tilorone (20, 40, 60, and 80 µM), ADQ (20, 40, 60, and 80 µM), QC (10, 12.5, 15, and 17.5 µM), cGAMP (5 µg/mL), or LPS (100 ng/mL) for 20 h. The chemical structural formulas of IFN-β inducers are shown above each column. d Immunoblot analysis for indicated proteins in GM-BMCs treated with the indicated concentrations of HCQ or cGAMP (5 µg/mL) for 6 h. e – g IFN-β concentration in the culture supernatant of control or gene-deficient GM-BMCs ( e ; Tnf -/- /Tbk1 -/- , f ; Sting1 -/- , g ; Cgas -/- ) treated with the indicated concentration of HCQ, cGAMP (5 µg/mL), or poly(dA:dT) (5 µg/mL) for 16 h. h RNA-seq analysis of mRNA expression in wild-type (WT) or Sting1 -/- GM-BMCs treated with HCQ (100 µM) for 6 h. i . IFN-β concentration in the culture supernatant of WT or Cgas -/- GM-BMCs treated with IFN-β inducers as in c for 16 h. j , k Pharmacophore model constructed from IFN-β inducers. Features without parentheses indicate features all compounds have in common, while features in parentheses indicate features some compounds have in common ( j ). Superimposition of IFN-β inducers and pharmacophore are shown in k. Data in b are expressed as the mean ± SD of biological triplicates. Data in ( c , e – g ), and i are expressed as the mean ± SD of n = 3 replicates of each in vitro culture condition in one representative experiment (each experiment was repeated three times). Data in d are representative of one experiment (experiment was repeated three times). See also Supplementary Fig. .

Article Snippet: Cells were suspended in medium at a density of 2.5 × 10 6 cells/mL and added to each chamber (5 mm diameter) of a 4-condition LCI-S chip (LCI-SPQ002; Live Cell Diagnosis, Saitama, Japan) at 50 μL, where the anti-IFN-β antibody (capture antibody from the Mouse IFN-beta DuoSet ELISA, DY8234-05; R&D Systems) was immobilised.

Techniques: RNA Sequencing, Expressing, Concentration Assay, Western Blot, Control, Construct, In Vitro

Journal: Nature Communications

Article Title: cGAS-IFN-I responses by extracting nuclear DNA from dying cells via nucleocytosis

doi: 10.1038/s41467-026-68839-w

Figure Lengend Snippet: a , b LCI-S of GM-BMCs treated with cGAMP (a; 5 µg/mL) or HCQ (b; 100 µM). IFN-β secretion (integral value at indicated time point from the previous time point), DNA staining, and bright field are shown in the indicated sets of colours. c High-resolution pictures of cGAMP (upper; 5 µg/mL) or HCQ (lower; 100 µM)-treated GM-BMCs. IFN-β secretion (integral value, middle panel; total value, upper panel) and bright field are shown in the indicated sets of colours. d Quantitative analysis of IFN-β-secreting cells and dying cells. Right: number of IFN-β spots at each time point (HCQ; n = 197, cGAMP; n = 396). A line graph of DNA intensity over time in 169 fields of view is shown (left). The coloured line graph shows the average. e – h scRNA-seq analysis of GM-BMCs treated with HCQ (100 µM) or cGAMP (5 µg/mL) for 6 h. e UMAP plots showing cell types (left three panels) annotated by the expression of key marker genes (Z-score) (right panel). f UMAP plots showing the expression level (left) and density (right) of Ifnb1 -expressing cells. Number of Ifnb1 -expressing cells (unique molecular identifier > 1) and all cells: HCQ; n = 197 and 3856, cGAMP; n = 1,352 and 6,158. g Violin plots showing the expression of Ifnb1 (HCQ; n = 197, cGAMP; n = 1352) and mitochondria genes (HCQ; n = 1,112, cGAMP; n = 894). UMAP plots showing the percentage (left) and density (right) of mitochondrial genes. Number of injured cells (percentage of mitochondrial genes > 10%): HCQ; 1,112, cGAMP; 894. i Lactate dehydrogenase (LDH) activity and IFN-β concentration in the culture supernatant of GM-BMCs treated with indicated concentrations of HCQ, cGAMP (5 µg/mL), or LPS (100 ng/mL) for 16 h. Data in a–c are representative pictures from one experiment, which was repeated three times. Data in g, the box plot within the violin plot shows the median (centre line), and the interquartile range (IQR) (the box boundaries, spanning the 25th to 75th percentiles). The whiskers extend to data points within 1.5 times the IQR, defining the non-outlier range. Points outside the whiskers are outliers. Data in i are expressed as the mean ± SD of n = 3 replicates of each in vitro culture condition in one representative experiment (experiment was repeated three times). Statistical differences in g were examined using the two-sided Wilcoxon rank sum test. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s., not significant. See also Supplementary Fig. .

Article Snippet: Cells were suspended in medium at a density of 2.5 × 10 6 cells/mL and added to each chamber (5 mm diameter) of a 4-condition LCI-S chip (LCI-SPQ002; Live Cell Diagnosis, Saitama, Japan) at 50 μL, where the anti-IFN-β antibody (capture antibody from the Mouse IFN-beta DuoSet ELISA, DY8234-05; R&D Systems) was immobilised.

Techniques: Staining, Expressing, Marker, Activity Assay, Concentration Assay, In Vitro

Journal: Nature Communications

Article Title: cGAS-IFN-I responses by extracting nuclear DNA from dying cells via nucleocytosis

doi: 10.1038/s41467-026-68839-w

Figure Lengend Snippet: a Image cytometry with GM-BMCs treated with HCQ (100 µM) for 6 h. SYTOX green-positive population (circles; upper panel) and “multiples” population (indicated region; lower panel) are shown. Statistical analyses of both populations is shown in the upper and lower right panels ( n = 3). b SYTOX green-positive population within the “multiplets” population is shown. Upper right panel: statistical analysis of SYTOX green higher population ( n = 3). Lower panels: images of multiplets in SYTOX green higher and lower populations. c (left) Schematic of the experiment. (right) IFN-β concentration in the culture supernatant of WT or cell mixture (WT/ Sting1 -/- , WT/ Cgas -/- , Sting1 -/- / Cgas -/- ) of GM-BMCs treated with HCQ (100 µM), cGAMP (5 µg/mL), or poly(dA:dT) (5 µg/mL) for 16 h. As this experiment was done with the data in Fig. , data with WT cells are shared. d – h . Microscopic analysis of HCQ-treated GM-BMCs. d Snapshot analysis of GM-BMCs treated with HCQ (80 µM) for indicated time periods. Bright field with DNA staining is shown in both maximum intensity projection (MIP) and Z-slice images. e Time-lapse analysis of GM-BMCs treated with HCQ (80 µM). Bright field with DNA staining is shown in the left five panels. DNA intensity is summarised in the middle. The Z-slice picture at the indicated time point is shown on the right. f Holotomographic analysis of GM-BMCs treated with HCQ (80 µM). Snapshot images in indicated time points are shown in both MIP and Z-slice pictures. g , h Holotomographic time-lapse analysis of GM-BMCs treated with HCQ (100 µM) for the indicated time periods. DNA staining is shown in red in MIP images. Z-slice images at the indicated time points are shown in ( h ). i Concentration of IFN-β in the culture supernatant of GM-BMCs treated with HCQ (100 µM), or cGAMP (5 µg/mL) in the presence of the indicated concentration of inhibitor. Both panels were performed together, sharing unstimulated controls. Data in c and i are expressed as the mean ± SD of n = 3 replicates of each in vitro culture condition in one representative experiment (each experiment was repeated three times). Yellow arrows indicate the protrusion-like structure. Data in a, b are expressed as the mean ± SD of n = 3 replicates of each in vitro culture condition in one representative experiment (experiment was repeated two times). See also Supplementary Fig. .

Article Snippet: Cells were suspended in medium at a density of 2.5 × 10 6 cells/mL and added to each chamber (5 mm diameter) of a 4-condition LCI-S chip (LCI-SPQ002; Live Cell Diagnosis, Saitama, Japan) at 50 μL, where the anti-IFN-β antibody (capture antibody from the Mouse IFN-beta DuoSet ELISA, DY8234-05; R&D Systems) was immobilised.

Techniques: Cytometry, Concentration Assay, Staining, In Vitro

Journal: Nature Communications

Article Title: cGAS-IFN-I responses by extracting nuclear DNA from dying cells via nucleocytosis

doi: 10.1038/s41467-026-68839-w

Figure Lengend Snippet: a LCI-S of GM-BMCs treated with HCQ (100 µM). IFN-β secretion (blue) and DNA staining (magenta) are shown. DNA intensity is summarised in the right. b Representative images of SYTOX Green-positive duplets analysed in Fig. . Six representative images from each population are shown. (-)P2: P2 population without HCQ treatment (Fig. ). HCQ P1: P1 population with HCQ treatment (Fig. ). c Quantitative analysis of DNA accumulation in the boundary region. Left: schematic of calculation. Right: DNA ratios within the cells in the unstimulated P2 and HCQ-stimulated P1 population in Fig. . d–g Confocal microscopy of GM-BMCs treated with HCQ (100 µM). d Calreticulin (green) and DNA (blue) staining. e Intensity at the site of white dashed lines. The X-axis indicates the distance from the left edge of the white dashed lines. f 3D pictures based on sectional 2D pictures. g Statistical analyses of nuclear calreticulin intensity in each cell. Upper panel: schematic of the calculation. Data are presented as the mean ± SEM (0 h; n = 33, 6 h; n = 42) h–k Image cytometry of GM-BMCs treated with HCQ (100 µM) for 6 h. h Singlet cells are shown in dot plots. Calreticulin high and low populations are gated within live and dead cell populations. Right: proportions of the P1 and P2 populations. i Mean fluorescence intensity of calreticulin in dead or live cells. Gating strategy for both h and i are shown in Supplementary Fig. . j Representative images of each population. Merged regions are shown in yellow. Based on the corrected images, correlation values between calreticulin and the nucleus were calculated and are presented in panel ( k ). l LCI-S of GM-BMCs treated with HCQ (80 µM). IFN-β secretion (blue) and DNA staining (magenta) are shown. m Concentration of IFN-β (left) and LDH activity (right) in the culture supernatant of GM-BMCs treated with HCQ (100 µM) in the presence of the indicated concentrations of inhibitors. n LCI-S of GM-BMCs treated with HCQ (100 µM) in the presence or absence of Fasudil (100 µM). Representative image of endpoint (12 h) is shown in left. Quantitative analysis of three independent experiments are shown on the right with the number of IFN-β spots detected. Data in a and l are representative pictures from one experiment (experiment was repeated three times). Data in ( h , i , k , and m ) are expressed as the mean ± SD of n = 3 replicates of each in vitro culture condition in one representative experiment (each experiment was repeated two to three times). Statistical analysis was performed using two-sided Wilcoxon test ( c ), two-sided Welch’s t test ( g ). See Supplementary Fig. .

Article Snippet: Cells were suspended in medium at a density of 2.5 × 10 6 cells/mL and added to each chamber (5 mm diameter) of a 4-condition LCI-S chip (LCI-SPQ002; Live Cell Diagnosis, Saitama, Japan) at 50 μL, where the anti-IFN-β antibody (capture antibody from the Mouse IFN-beta DuoSet ELISA, DY8234-05; R&D Systems) was immobilised.

Techniques: Staining, Confocal Microscopy, Cytometry, Fluorescence, Concentration Assay, Activity Assay, In Vitro

Journal: Nature Communications

Article Title: cGAS-IFN-I responses by extracting nuclear DNA from dying cells via nucleocytosis

doi: 10.1038/s41467-026-68839-w

Figure Lengend Snippet: a IFN-β concentration in the culture supernatant of GM-BMCs treated with the indicated concentrations of DC661, cGAMP (5 µg/mL), or LPS (100 ng/mL) for 16 h. b IFN-β concentration in the culture supernatant of GM-BMCs treated with HCQ (100 µM) in the presence of indicated concentrations of N-(tert-Butyl) hydroxylamine (10 mM) for 16 h. c , d In silico analysis of the interaction between human PPT1 and compounds. Yellow circle: binding pocket of palmitate. An enlarged picture of the binding pocket with detailed information is shown in ( c ). e In silico analysis of the interaction between human PPT1 and HCQ lacking each sub-structure. Left: schematic of HCQ with conserved pharmacophore features. Red, conserved pharmacophore features. Right: binding energy of HCQ lacking each pharmacophore feature of human PPT1. f , g In silico analysis of the interaction between human PPT1 and compounds. Graphical images are shown in e. The binding energy of the indicated compound to human PPT1 is shown in f. Binding energy is calculated under near-neutral conditions (pH 7.4). h Correlation analysis between IFN-β-inducing activity and binding energy to human PPT1. i , j In silico analysis of the interaction between human PPT1 and compounds under acidic conditions. The binding energy of the indicated compound to is shown in h. Graphical images of the interaction are shown in i. Interactions between each compound and H 3 O + were also examined and shown in each right panel. k IFN-β concentration in the culture supernatant of WT or Cgas -/- GM-BMCs treated with the indicated concentration of DC661 in combination with Baf A (15 nM) for 16 h. l LCI-S of GM-BMCs treated with DC661 (3 µM) in combination with Baf A (15 nM). IFN-β secretion (blue) and DNA staining (magenta) are shown. DNA intensity is summarised in the lower right. Data in ( a , b and k ) are expressed as the mean ± SD of n = 3 replicates of each in vitro culture condition in one representative experiment (each experiment was repeated three times). See also Supplementary Fig. .

Article Snippet: Cells were suspended in medium at a density of 2.5 × 10 6 cells/mL and added to each chamber (5 mm diameter) of a 4-condition LCI-S chip (LCI-SPQ002; Live Cell Diagnosis, Saitama, Japan) at 50 μL, where the anti-IFN-β antibody (capture antibody from the Mouse IFN-beta DuoSet ELISA, DY8234-05; R&D Systems) was immobilised.

Techniques: Concentration Assay, In Silico, Binding Assay, Activity Assay, Staining, In Vitro

Journal: Nature Communications

Article Title: cGAS-IFN-I responses by extracting nuclear DNA from dying cells via nucleocytosis

doi: 10.1038/s41467-026-68839-w

Figure Lengend Snippet: a Schematic of the experiment. b IFN-β concentration in the culture supernatant of GM-BMCs mixed with the indicated cell lines. Tumour cell lines were treated with the indicated concentrations of HCQ before being mixed with GM-BMCs. c Confocal microscopy of B16 cells treated with HCQ (250 µM) for the indicated periods. Left: intracellular calreticulin (green) and DNA staining (blue). Intensities of calreticulin and DNA staining were calculated at the site of the white dashed lines and shown in the upper left panel. The X-axis indicates the distance from the left edge of the white dashed lines. Schematic of the calculation is shown in the lower right. Data are presented as the mean ± SEM (0 h; n = 39, 6 h; n = 36). d Schematic of the in vivo experiment. e – i scRNA-seq analysis of lung cells from mice treated with HCQ. e UMAP plots showing cluster numbers and cell types (left two panels) annotated by the expression of key marker genes (Z-score) (right panel). f Violin plots of the sting1 gene in each cell clusters. g Dot plots showing enriched gene sets for each cell type in WT-HCQ compared with that in WT-water (left) or Sting − / − -HCQ (right) in GSEA. NES, normalised enrichment score. The top 10 gene sets with high NES and the bottom 10 with low NES are shown. h , i Violin plots of indicated genes ( h ) and ISG signature ( i ) in each cell type. Data in b are expressed as the mean ± SD of n = 3 replicates of each in vitro culture condition in one representative experiment (each experiment was repeated three times). Data in ( h and i ), the box plot within the violin plot shows the median (centre line), and the interquartile range (IQR) (the box boundaries, spanning the 25th to 75th percentiles). The whiskers extend to data points within 1.5 times the IQR, defining the non-outlier range. Points outside the whiskers are outliers. Statistical differences in ( c ) was examined using Welch’s t test. Statistical differences in ( h , i ) were examined using the two-sided Wilcoxon rank sum test. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s., not significant. See also Supplementary Fig. .

Article Snippet: Cells were suspended in medium at a density of 2.5 × 10 6 cells/mL and added to each chamber (5 mm diameter) of a 4-condition LCI-S chip (LCI-SPQ002; Live Cell Diagnosis, Saitama, Japan) at 50 μL, where the anti-IFN-β antibody (capture antibody from the Mouse IFN-beta DuoSet ELISA, DY8234-05; R&D Systems) was immobilised.

Techniques: Concentration Assay, Confocal Microscopy, Staining, In Vivo, Expressing, Marker, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: Elongator is required for pattern recognition receptor and type I interferon signaling in macrophages

doi: 10.1016/j.jbc.2025.110916

Figure Lengend Snippet: Differential protein expression in Elp3 −/− BMDMs relative to WT cells . Volcano plots showing differentially expressed proteins in unstimulated ( A ), 6 h LPS-treated ( B ) or 12 h LPS-treated ( C ) Elp3 −/− lysates versus lysates from WT cells. To capture a broad range of biological processes potentially influenced by loss of ELP3 an enrichment cut-off of a –log10 ( p -value) of 1.3 was considered significant. Proteins associated with IFN signalling by gene ontology biological process terms are labelled as red dots . IFN, interferon; LPS, lipopolysaccharide.

Article Snippet: ELISA plates were coated with a monoclonal rat anti-mouse IFNβ capture antibody (Santa Cruz, Cat#SC-57201) in carbonate buffer overnight at 4 °C.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Elongator is required for pattern recognition receptor and type I interferon signaling in macrophages

doi: 10.1016/j.jbc.2025.110916

Figure Lengend Snippet: Impaired LPS-IFN I signalling axis in Elp3 −/− BMDMs . A , schematic of LPS- IFN-I signalling axis. Figure created in BioRender ( B – K ) Label free quantification intensity values detected by mass spectrometry in WT and Elp3 −/− cells stimulated for 0, 6 or 12 h with LPS for peptides from transcription factor proteins IRF3 ( B ), IRF5 ( C ), IRF7 ( D ), IRF9 ( E ), RELA ( F ), NFKB1 ( G ), NFKB2 ( H ), STAT1 ( I ), STAT3 ( J ) and STAT6 ( K ). ND, not detected. Data are mean ± SD for quadruplicate (or triplicate for WT 0 h sample) measurements. ∗ p < 0.05 compared to WT, based on Mann-Whitney test. L – N , WT and Elp3 −/− cells were stimulated with LPS (100 ng/ml) for 3, 6 and 24 h. Ifnb ( L ), Irf7 ( M ) and Ifna ( N ) mRNA were assayed by qRT-PCR, expressed relative to the untreated WT control and normalized to the housekeeping gene β-actin. Data are mean ± SD of three independent experiments. Data significance was tested with a 2-way ANOVA using Šídák's multiple comparisons test. ∗ p < 0.05 and ∗∗ p < 0.0001 compared to WT. O , cells were stimulated with LPS for the indicated times and supernatants assayed by ELISA for IFN-β. Data are mean ± SD for three independent experiments, each performed on three WT or Elp3 −/− clones in triplicate. ∗∗∗ p < 0.001. LPS, lipopolysaccharide.

Article Snippet: ELISA plates were coated with a monoclonal rat anti-mouse IFNβ capture antibody (Santa Cruz, Cat#SC-57201) in carbonate buffer overnight at 4 °C.

Techniques: Quantitative Proteomics, Mass Spectrometry, MANN-WHITNEY, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Clone Assay

Journal: The Journal of Biological Chemistry

Article Title: Elongator is required for pattern recognition receptor and type I interferon signaling in macrophages

doi: 10.1016/j.jbc.2025.110916

Figure Lengend Snippet: ELP3 is required for IFN I and TYK 2-dependent signaling . A , WT and Elp3 −/− BMDMs were stimulated with 1000 U/ml IFNβ for the indicated times. Cells were harvested and immunoblotted for phosphorylated STAT1, STAT1 and β-actin. Representative of three independent experiments. B – D , WT and Elp3 −/− BMDMs were stimulated with 1000 U/ml IFNβ for the indicated times. RNA was isolated and mRNA expression of Irf7 ( B ), Isg15 ( C ), and Stat1 ( D ) were assayed by qRT-PCR, expressed relative to the untreated WT control and normalized to β-actin. E , WT and Elp3 −/− iBMDMs were stimulated with either LPS (100 ng/ml), IFNβ (1000 U/ml) or IFNγ (25 ng/ml) for 90 min. Cells were harvested and expression of the indicated proteins was assessed by immunoblot. Representative of 3 independent experiments. F , WT and Elp3 −/− iBMDMs were stimulated with either LPS (100 ng/ml), IFNβ (1000 U/ml) or IFNγ (25 ng/ml) for 3 h. Irf1 gene expression was assessed by qRT-PCR. mRNA levels are represented relative to β-actin, with each WT sample set to 100% and Elp3 −/− mRNA levels expressed as a percentage of their WT stimulated counterpart. G , WT and Elp3 −/− iBMDMs were stimulated with IL-10 (10 ng/ml) for 3 h. Socs3 mRNA was assayed by qRT-PCR, expressed relative to the untreated WT control and normalized to the housekeeping gene β-actin. H , WT and Elp3 −/− iBMDMs were stimulated with IL-4 (10 ng/ml) for 3 h. Arg1 mRNA was assayed by qRT-PCR, expressed relative to the untreated WT control and normalized to the housekeeping gene β-actin. For ( B – D and F – H ), data are mean ± SD of 3 independent experiments, each performed in triplicate. Data significance was tested with a 2-way ANOVA using Šídák's multiple comparisons test. ∗ p < 0.05 and ∗∗ p < 0.01 and ∗∗∗ p < 0.001 compared to WT. iBMDM, immortalised bone marrow–derived macrophage; LPS, lipopolysaccharide.

Article Snippet: ELISA plates were coated with a monoclonal rat anti-mouse IFNβ capture antibody (Santa Cruz, Cat#SC-57201) in carbonate buffer overnight at 4 °C.

Techniques: Isolation, Expressing, Quantitative RT-PCR, Control, Western Blot, Gene Expression, Derivative Assay