interferon ifn β  (R&D Systems)

Journal: eBioMedicine

Article Title: Autoantibodies against type I interferons in patients with zoonotic H7N9 influenza: an observational case–control study

doi: 10.1016/j.ebiom.2026.106387

Figure Lengend Snippet: Autoantibodies binding to IFNα 2 , IFNβ 1b and IFNω in patients with H7N9 infection and healthy controls. (A) Age and sex distribution of the three study groups. For each age group, the number of individuals positive for autoantibodies neutralising at least one tested IFN-I (IFNα 2 , IFNβ 1b , IFNω) at low concentrations is indicated in red. M, male; F, female; nAb+, positive for IFN-I-neutralising autoantibodies. (B) Detection of IgG autoantibodies binding to IFNα 2 , IFNβ 1b or IFNω in serum samples by multiplex bead-based assay. Samples with a Z-score >7 were considered positive for IFN-I-binding autoantibodies. Measurements were performed without technical replicates because of limited sample availability. (C) Prevalence of IFN-I-binding autoantibodies by IFN type and study group. IFNα 2 ± IFNβ 1b ± IFNω, positive for autoantibodies binding to at least one of the tested IFN-I; IFNα 2 + IFNω, positive for autoantibodies binding to both IFNα 2 and IFNω.

Article Snippet: Serum was diluted 1:50 in DMEM supplemented with 10% FCS and penicillin/streptomycin, and pre-incubated for 1 h at room temperature with one of the following IFN concentrations: (i) IFNα 2 (Novus Biologicals, NBP2-34971) at 10 ng/ml or 0.5 ng/ml; (ii) IFNβ 1b (PBL Assay Science, 11420-1) at 1 ng/ml or 0.25 ng/ml; (iii) IFN-ω (Novus Biologicals, NBP2-35893) at 10 ng/ml or 0.2 ng/ml.

Techniques: Binding Assay, Infection, Multiplex Assay, Bead-based Assay

Journal: eBioMedicine

Article Title: Autoantibodies against type I interferons in patients with zoonotic H7N9 influenza: an observational case–control study

doi: 10.1016/j.ebiom.2026.106387

Figure Lengend Snippet: Autoantibodies neutralising IFN-I in patients with H7N9 infection and healthy controls. (A) Luciferase-based reporter assay to assess the capacity of autoantibody positive sera to neutralise IFNα 2 (10 or 0.5 ng/ml), IFNβ 1b (1 or 0.25 ng/ml) or IFNω (10 or 0.2 ng/ml). Each sample was tested in biological duplicates and the mean values are shown. Samples were classified as neutralising if the mean of the relative luciferase activities was below 25% (dotted line) of the mean of the negative pool (four autoantibody-negative control sera). All sera positive for IFN-I-binding autoantibodies were tested; numbers are indicated above the graphs. Lines connect measurements of neutralising activity from the same serum sample at low and high IFN concentrations. (B) Prevalence of autoantibodies neutralising low IFN concentrations (IFNα 2 : 0.5 ng/ml, IFNβ 1b : 0.25 ng/ml; IFNω: 0.2 ng/ml) by IFN type and study group. IFNα 2 ± IFNβ 1b ± IFNω, positive for autoantibodies neutralising at least one tested IFN-I; IFNα 2 + IFNω, positive for autoantibodies neutralising both IFNα 2 and IFNω. (C) Area-proportional Venn diagrams illustrating the absolute numbers of samples with autoantibodies neutralising high and low concentrations of IFNα 2 (10 or 0.5 ng/ml), IFNβ 1b (1 or 0.25 ng/ml) or IFNω (10 or 0.2 ng/ml). Venn diagrams were created with BioVenn ( https://www.biovenn.nl/index.php ).

Article Snippet: Serum was diluted 1:50 in DMEM supplemented with 10% FCS and penicillin/streptomycin, and pre-incubated for 1 h at room temperature with one of the following IFN concentrations: (i) IFNα 2 (Novus Biologicals, NBP2-34971) at 10 ng/ml or 0.5 ng/ml; (ii) IFNβ 1b (PBL Assay Science, 11420-1) at 1 ng/ml or 0.25 ng/ml; (iii) IFN-ω (Novus Biologicals, NBP2-35893) at 10 ng/ml or 0.2 ng/ml.

Techniques: Infection, Luciferase, Reporter Assay, Negative Control, Binding Assay, Activity Assay

Journal: eBioMedicine

Article Title: Autoantibodies against type I interferons in patients with zoonotic H7N9 influenza: an observational case–control study

doi: 10.1016/j.ebiom.2026.106387

Figure Lengend Snippet: Association between the presence of IFN-I-neutralising autoantibodies and H7N9 infection. (A) The association between age, sex and IFN-I-neutralising autoantibodies in patients with H7N9 infection or in the two control groups combined (poultry workers + close contacts) was assessed using Firth's penalised logistic regression. Predicted probabilities for the presence of autoantibodies with 95% confidence intervals (CIs, shaded areas around the curve) are shown across participant age for men and women. To visualise the modelled probabilities in relation to the underlying data, we overlaid sex-specific age density distributions beneath the predicted probability curves. (B) Odds ratios (OR) with 95% CIs for the presence of autoantibodies neutralising low IFN concentrations in patients compared to healthy controls, adjusted for age and sex, determined by Firth’s penalised logistic regression models. See also for the results of the logistic regression analyses and for unadjusted estimates. IFNα 2 ± IFNω ± IFNβ 1b , positive for autoantibodies neutralising at least one of the tested IFN-I; IFNα 2 ± IFNω, positive for autoantibodies neutralising IFNα 2 and/or IFNω; ∗∗∗∗, p < 0.0001 (Firth’s penalised logistic regression).

Article Snippet: Serum was diluted 1:50 in DMEM supplemented with 10% FCS and penicillin/streptomycin, and pre-incubated for 1 h at room temperature with one of the following IFN concentrations: (i) IFNα 2 (Novus Biologicals, NBP2-34971) at 10 ng/ml or 0.5 ng/ml; (ii) IFNβ 1b (PBL Assay Science, 11420-1) at 1 ng/ml or 0.25 ng/ml; (iii) IFN-ω (Novus Biologicals, NBP2-35893) at 10 ng/ml or 0.2 ng/ml.

Techniques: Infection, Control

Journal: eBioMedicine

Article Title: Autoantibodies against type I interferons in patients with zoonotic H7N9 influenza: an observational case–control study

doi: 10.1016/j.ebiom.2026.106387

Figure Lengend Snippet: Neutralising sera block the antiviral effect of IFNα 2 in cell culture infected with IAV. Antiviral activity of IFNα 2 (5 ng/ml) against IAV (PR8-GFP, MOI 1) alone or in the presence of serially diluted IFN-I-neutralising sera (n = 19), autoantibody-negative sera (n = 4), or a monoclonal anti-IFNα 2 antibody in A549 cells. Infection rates (GFP + /DAPI + cells) at 7 h post-infection were normalised to untreated, infected cells. The dotted line indicates the reduction of infected cells after IFN treatment alone. If possible, the mean of two independent experiments is shown. Sufficient material was available for 12 out of 19 samples.

Article Snippet: Serum was diluted 1:50 in DMEM supplemented with 10% FCS and penicillin/streptomycin, and pre-incubated for 1 h at room temperature with one of the following IFN concentrations: (i) IFNα 2 (Novus Biologicals, NBP2-34971) at 10 ng/ml or 0.5 ng/ml; (ii) IFNβ 1b (PBL Assay Science, 11420-1) at 1 ng/ml or 0.25 ng/ml; (iii) IFN-ω (Novus Biologicals, NBP2-35893) at 10 ng/ml or 0.2 ng/ml.

Techniques: Blocking Assay, Cell Culture, Infection, Activity Assay

Journal: mBio

Article Title: A dominant role of cell death in limiting Chandipura virus propagation at cell-saturating high multiplicity of infection

doi: 10.1128/mbio.01013-26

Figure Lengend Snippet: Comparing CHPV propagation in cultured cells infected at different MOIs. ( a ) Schema summarizing cell-infection experiments performed in this study. ( b ) Barplot revealing progeny virus titer in the culture supernatant of MEFs at 24 h post-infection with CHPV at the indicated input MOI. ( c ) Similarly, progeny virus yield was measured at 12 h and 36 h post-infection for cells infected at MOI 2 and 20. ( d ) ELISA showing accumulation of IFNβ in a time course in the culture supernatant of cells infected with CHPV at the indicated MOI. ( e ) CHPV-mediated cell death was measured at 24 h post-infection by crystal violet staining. The abundance of viable cells at various MOI was determined relative to corresponding uninfected MEFs and presented as bargraphs. ( f ) Infection-induced cell death was similarly measured at 12 h and 36 h post-infection. Data represent the means of three biological replicates ± SEM. The statistical significance was determined using two-way ANOVA test. ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: The abundance of IFNβ in the culture supernatant of infected MEFs was determined using a mouse IFNβ ELISA kit (PBL Assay Science, Piscataway, NJ) adhering to manufacturer’s protocol.

Techniques: Cell Culture, Infection, Virus, Enzyme-linked Immunosorbent Assay, Staining

Journal: mBio

Article Title: A dominant role of cell death in limiting Chandipura virus propagation at cell-saturating high multiplicity of infection

doi: 10.1128/mbio.01013-26

Figure Lengend Snippet: Mathematically probing CHPV multiplication and host responses at cell-saturating MOIs. ( a ) A cartoon depicting key processes involved in CHPV perpetuation at cell-saturating MOIs that were considered for mathematical analyses. Kinetic rate parameters associated with each of the reactions have been indicated. Right, a set of mathematical equations used for describing the relationship between number of infected live cells, IFN concentration, and progeny virus titer has been indicated. IFN( t ) and V ( t ) have been used to represent IFN concentration in pg/mL and viral titer in pfu/mL at time t . The fraction of live, infected cells at time t has been described as C ( t ). Other parameters have been described in . ( b – d ) Fitting mathematical equations with experimental time course data related to progeny virus titer ( b ), cell-produced IFNβ levels ( c ), and infection-inflicted cell death ( d ) observed at MOI 2 and MOI 20. Experimental data points derived from biological replicates are indicated in black circles, and solid lines represent the fitted mean values for viral titer, IFN-β levels, or fraction of live cells. Shaded regions around the solid lines indicate the corresponding 95% confidence intervals. ( e ) Bar chart comparing the values of cell death , IFN pdn , and virus pdn at MOI 2 and MOI 20 extracted from fitting exercise. Values of these rate parameters were independently determined using data from experimental replicates. Unpaired t -test was performed to determine the statistical significance. ( f ) Simulating virus yield as a function of time in the MOI 20 regime using the virus pdn , IFN pdn , or cell death rate constant values linked to MOI 2. Data represent the means of three biological replicates ± SEM. *** P ≤ 0.001; ns, not significant, ≥0.05.

Article Snippet: The abundance of IFNβ in the culture supernatant of infected MEFs was determined using a mouse IFNβ ELISA kit (PBL Assay Science, Piscataway, NJ) adhering to manufacturer’s protocol.

Techniques: Infection, Concentration Assay, Virus, Produced, Derivative Assay

Journal: mBio

Article Title: A dominant role of cell death in limiting Chandipura virus propagation at cell-saturating high multiplicity of infection

doi: 10.1128/mbio.01013-26

Figure Lengend Snippet: Charting CHPV growth in Ifnar1 −/− MEFs infected at different MOI. ( a ) Barplot comparing virus yields in WT and Ifnar1 −/− MEFs determined at 24 h post-infection at the indicated MOIs. ( b ) ELISA revealing the abundance of IFNβ in the culture supernatant of Ifnar1 −/− cells infected with CHPV at the indicated MOIs. ( c ) Barplot showing cell death in WT and Ifnar1 −/− MEFs at 24 h post-infection with CHPV at the indicated MOIs. ( d – f ) Line plots depicting fitting of mathematical equations with experimental time course data on CHPV propagation in Ifnar1 −/− cells (left). Shaded regions represent 95% confidence intervals. Extracted values for various rate parameters are indicated in the accompanying bar chart (right). Data represent the means of three biological replicates ± SEM. The statistical significance was determined using two-way ANOVA test for panels a through c and using unpaired t -test for panel d ** P ≤ 0.01; *** P ≤ 0.001; ns, not significant, ≥0.05.

Article Snippet: The abundance of IFNβ in the culture supernatant of infected MEFs was determined using a mouse IFNβ ELISA kit (PBL Assay Science, Piscataway, NJ) adhering to manufacturer’s protocol.

Techniques: Infection, Virus, Enzyme-linked Immunosorbent Assay

Journal: mBio

Article Title: A dominant role of cell death in limiting Chandipura virus propagation at cell-saturating high multiplicity of infection

doi: 10.1128/mbio.01013-26

Figure Lengend Snippet: Studying CHPV propagation in Nfkbia −/− MEFs infected at different MOI. ( a, b, c ) Comparing WT and Nfkbia −/− MEFs infected at various MOIs for cell death ( a ), IFNβ production ( b ), and CHPV yield ( c ). ( d – f ) Fitting experimental time course data on CHPV propagation in Nfkbia −/− cells in our mathematical model (left). Shaded regions represent 95% confidence intervals. Extracted values for various rate parameters are also indicated (right). ( g ) Line plot charting the simulated progeny virus titer estimated in the Nfkbia −/− settings at MOI 20 using altered virus pdn , cell death , and IFN pdn values that preserved the quantum of changes in these rates upon raising MOI from 2 to 20 to those determined in WT cells. Data represent the means of three biological replicates ± SEM. The statistical significance was determined using two-way ANOVA test for panels a through c and using unpaired t -test for panel d. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: The abundance of IFNβ in the culture supernatant of infected MEFs was determined using a mouse IFNβ ELISA kit (PBL Assay Science, Piscataway, NJ) adhering to manufacturer’s protocol.

Techniques: Infection, Virus

Journal: Advanced Science

Article Title: RNA‐Binding Protein RBM25 Targets the mRNA Stability of GTPase Rab22a to Restrict Viral Entry and Infection

doi: 10.1002/advs.76160

Figure Lengend Snippet: RBM25 mediates antiviral protection independent of the IFN‐I signaling pathway. (A) A scheme of RNA‐seq analysis of WT and Rbm25 ‐cKO peritoneal macrophages infected with VSV for 4 h. (B) Heatmap of differentially expressed genes (DEGs) in Rbm25 ‐deficient and WT peritoneal macrophages infected with VSV for 4 h. (C) GSEA showing enrichment of DEGs in the pathways involving in the response to IFN‐α, IFN‐β production, JAK‐STAT3, IFN‐I receptor binding, toll like receptor, and cGAS target gene signaling pathways. (D) RT‐qPCR analysis of IFN‐I ( Ifna4, Ifnb1 ) mRNA in the lung tissues from WT and Rbm25 ‐cKO mice infected with PR8 influenza virus with a sublethal dose (50 PFU., n = 5 per group) at 7 dpi. (E) RT‐qPCR analysis of Cxcl10, Mx1, Isg15 , and Mx2 mRNA in the lung tissues from WT and Rbm25 ‐cKO mice treated as in (D). (F) ELISA of IFN‐β in serum from Rbm25 ‐cKO and WT mice infected with PR8 influenza virus, as in (D). (G) RT‐qPCR analysis of IFN‐I ( Ifna4, Ifnb1 ) mRNA in peritoneal macrophages from Rbm25 ‐cKO and WT mice infected with PR8 influenza virus for 12 h. (H) RT‐qPCR analysis of Cxcl10, Isg15 , and Rsad2 mRNA in peritoneal macrophages from WT and Rbm25 ‐cKO mice treated as in (G). (I) Immunoblots analysis of RBM25, RIG‐I, MAVS, p‐TBK1, TBK1, p‐IRF3, and IRF3 in peritoneal macrophages from WT and Rbm25 ‐cKO mice infected with PR8 influenza virus for the indicated times. β‐ACTIN was used as a loading control. (J) A scheme of experiment analyzing the infection of viruses in peritoneal macrophages from Irf3 −/− mice transfected with control siRNA or siRNA targeting Rbm25 . (K) RT‐qPCR analysis of PR8 HA and IAV M1, HSV‐1 TK and ICP0, VSV‐G mRNA in peritoneal macrophages from Irf3 −/− mice transfected with control siRNA or siRNA targeting Rbm25 followed by infection with PR8 influenza virus for 12 h, HSV‐1 for 12 h, or VSV for 8 h, respectively. (L, M) RT‐qPCR analysis of IFN‐I ( Ifna4, Ifnb1 ) mRNA in Rbm25 ‐cKO and WT peritoneal macrophages transfected with poly (I:C) for 4 h (L) or stimulated with UV‐VSV for 18 h (M). (N) Immunoblots analysis of p‐STAT1 and STAT1 in peritoneal macrophages from WT and Rbm25 ‐cKO mice infected with PR8 influenza virus for the indicated times. β‐ACTIN was used as a loading control. Data are presented as the mean ± SD. Unpaired two‐tailed Student's t ‐test (D–H, K–M). ns, not significant.

Article Snippet: IFN‐β levels in the supernatants or sera were measured using a mouse IFN‐β ELISA kit (PBL Biomedical Laboratories) according to the manufacturer's instructions.

Techniques: RNA Sequencing, Infection, Binding Assay, Protein-Protein interactions, Quantitative RT-PCR, Virus, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: Spatiotemporal dynamics of adoptively transferred stem-like CD8 + T cells in the tumor microenvironment following vaccination

doi: 10.64898/2026.05.12.724323

Figure Lengend Snippet: (A) Tumor curve following treatment of Tscm cells with various vaccine conjugates (n=7). Statistics accessed by two-way ANOVA. (B) Survival curve following treatment of Tscm cells with various vaccine conjugates (n=35). Statistics accessed by log-rank test. (C) Bar graphs showing the frequency of CD45.2 + transferred cells in the tumor, tdLN, and spleen by treatment group (n=3). Statistics accessed by ANOVA. (D) Stacked bar graphs indicating the percentages of PD1 and TCF1 subpopulations in tumor, tdLN, and spleen (n=3). Statistics assessed by ANOVA. (E) Bar graph showing the serum cytokine levels (pg/mL) of IFN-α 6 hours post-vaccination in indicated treatment groups (n=5). Statistics assessed by ANOVA. (F) Bar graph showing the serum cytokine levels (pg/mL) of IFN-β 6 hours post-vaccination in indicated treatment groups (n=5). Statistics assessed by ANOVA. (G) Bar graph showing the serum cytokine levels (pg/mL) of IP-10 6 hours post-vaccination in indicated treatment groups (n=5). Statistics assessed by ANOVA. (H) Heatmap of Z-scored serum cytokine data taken 6 hours post-vaccination in indicated treatment groups (n=5). (I) Bar graph showing the serum cytokine levels (pg/mL) of IFN-γ 6 hours post-vaccination in indicated treatment groups (n=5). Statistics assessed by ANOVA. (J) Bar graph showing frequency by flow cytometry of IFN-γ + cells among live, CD8 + T cells in the spleen three days following vaccination (n=3). Statistics assessed by ANOVA.

Article Snippet: ELISA kits were used to measure IFN-α and IFN-β (PBL Assay Science) and ran in accordance with manufacturer’s instructions.

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: Gene regulatory networks define human airway epithelial cell types and their distinct responses to type I interferon

doi: 10.64898/2026.05.09.724010

Figure Lengend Snippet: ( A ) ( i - v ) Study design, ( ii - iv ) UMAP visualization of ( ii ) scRNA-seq, ( iii ) snATAC-seq and ( iv ) sn-multiome-seq cell type annotations. ( B ) Principal component analysis ( PCA ) of pseudobulk (cell type, time point, donor) gene expression data. In the scores plots, pseudobulk samples are colored by cell type, with timepoint indicated (“0”, “2”, or “6” hours post-IFN) for each of four donors. Ellipses represent 95% confidence intervals for each cell type. Loadings plots depict gene contributions to the PCs, with select genes labeled. Axis labels include percent variance explained by each PC in parentheses. ( C ) Normalized pseudobulk gene expression (RNA, z-scaled) of 9,925 signature genes (P adj < 0.1, |log 2 (FC)| > 0.58, Methods ) distinguishing the major epithelial cell types; labels indicate cell type marker genes described in Hewitt and Lloyd (4 donors per cell type and 3 timepoints). ( D ) Normalized pseudobulk (cell type, time point, donor) chromatin accessibility (z-scaled) of 166,968 differential peaks (|log 2 (FC)| > 0.58) distinguishing five HAE cell types (3 donors per cell type and 3 timepoints). ( E ) Chromatin accessibility (from snATAC-seq assay) at select cell type marker gene loci across HAE cell types. Box colors indicate the associated cell type for each marker. Coverage signal normalized per million snATAC-seq fragments. ( F ) Chromatin accessibility (from sn-multiome-seq) of select deuterosomal cell marker genes across the major epithelial cell populations. Coverage signal normalized per million snATAC-seq fragments. ( G ) Normalized pseudobulk (cell type, time point, donor) chromatin accessibility of deuterosomal cell peaks (from sn-multiome-seq, n = 2 donors) across HAE cell types. Each cluster is annotated (right-hand-side) by TF families whose motifs are significantly enriched in that cluster (FDR = 5%, Fisher’s exact test). ( H ) Enrichment of accessible chromatin regions detected in deuterosomal cells (from G ) proximal to nominally detected genes (±2kb of TSS) in cell type signature gene sets (from C ) (P adj < 0.05 indicated by black circle outline, Fisher’s exact test). ( I ) PCA of pseudobulk ATAC-seq signal from sn-multiome-seq; pseudobulks were down-sampled to eliminate library-size (technical) variability. ( J ) GRN-estimated protein TF activities ( TFAs ) of “core” TFs predicted to control cell type-specific gene signatures at steady-state. HAE schematics in ( A ) generated with BioRender.

Article Snippet: After imaging at the 0h timepoint, cultures were maintained with or without IFNβ (2 nM; PBL Assay Science, Cat #: 11415-1) in the basolateral compartment, along with CD38 inhibitor (78C; 1 mM; TOCRIS Bioscience, cat #:6391) or dimethylsulfoxide (DMSO (vehicle control); 1 mM; ATCC, cat. # 4-X) in both the apical and basolateral compartments.

Techniques: Gene Expression, Labeling, Marker, Control, Generated

Journal: bioRxiv

Article Title: Gene regulatory networks define human airway epithelial cell types and their distinct responses to type I interferon

doi: 10.64898/2026.05.09.724010

Figure Lengend Snippet: ( A ) Enrichment of IRG clusters from in genes proximal (±2kb of TSS) to cell-type signature peaks (left) or signature genes (right), derived from steady state (i.e., from Fig. 1C -D ). For IRG clusters containing both IIGs and IDGs, the enrichment was limited to the dominant IIG or IDG pattern, and predominantly IDG clusters are highlighted with a light blue strip (Fisher’s exact test, *P adj < 0.05). ( B ) Data supporting the top-predicted “ dynamic” TF regulators of IRG patterns, a subset (bolded text) are additionally “baseline” TFs, predicted to prime chromatin proximal to cell type-associated IRGs at steady state. We report ( i ) “within-cell type” predicted activator role (red: enrichment of the TF’s activating gene targets in a cell type’s IFN-upregulated genes at 2 or 6h relative to 0h) and/or a predicted repressor role (blue: enrichment of the TF’s repressed gene targets in a cell type’s IFN-downregulated genes at 2 or 6h relative to 0h), ( ii ) TFA dynamics across cell types and time points, ( iii ) potential role in priming of the IRG clusters: asterisks indicate that TFBS (from maxATAC or motif scanning) are enriched proximally to genes within the IRG cluster; Fisher exact test, P adj < 0.05; while box shading indicates predicted activator or repressor role, based on TF’s target enrichments in the IIGs or IDGs of each IRG cluster, and ( iv ) the number of activating or repressing IRG targets per TF. ( C ) Select regulatory interactions between shared or cell type-associated dynamic TFs and shared IIGs. Node colors represent the z-scored gene expression (target nodes) or TFA (TF nodes) over time, averaged across basal, ciliated and secretory cells. TFs are grouped based on their cell type-associated activities and dynamics. ( D ) Top baseline TF regulators of cell type-associated expression of IRG clusters (see Methods for inclusion criteria), annotated as in ( B ). ( E - G ) Network diagrams showing the regulation of IRG clusters by baseline and/or IFN-responsive dynamic TFs. TFs are shaded according to z-scored TFA (donors averaged per time point) in the represented cell type. Edges are colored based on the adjusted p-value of TF target enrichment from panel iii in 6B, D .

Article Snippet: After imaging at the 0h timepoint, cultures were maintained with or without IFNβ (2 nM; PBL Assay Science, Cat #: 11415-1) in the basolateral compartment, along with CD38 inhibitor (78C; 1 mM; TOCRIS Bioscience, cat #:6391) or dimethylsulfoxide (DMSO (vehicle control); 1 mM; ATCC, cat. # 4-X) in both the apical and basolateral compartments.

Techniques: Derivative Assay, Stripping Membranes, Gene Expression, Expressing

Journal: bioRxiv

Article Title: The COX2-PGE2-PKA Axis Suppresses Antiviral Immunity by Inhibiting mtDNA-Dependent STING Activation

doi: 10.64898/2026.04.03.716411

Figure Lengend Snippet: (A) Schematic of the antiviral property of cGAS/STING-mediated type I IFN during HSV-1 infection. (B) Representative images of bone marrow-derived macrophages infected with GFP-expressing HSV-1 (MOI = 1) for 24 hours in the presence or absence of 1 µM PGE 2 . (C) Bone marrow-derived macrophages were infected with HSV-1 (MOI = 1) in the presence or absence of 1 µM PGE 2 . At 24 h.p.i, cell lysates were collected and subjected to qPCR to quantify HSV-1 UL30 genomic abundance (n = 3). Data are shown as ΔΔCt values. (D) Schematic of the COX2/PGE 2 signaling axis. (E) Bone marrow-derived macrophages were infected with HSV-1 (MOI = 1) in the presence or absence of 1 µM celecoxib. At 24 h.p.i, cell lysates were collected and subjected to qPCR to quantify HSV-1 UL30 genomic abundance (n = 3). Data are shown as ΔΔCt values. (F) Schematic illustrates experimental design of bulk RNA-seq workflow in THP-1 macrophages under four conditions: vehicle (Veh), PGE 2 alone (PGE 2 ), infected with HSV-1 for 16 hours in the absence (HSV-1+Veh) or presence of exogenous PGE 2 (HSV-1+PGE 2 ) to study mechanisms in which PGE 2 controls innate immunity in response to HSV-1 infection. (G-H) Gene set enrichment analysis (GSEA) of RNA-seq data comparing HSV-1 + PGE2 versus HSV-1 + Veh. (G) Tables summarize enriched gene sets and associated statistics. (H) Representative enrichment plots for the indicated pathways. See also Fig. S1E. NES, normalized enrichment score; FDR, false discovery rate. (I) THP-1 macrophages expressing ISRE-Luciferase were mock-infected and infected with HSV-1 (MOI = 1) in the presence or absence of 1 µM PGE 2 . At 16 h.p.i, cell lysates were collected and subjected to a luciferase reporter assay to assess ISRE promoter activity (n = 3). (J) THP-1 macrophages were mock-infected and infected with HSV-1 (MOI = 1) in the presence or absence of 1 µM PGE 2 . At 16 h.p.i, cell lysates were collected and subjected to RT-qPCR to measure mRNA levels of representative type I ISGs (n = 3). Data are shown ΔΔCt values. (K) THP-1 macrophages were mock-infected and infected with HSV-1 (MOI = 1) in the presence or absence of 1 µM PGE 2 . At 24 h.p.i, supernatants were collected and analyzed by ELISA to measure secreted levels of IFNβ (n = 3). All experiments were performed with three independent biological replicates and repeated at least twice with reproducible results. Data are presented as mean ± s.e.m. Statistical significance was determined by unpaired, two-tailed Student’s t-test (C, E) or one-way ANOVA followed by Sidak’s multiple comparisons test (I-K); p- values are indicated.

Article Snippet: Concentration of secreted IFNβ was measured using Human IFN-Beta TCM ELISA, with high sensitivity (PBL Assay Science, 41435) according to the manufacturer’s instructions.

Techniques: Infection, Derivative Assay, Expressing, RNA Sequencing, Luciferase, Reporter Assay, Activity Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: bioRxiv

Article Title: The COX2-PGE2-PKA Axis Suppresses Antiviral Immunity by Inhibiting mtDNA-Dependent STING Activation

doi: 10.64898/2026.04.03.716411

Figure Lengend Snippet: (A) A schematic illustrates the activation of STING, TBK1, and IRF3 in response to double-stranded DNA (dsDNA) in the cytosol by cGAS. (B-D) THP-1 macrophages were mock-infected or infected with HSV-1 in the presence or absence of 1 µM PGE 2 . At the indicated h.p.i, cell lysates were collected and subjected to immunoblotting with the indicated antibodies (B) . Band intensity of phosphorylated TBK1 (C) and IRF3 (D) was quantified and normalized to total TBK1 and IRF3, respectively (n = 3). (E) THP-1 macrophages expressing ISRE-Luciferase were transfected with viral DNA at indicated concentrations in the presence or absence of 1 µM PGE 2 for 16 hours. Cell lysates were collected and subjected to a luciferase reporter assay to assess ISRE promoter activity (n = 3). (F) THP-1 macrophages were mock-infected or infected with HSV-1 (MOI = 1) for the indicated times. Cell lysates were collected and subjected to immunoblotting with the indicated antibodies. Samples transfected with either scramble siRNA (siCTRL) or TFAM siRNA (siTFAM) were included as controls. (G) THP-1 macrophages were mock-infected or infected with HSV-1 (MOI = 1). At 16 h.p.i, cell lysates were collected and subjected to qPCR to assess levels of total mt-D-loop and mt-ND1 regions (n = 3). (H) Representative Airyscan live-cell imaging of THP-1 macrophages mock-infected or infected with HSV-1 for 3 hours. MitoTracker DeepRed (magenta): mitochondria, PicoGreen (yellow): DNA. Scale bars, 2 µm. (I) Quantification of cytosolic PicoGreen in THP-1 macrophages mock-infected or infected with HSV-1 (MOI = 1) for 3 hours (n = 10). (J-K) THP-1 macrophages were infected with HSV-1 in the presence or absence of the mtDNA replication inhibitor, ddC. At 24 h.p.i, cell supernatants were collected and subjected to ELISA to measure levels of secreted IFNβ (n = 3) (J) , and cell lysates were collected and subjected to qPCR to quantify HSV-1 UL30 genomic abundance (n = 3) (K) . (L) THP-1 macrophages expressing ISRE-Luciferase were transfected with either scramble siRNA (siCTRL) or TFAM siRNA (siTFAM) in the presence or absence of 1 µM PGE 2. At 16 hours post stimulation, cell lysates were collected and subjected to a luciferase reporter assay to assess ISRE promoter activity (n = 3). (M) THP-1 macrophages were transfected with either scramble siRNA (siCTRL) or TFAM siRNA (siTFAM) in the presence or absence of 1 µM PGE 2 . At 16 hours post-stimulation, cell lysates were collected and subjected to RT-qPCR to assess the mRNA levels of representative type I ISGs (n = 3). All experiments were performed with three independent biological replicates and repeated at least twice with reproducible results. Data are presented as mean ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test ( C-E, L-M ) or unpaired, two-tailed Student’s t-test ( G, I-K ). p -values are indicated.

Article Snippet: Concentration of secreted IFNβ was measured using Human IFN-Beta TCM ELISA, with high sensitivity (PBL Assay Science, 41435) according to the manufacturer’s instructions.

Techniques: Activation Assay, Infection, Western Blot, Expressing, Luciferase, Transfection, Reporter Assay, Activity Assay, Live Cell Imaging, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: The COX2-PGE2-PKA Axis Suppresses Antiviral Immunity by Inhibiting mtDNA-Dependent STING Activation

doi: 10.64898/2026.04.03.716411

Figure Lengend Snippet: (A) Heatmap showing relative expression of COX2 and type I ISGs in mice treated with either vehicle (Veh) or doxorubicin (Doxo) (GEO: GSE223698). (B) Heatmap showing relative expression of COX2 and type I ISGs in proliferating cells (Prof) and senescence cells (Sen) (GEO: GSE196610). (C) THP-1 macrophages were mock-infected or infected with HSV-1 (MOI = 1) for either 24 or 48 hours. Cell lysates were subjected to RT-qPCR to assess mRNA levels of COX2 (n = 3). (D) THP-1 macrophages were mock-infected or infected with HSV-1 (MOI = 1) for either 24 or 48 hours in the presence or absence of 1 µM celecoxib (COX2i). Cell lysates were collected and analyzed by ELISA to measure extracellular PGE 2 levels (n = 3). (E) A schematic illustrates PGE 2 -cAMP-PKA signaling. (F) THP-1 macrophages were treated with 1 µM PGE 2 in the presence or absence of 1 µM EP4 inhibitor (EP4i) for 16 hours. Cell lysates were collected and analyzed by ELISA to measure intracellular cAMP levels (n = 3). (G) THP-1 macrophages were treated with 1 µM PGE 2 at indicated concentrations in the presence or absence of 1 µM EP4 inhibitor (EP4i) for 16 hours. Cell lysates were collected and subjected to immunoblotting with the indicated antibodies. (H) THP-1 macrophages expressing ISRE-Luciferase were mock-infected or infected with HSV-1 (MOI = 1) in the presence of either 1 µM PGE 2 or 1 µM forskolin and 50 µM IBMX. At 16 h.p.i, cell lysates were collected and subjected to a luciferase reporter assay to assess ISRE promoter activity (n = 3). (I-J) THP-1 macrophages were mock-infected or infected with HSV-1 (MOI = 1), followed by 1 µM PGE 2 stimulation in the presence of either 1 µM EP4 inhibitor (EP4i) or 1 µM PKA inhibitor (PKAi). At 24 h.p.i, cell lysates were subjected to RT-qPCR to assess mRNA levels of IFNβ (n = 3) (I) , and cell supernatants were subjected to ELISA to measure secreted levels of IFNβ (n = 3) (J) . (K) THP-1 macrophages expressing ISRE-Luciferase were mock-infected or infected with HSV-1 (MOI = 1), followed by 1 µM PGE 2 stimulation in the presence of either 1 µM EP4 inhibitor (EP4i) or 1 µM PKA inhibitor (PKAi). At 16 h.p.i, cell lysates were collected and subjected to a luciferase reporter assay to assess ISRE promoter activity (n = 3). (L) THP-1 macrophages were infected with HSV-1 (MOI = 1), followed by 1 µM PGE 2 stimulation in the presence of either 1 µM EP4 inhibitor (EP4i) or 1 µM PKA inhibitor (PKAi). At 16 h.p.i, cell lysates were collected and subjected to RT-qPCR to assess HSV-1 UL30 genomic abundance (n = 3). Data are presented as mean ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test. P -values are indicated.

Article Snippet: Concentration of secreted IFNβ was measured using Human IFN-Beta TCM ELISA, with high sensitivity (PBL Assay Science, 41435) according to the manufacturer’s instructions.

Techniques: Expressing, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Luciferase, Reporter Assay, Activity Assay