Journal: Journal of Advanced Research

Article Title: Enhancement of NK cell activity via DNA-sensing inhibition by Poxin transgene

doi: 10.1016/j.jare.2025.05.058

Figure Lengend Snippet: Poxin transgene inhibits DNA sensing-induced innate immune response. A. Schematic of cloning process. B-G. Cells were transfected with plasmid DNA (pDNA) using TransIT-2020. B. 2.5 μg and 7.5 μg of pDNA encoding Poxin or mutant Poxin (mtPoxin) were transfected into 293T cells, respectively, and FLAG-tagged proteins were detected. C. Activation of STING, TBK1, and IRF3 was analyzed by western blotting. Quantitative analysis of p-IRF3 expression level is shown. D. H151 was pre-treated 1 h before pDNA transfection, and protein expression levels were assessed after 24 h. E. Cells were transfected with pEGFP-N1, and protein expression levels were detected. F. mRNA levels of IFNB1 and ISG15 were analyzed by RT-qPCR (n = 3). G. IFN-β secretion levels were quantitatively analyzed using ELISA. (Representative western blots are shown.).

Article Snippet: Quantitative analysis of secreted human IFN-β was performed following manufacturers' protocol (luex-hifnbv2, Invivogen).

Techniques: Cloning, Transfection, Plasmid Preparation, Mutagenesis, Activation Assay, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Advanced Research

Article Title: Enhancement of NK cell activity via DNA-sensing inhibition by Poxin transgene

doi: 10.1016/j.jare.2025.05.058

Figure Lengend Snippet: Poxin protects NK-92 cells from pDNA transfection-induced innate immunity. A. Cells were transfected by electroporation with 10 μg pDNA, and mRNA dynamics were analyzed by RT-qPCR (n = 3). B. IFN-β secretion levels were quantitatively analyzed using ELISA. C. Expression levels of innate immune signaling proteins were detected by western blotting. (Representative data shown). D. Quantitative analysis of p-STING and p-TBK1 expression levels.

Article Snippet: Quantitative analysis of secreted human IFN-β was performed following manufacturers' protocol (luex-hifnbv2, Invivogen).

Techniques: Transfection, Electroporation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Cytosolic nucleic acid sensing triggers type I interferon activation via the Hippo kinase LATS1

doi: 10.1016/j.jbc.2026.111204

Figure Lengend Snippet: Pharmacological Inhibition of LATS impedes cytosolic nucleic acid sensing PRR activation of IFN-I. A , immunoblot analysis ( left panel ) and relative quantification ( right panel ) of IRF3 (S396) phosphorylation in MEF cells pretreated with vehicle control (DMSO) or LATS inhibitor (20 μM; 2.5 h) then transfected with pI:C or ISD (2 μg/ml each; 3 h). B , qPCR analysis of Ifnb mRNA in MEF cells pretreated with DMSO or LATS inhibitor as in A , followed by pI:C transfection (2 μg/ml; 3.5 h) ( left panel ) or ISD transfection (2 μg/ml; 4 h) ( right panel ). C and D , qPCR of Cxcl10, Ccl5, and Isg15 mRNA in MEF cells pretreated with DMSO or LATS1 inhibitor followed by pI:C transfection as in B , left panel ( C ) or ISD as in B , right panel ( D ). Statistical significance was determined using student’s t test (∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05).

Article Snippet: Forskolin, Vadimezan (DMXAA), and recombinant murine IFNβ were purchased from MedChemExpress.

Techniques: Inhibition, Activation Assay, Western Blot, Quantitative Proteomics, Phospho-proteomics, Control, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Cytosolic nucleic acid sensing triggers type I interferon activation via the Hippo kinase LATS1

doi: 10.1016/j.jbc.2026.111204

Figure Lengend Snippet: LATS1 is required for IFN-I activation. A and B , qPCR analysis of Ifnb mRNA expression in WT and Lats1 −/− MEF cells infected with VSV (MOI 0.5) ( A ) or HSV-1 (MOI 0.1) for 24 h. C and D , qPCR analysis of Cxcl10 and Ccl5 mRNA in WT and Lats1 −/− MEF cells infected with VSV as in panel A ( C ) or HSV-1 as in panel B ( D ). E and F , immunoblot analysis ( left panels ) and relative quantification ( right panels ) of IRF3 (S396) phosphorylation in WT and Lats1 −/− MEF cells transfected with pI:C ( E ) or ISD ( F ) (2 μg/ml each) for the indicated times. G and H , qPCR analysis of Ifnb mRNA expression in WT and Lats1 −/− MEF cells transfected with pI:C ( G ) or ISD ( H ) (2 μg/ml each) for the indicated times. I and J , ELISA for IFN-β secretion in WT and Lats1 −/− MEF cells transfected with pI:C ( I ) or ISD ( J ) (2 μg/ml each; 6 h). K and L , qPCR analysis of Cxcl10, Ccl5, and Isg15 mRNA in WT and Lats1 −/− MEF cells transfected with pI:C ( K ) or ISD ( L ) (2 μg/ml each) for the indicated times. Statistical significance was determined using Student’s t test (∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05).

Article Snippet: Forskolin, Vadimezan (DMXAA), and recombinant murine IFNβ were purchased from MedChemExpress.

Techniques: Activation Assay, Expressing, Infection, Western Blot, Quantitative Proteomics, Phospho-proteomics, Transfection, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Cytosolic nucleic acid sensing triggers type I interferon activation via the Hippo kinase LATS1

doi: 10.1016/j.jbc.2026.111204

Figure Lengend Snippet: LATS1 operates at the level of TBK1. A , immunoblot analysis of LATS1 expression in HEK 293T cells treated with control siRNA or siRNAs targeting LATS1 for 24 h. B , IFN-β luciferase reporter assay in HEK 293T cells treated with control siRNA or siRNAs targeting LATS1 as in A , followed by transfection of plasmids encoding RIG-I, MAVS, STING, TBK1, and IRF3. C , co-immunoprecipitation and immunoblot of FLAG-LATS1 and HA-TBK1 co-transfected in HEK 293T cells. D and E , immunoblot analysis of TBK1-LATS1 interactions in MEF cells transfected with pI:C ( D ) or ISD ( E ) (2.5 μg/ml each; 2 h). Statistical significance was determined using student’s t test (∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05).

Article Snippet: Forskolin, Vadimezan (DMXAA), and recombinant murine IFNβ were purchased from MedChemExpress.

Techniques: Western Blot, Expressing, Control, Luciferase, Reporter Assay, Transfection, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Cytosolic nucleic acid sensing triggers type I interferon activation via the Hippo kinase LATS1

doi: 10.1016/j.jbc.2026.111204

Figure Lengend Snippet: LATS1 facilitates TBK1 activation and signaling events. A and B , immunoblot analysis ( left panels ) and relative quantification ( right panels ) of TBK1 (S172) phosphorylation in WT and Lats1 −/− MEF cells transfected with pI:C ( A ) or ISD ( B ) (2 μg/ml each) for the indicated times. C , immunoblot analysis of TBK1 (S172) phosphorylation in HEK 293T cells co-transfected with plasmids encoding WT TBK1 or kinase dead (KD) TBK1 (K38A) in the absence or presence of LATS1. D , IFN-β luciferase reporter assay in HEK 293T cells co-transfected with plasmids encoding TBK1 in the presence of WT LATS1 or kinase dead (KD) LATS1 (D846A). E , co-immunoprecipitation and immunoblot ( left panel ) and relative quantification ( right panel ) of WT LATS1-TBK1 and KD LATS1-TBK1 interactions in HEK 293T cells co-transfected with the indicated plasmids. F , co-immunoprecipitation and immunoblot ( left panel ) and relative quantification ( right panel ) of TBK1-IRF3 interactions in the absence or presence of LATS1 in HEK 293T cells co-transfected with the indicated plasmids. G and H , immunoblot analysis of TBK1-IRF3 interactions in WT and Lats1 −/− MEF cells transfected with pI:C ( G ) or ISD ( H ) (2 μg/ml each; 2.5 h). Statistical significance was determined using student’s t test (∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05).

Article Snippet: Forskolin, Vadimezan (DMXAA), and recombinant murine IFNβ were purchased from MedChemExpress.

Techniques: Activation Assay, Western Blot, Quantitative Proteomics, Phospho-proteomics, Transfection, Luciferase, Reporter Assay, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Cytosolic nucleic acid sensing triggers type I interferon activation via the Hippo kinase LATS1

doi: 10.1016/j.jbc.2026.111204

Figure Lengend Snippet: Model of Hippo kinase LATS1-dependent regulation of IFN-I in cytosolic nucleic acid sensing PRR signaling pathways. Under basal conditions, YAP operates as a steady-state negative regulator of TBK1-IRF3 signaling in part by associating with TBK1. Upon the detection of RNA or DNA virus genomes by distinct cytosolic PRRs, the Hippo kinase LATS1 undergoes phosphorylation to trigger the phospho-inhibition of YAP, abrogating its ability to antagonize TBK1. Activated LATS1 further associates with TBK1 and licenses TBK1 dependent signaling to IRF3, resulting in the subsequent induction of IFN-I. Figure created with BioRender.

Article Snippet: Forskolin, Vadimezan (DMXAA), and recombinant murine IFNβ were purchased from MedChemExpress.

Techniques: Protein-Protein interactions, Virus, Phospho-proteomics, Inhibition

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: HSPA6 is induced by RIG-I-like receptors and negatively regulates type-I interferon signaling

doi: 10.1007/s00018-026-06147-8

Figure Lengend Snippet: HSPA6 is upregulated in expression by IFN signaling-competent MDA5. A . Volcano plot of RNA-seq data comparing gene expression between WT and GOF MDA5 expressed HEK293 cells. B . HEK293T cells were transfected with plasmids expressing WT or GOF MDA5. 24 h later, cells were lysed for RNA detection by RT-qPCR and protein detection by western blotting. EV, empty vector. C . HEK293T cells were mock-treated or treated with recombinant IFNβ; at a final concentration of 10 ng/ml for 24 h. RNA was purified, and gene expression was tested by RT-qPCR. D . HEK293T cells were transfected with plasmids expressing MDA5. 24 h later, cells were lysed for RNA detection. The qPCR primer-targeting regions on AL590385.23 was indicated in green. E . HEK293T cells were transfected with MDA5 expressing plasmid. 6 h post transfection, cells were transfected with poly I: C at a final concentration of 0.8 ng/μ;l. 18 h later, cells were lysed for RNA measurement. F . Cells were transfected with GOF MDA5 expressing plasmid and harvested at indicated hours post transfection. RNA was measured by RT-qPCR. G , H . Act.D was added at a final concentration of 5 μ;g/ml 24 h post transfection of plasmids indicated. 24 h post treatment, cells were lysed for RNA measurement. I , J . HEK293T cells were infected with EMCV at an MOI of 0.01. 24 h post infection, cells were lysed for RNA measurement. K . RNA levels of IFNβ; and HSPA6 in epithelial cells were shown upon SARS-CoV infection. The data was obtained from GEO database (accession number: GSE17400 ). For B-E, G-J, data are means ± SD of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001. For F, data are means ± SD of two independent experiments

Article Snippet: The recombinant human IFNβ (Sino Biological, 10704-HNAS) was dissolved in PBS at a stock concentration of 1.0 mg/ml as the manufacture’s instruction suggested.

Techniques: Expressing, RNA Sequencing, Gene Expression, Transfection, RNA Detection, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Recombinant, Concentration Assay, Purification, Infection