thp 1 blue isg cells  (InvivoGen)

Journal: npj Viruses

Article Title: The soluble G protein of respiratory syncytial virus promotes viral dissemination via TLR2-mediated NLRP3 priming and pyroptosis

doi: 10.1038/s44298-026-00172-x

Figure Lengend Snippet: a NF-κB reporter assay in THP-1-Blue cells treated with rsG WT, rsG CX₃Cmut, rBSA, or LTA in the presence or absence of TLR2 or MyD88 inhibitors. SEAP secretion was measured at 550 nm. b Luminex multiplex analysis of IL-6, IL-8, VEGF and CCL2 in A549 cell supernatants after treatment. The data represent the means ± SDs from three independent experiments. Statistical analysis: a Two-way ANOVA with Tukey’s post hoc test. b Kruskal‒Wallis test with Dunn’s post hoc test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: THP-1 blue NF-κB cells (InvivoGen) were seeded in 96-well plates and treated with 50 or 150 nM rBSA, rsG WT, or the rsG CX3C mutant, with or without 200 nM C29 (MedChemExpress HY-100461) or 50 nM TJ-M2010-5 (MedChemExpress HY-139397) for 1 h. After 18 h, SEAP activity was measured by transferring the supernatant to Quanti-BlueTM reagent (InvivoGen) and reading the absorbance at 650 nm after 2 h.

Techniques: Reporter Assay, Luminex, Multiplex Assay

Journal: npj Viruses

Article Title: The soluble G protein of respiratory syncytial virus promotes viral dissemination via TLR2-mediated NLRP3 priming and pyroptosis

doi: 10.1038/s44298-026-00172-x

Figure Lengend Snippet: a RT‒qPCR analysis of NLRP3 mRNA expression in A549 cells after treatment with sG, rBSA, RSV or LTA at the indicated time points. b Caspase-1 activity in A549 cells pretreated with sG and superinfected with RSV, with or without the NLRP3 inhibitor MCC950. c Western blot detection of full-length and cleaved gasdermin D (GSDMD) in cell lysates. Lanes 1–4 and 5–8 represent untreated (1/5), rBSA 150 nM (2/6), rsG WT (3/7) and rsG CX3Cmut (4/8). Lane 9 represents in both panels (left (L)/right (R)) a PMA + LPS-treated THP-1 positive control. a , b Data represent the mean ± SD from three independent experiments. Statistical analysis: two-way ANOVA with Tukey’s post hoc test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: THP-1 blue NF-κB cells (InvivoGen) were seeded in 96-well plates and treated with 50 or 150 nM rBSA, rsG WT, or the rsG CX3C mutant, with or without 200 nM C29 (MedChemExpress HY-100461) or 50 nM TJ-M2010-5 (MedChemExpress HY-139397) for 1 h. After 18 h, SEAP activity was measured by transferring the supernatant to Quanti-BlueTM reagent (InvivoGen) and reading the absorbance at 650 nm after 2 h.

Techniques: Expressing, Activity Assay, Western Blot, Positive Control

Journal: npj Viruses

Article Title: The soluble G protein of respiratory syncytial virus promotes viral dissemination via TLR2-mediated NLRP3 priming and pyroptosis

doi: 10.1038/s44298-026-00172-x

Figure Lengend Snippet: a Western blot analysis of TLR2 and CX3CR1 expression in the indicated cell lines. b GFP fluorescence imaging of A549, HEp-2, HULEC-5a, THP-1 and U937 cells after RSV-A-0594-eGFP infection. c Quantification of LDH release into the supernatants of RSV-A-0594-infected recombinant protein-treated cells treated with or without the NLRP3 inhibitor MCC950. The data represent the means ± SDs from three independent experiments. Statistical analysis: linear regression and two-way ANOVA with Tukey’s post hoc test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: THP-1 blue NF-κB cells (InvivoGen) were seeded in 96-well plates and treated with 50 or 150 nM rBSA, rsG WT, or the rsG CX3C mutant, with or without 200 nM C29 (MedChemExpress HY-100461) or 50 nM TJ-M2010-5 (MedChemExpress HY-139397) for 1 h. After 18 h, SEAP activity was measured by transferring the supernatant to Quanti-BlueTM reagent (InvivoGen) and reading the absorbance at 650 nm after 2 h.

Techniques: Western Blot, Expressing, Fluorescence, Imaging, Infection, Recombinant

Journal: medRxiv

Article Title: A highly prevalent lupus risk haplotype increases IRF7-dependent induction of IFN-α, enhancing antiviral defense and exacerbating autoimmunity

doi: 10.64898/2026.01.21.26344474

Figure Lengend Snippet: (A-C) Genome-wide association signal of genetic variants in the IRF7-PHRF1 locus. Each variant is represented as a data point in the context of its genomic location within the locus and is colored based on its P-value, with red dots representing genome-wide significant genetic variants (P<5×10-8) and blue dots signifying variants that do not meet this threshold. rs1131665 and rs1061502, which are characterized in this study, are identified in A-C. The variants were assessed in a logistic regression model using admixture estimates as a covariate. Genomic position is provided using GRCh37 (hg19) coordinates. (A) European Ancestry, (B) African Ancestry, (C) Trans-ancestral Meta-Analysis. (D) Schematic of IRF7 (isoform A) protein domains: DBD: DNA Binding Domain; CAD: Constitutive Activation Domain; VAD: Virus-Activated Domain; ID: Inhibitory Domain; SRD: Signal Response Domain. (E) Heatmap showing variants in the IRF7 haplotype assessed in the trans-ancestral meta-analysis demonstrating perfect linkage disequilibrium of IRF7-PHRF1 variants; (F) Pie charts quantifying the frequency of the lupus risk (“T”) and protective (“C”) haplotypes globally and in specified ancestries (Ensembl database). Ancestry abbreviations (defined by Ensembl Gene): ALL = Global Population; EUR = European Ancestry; AFR = African Ancestry; AMR = American Ancestry (native people of the Americas); EAS = East Asian Ancestry; SAS = South Asian Ancestry.

Article Snippet: THP-1 IRF7 deficient and HEK Blue hTLR7 cells were purchased from InvivoGen.

Techniques: GWAS, Variant Assay, Genome Wide, Binding Assay, Activation Assay, Virus

Journal: medRxiv

Article Title: A highly prevalent lupus risk haplotype increases IRF7-dependent induction of IFN-α, enhancing antiviral defense and exacerbating autoimmunity

doi: 10.64898/2026.01.21.26344474

Figure Lengend Snippet:

Article Snippet: THP-1 IRF7 deficient and HEK Blue hTLR7 cells were purchased from InvivoGen.

Techniques: Blocking Assay, Variant Assay

Journal: medRxiv

Article Title: A highly prevalent lupus risk haplotype increases IRF7-dependent induction of IFN-α, enhancing antiviral defense and exacerbating autoimmunity

doi: 10.64898/2026.01.21.26344474

Figure Lengend Snippet: (A) Schematic of IRF7 activation, nuclear localization, and DNA binding downstream of multiple pattern recognition receptor pathways. (B) Overview of experimental approach employed in HEK Blue hTLR7 cells. C) Representative Western blot image of HEK Blue hTLR7 nuclear lysates following stimulation with R848 for 3-hours. β-actin and total H3 are included as loading controls. (D) Densitometry analysis of band intensities for risk and protective haplotype bands based on the quantitative Western blot presented in Figure S2. IRF7 bands were normalized to a total protein stain to calculate normalized signal as described in Methods. Welch’s t-test was used to determine significance. (E) Normalized ISRE luciferase activity (ratio of ISRE firefly to Renilla luciferase relative light units (RLU)) following 24-hours of R848 stimulation. Welch’s t-test was used to determine significance. (F) IFN-α measured in cell-free supernatants from cells stimulated with R848 for 3– and 24-hours. Data are mean ± SEM of triplicate samples (unless otherwise indicated) and are representative of at least three independent experiments. Significance was assessed with two-way ANOVA with a Holm-Šidak’s multiple comparisons correction. *P<0.05; ***P<0.005; ns indicates non-significant (P>0.05). ISRE: Interferon stimulated response element; EV: Empty Vector; R848: Resiquimod.

Article Snippet: THP-1 IRF7 deficient and HEK Blue hTLR7 cells were purchased from InvivoGen.

Techniques: Activation Assay, Binding Assay, Western Blot, Staining, Luciferase, Activity Assay, Plasmid Preparation

Journal: medRxiv

Article Title: A highly prevalent lupus risk haplotype increases IRF7-dependent induction of IFN-α, enhancing antiviral defense and exacerbating autoimmunity

doi: 10.64898/2026.01.21.26344474

Figure Lengend Snippet: (A) Schematic showing the generation of THP-1 monocytes stably expressing the lupus risk and protective haplotypes in IRF7 . (B) Quantitative PCR analysis of mRNA expression of IFN-α (subtypes IFNα2, IFNα25, and IFNα21) following transfection with Poly(I:C) at multiple time points in IRF7-deficient (KO) cells engineered to express the risk or protective haplotypes of IRF7 . (C) IFN-α measured in cell-free supernatants from cells transfected with Poly(I:C). (D) Differentially expressed genes in THP-1 monocytes expressing either the lupus risk or the lupus protective haplotypes 3-hours post-transfection of Poly(I:C). (E) Gene expression profiles [transcripts per million (TPM) values] of IRF3 and IRF7 in lupus risk and lupus protective monocytes. (FI) Gene expression profiles [TPM values] of IFNA subset genes differentially expressed in IRF7 deficient (KO), lupus protective, and lupus risk monocytes 3-hours post transfection with Poly(I:C). (J) Pathway enrichment analysis of differentially expressed genes from D. (K) Motif enrichment for a representative IRF7 DNA binding motif for genes differentially expressed in the analysis presented in D. The motif image is taken from CIS-BP data build 2.00. Data are mean ± SEM of triplicate samples and are representative of at least three independent experiments, except the RNA-seq experiment in which was performed once for all replicates. Statistical comparisons between risk and protective are based on a two-tailed unpaired Student’s t-test; *P<0.05; **P<0.01; ****P<0.0001; ns indicates non-significant (P>0.05).

Article Snippet: THP-1 IRF7 deficient and HEK Blue hTLR7 cells were purchased from InvivoGen.

Techniques: Stable Transfection, Expressing, Real-time Polymerase Chain Reaction, Transfection, Gene Expression, Binding Assay, RNA Sequencing, Two Tailed Test

Journal: medRxiv

Article Title: A highly prevalent lupus risk haplotype increases IRF7-dependent induction of IFN-α, enhancing antiviral defense and exacerbating autoimmunity

doi: 10.64898/2026.01.21.26344474

Figure Lengend Snippet: Comparison of DNA binding strength for the IRF7 lupus risk haplotype to purified IRF7 dimer (A) or the IRF7 protective haplotype (B). Binding for IRF7 lupus risk and protective haplotypes was determined via nextPBM assays using extracts from lupus risk and protective THP-1 monocytes following 3-hours of Poly(I:C) transfection. Data for the purified IRF7 dimer is from Andrilenas et al. 55, which used a mutant form of IRF7 that is constitutively dimeric. Binding is shown to 6,954 2-bp-spacer variant IRF7 binding sites (dark) and 3,456 3-bp-spacer variants (light) (see Methods) and is quantified by median PBM probe fluorescence values for each DNA sequence. (C) Increased binding of lupus risk haplotype IRF7 to the promoters of genes assessed on the PBM leads to increased gene expression (RNA-seq) of the 8 indicated genes. Log2 fold change values for these genes are plotted along with the adjusted P-value from the RNA-seq analysis . *Padj<0.005, **Padj<0.0005, ***Padj<1.0×10-5.

Article Snippet: THP-1 IRF7 deficient and HEK Blue hTLR7 cells were purchased from InvivoGen.

Techniques: Comparison, Binding Assay, Purification, Transfection, Mutagenesis, Variant Assay, Fluorescence, Sequencing, Gene Expression, RNA Sequencing

Journal: medRxiv

Article Title: A highly prevalent lupus risk haplotype increases IRF7-dependent induction of IFN-α, enhancing antiviral defense and exacerbating autoimmunity

doi: 10.64898/2026.01.21.26344474

Figure Lengend Snippet: Using CRISPR targeting and homologous recombination, we mutated C/G to T/A at position 141,263,661 on chromosome 7 (Genome Reference Consortium Mouse Build 38), resulting in an R334Q substitution in mouse IRF7 that is analogous to the rs1131665 SLE risk-variant R412Q substitution in human IRF7. (A) 100 micrograms of resiquimod (R848) was epicutaneously applied three times per week to the ears of genome edited C57BL/6 mice with the lupus risk or non-risk IRF7 genotype for a total of eight weeks. (B) Sera was collected and used to quantify anti-double-stranded DNA (ds-DNA) antibodies by ELISA. (C) Eightweek-old male mice with the lupus risk or non-risk IRF7 genotype were intranasally infected with 1 million plaque-forming units of VSV-NJ. (D) After 24 hours, virus was quantified in lung homogenates using viral plaque assays. (E) Model for the lupus risk genotype in IRF7 enhancing both viral responses (with the lupus risk genotype enhancing viral clearance) and autoimmunity (with the lupus risk genotype enhancing the production of autoantibodies).

Article Snippet: THP-1 IRF7 deficient and HEK Blue hTLR7 cells were purchased from InvivoGen.

Techniques: CRISPR, Homologous Recombination, Variant Assay, Enzyme-linked Immunosorbent Assay, Infection, Virus