Journal: Cell Death & Disease

Article Title: The TRIM3/TLR3 axis overrides IFN-β feedback inhibition to suppress NSCLC progression

doi: 10.1038/s41419-025-08265-w

Figure Lengend Snippet: A TRIM3 mRNA expression was analyzed by qPCR in H1299, PC-9, H2030, H2228, H1944, 95-D and A549 cells following stimulation with 100 ng/mL IFN-β (n = 4). Significance was assessed using Student’s t test. B – D TRIM3 protein and mRNA levels were measured in A549 and H1299 cells treated with IFN-β ± 20 ng/mL BMS-986165 or IFNAR1 and IFNAR2 knockout. Statistical significance was analyzed using one-way ANOVA. E – H IFNB1 mRNA levels were quantified by qPCR in A549 and H1299 cells with TRIM3 modulation (n = 4). Significance was assessed using Student’s t test. I – L Luciferase reporter assays were performed in A549 and H1299 cells transfected with IFNB1 promoter luciferase constructs (n = 4). Significance was assessed using Student’s t test. * p < 0.05; ** p < 0.01.

Article Snippet: Cultured cells were treated with IFN-β (TMPY-03145, TargetMol, Massachusetts, USA), poly(I:C) ( T12516 , TargetMol) and BMS-986165 ( T14687 , TargetMol).

Techniques: Expressing, Knock-Out, Luciferase, Transfection, Construct

Journal: Cell Death & Disease

Article Title: The TRIM3/TLR3 axis overrides IFN-β feedback inhibition to suppress NSCLC progression

doi: 10.1038/s41419-025-08265-w

Figure Lengend Snippet: A Co-IP coupled with mass spectrometry (IP‒MS) was performed in TRIM3-overexpressing H1299 cells. Immunoprecipitated proteins were quantified by unique peptide counts to identify the top 100 candidates against IFNB1 -associated proteins. B , C Co-IP analysis (IP-Flag) validated the physical interaction between TRIM3 and TLR3 in A549 and H1299 cells. D , E Co-IP analysis (IP-Myc) validated the physical interaction between TRIM3 and TLR3 in A549 and H1299 cells. F The interaction between TRIM3 and TLR3 (TRIM3/TLR3; red) was analyzed by PLA in A549 and H1299 cells. G Domain mapping of TRIM3 was pictured. Co-IP between TRIM3 deletion mutants (ΔRING, ΔB-box, ΔCC and ΔNHL) and TLR3 was performed in H1299 cells. H Ubiquitination assays using HA-ubiquitin and TRIM3 catalytic mutants (C22/25S) were performed in A549 and H1299 cells. I WB was performed to detect TBK1 (Ser172) and IRF3 (Ser396) phosphorylation in WT TRIM3 vs. mutant-expressing cells under 20 ng/mL poly(I:C) stimulation. J ELISA was performed to quantify TRIM3-dependent IFN-β induction in A549 and H1299 cells (n = 4). Statistical significance was analyzed using one-way ANOVA for multiple comparisons. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Cultured cells were treated with IFN-β (TMPY-03145, TargetMol, Massachusetts, USA), poly(I:C) ( T12516 , TargetMol) and BMS-986165 ( T14687 , TargetMol).

Techniques: Co-Immunoprecipitation Assay, Mass Spectrometry, Immunoprecipitation, Ubiquitin Proteomics, Phospho-proteomics, Mutagenesis, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: The TRIM3/TLR3 axis overrides IFN-β feedback inhibition to suppress NSCLC progression

doi: 10.1038/s41419-025-08265-w

Figure Lengend Snippet: A Ubiquitination assays were performed in H1299 cells cotransfected with TRIM3-Flag, TLR3-Myc, and linkage-specific ubiquitin constructs (K6, K11, K27, K33, K48, and K63). B Ubiquitination assays were performed to determine the dose-dependent effects of WT and mutant TRIM3 on the K63-linked ubiquitination of TLR3 in A549 and H1299 cells. C Ubiquitination assays were performed in A549 and H1299 cells cotransfected with TRIM3-Flag, TLR3-Myc, and linkage-specific ubiquitin constructs (K63, K63R). D Ubiquitination assays were performed to examine the enrichment of cleaved TLR3 in A549 and H1299 cells following TRIM3 overexpression and stimulation with or without 20 ng/mL poly(I:C). E Domain mapping of TLR3 was pictured. Co-IP between TLR3 deletion mutants (1-632, 633-904) and TRIM3 was performed in H1299 cells. F Ubiquitination assays were performed to verify the lysine sites of TLR3 ubiquitinated by TRIM3 in H1299 cells. G ELISA was performed to analyze IFN-β secretion levels in A549 and H1299 cells transfected with WT TLR3 or K808R TLR3 (n = 4). Statistical significance was analyzed using one-way ANOVA. * p < 0.05; ** p < 0.01.

Article Snippet: Cultured cells were treated with IFN-β (TMPY-03145, TargetMol, Massachusetts, USA), poly(I:C) ( T12516 , TargetMol) and BMS-986165 ( T14687 , TargetMol).

Techniques: Ubiquitin Proteomics, Construct, Mutagenesis, Over Expression, Co-Immunoprecipitation Assay, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Cell Death & Disease

Article Title: The TRIM3/TLR3 axis overrides IFN-β feedback inhibition to suppress NSCLC progression

doi: 10.1038/s41419-025-08265-w

Figure Lengend Snippet: A – F CCK-8 and colony formation assays were performed to validate the effect of the TRIM3/TLR3 axis on A549, H1299 and LLC cell proliferation following 10 ng/mL poly(I:C) stimulation (n = 4). Statistical significance was analyzed using one-way ANOVA. G Schematic diagram depicting the experimental workflow of the in vivo experiments. H – J Syngeneic mouse models were established to evaluate the effect of the TRIM3/TLR3 axis on tumor growth, with intraperitoneal poly(I:C) (1 mg/kg) injections administered every 3 days. Representative images ( H ) and tumor weights ( I ) and volumes ( J ) are shown (n = 8). Statistical significance was analyzed using one-way ANOVA. K IHC analysis was performed to evaluate IFN-β, CD4, CD49b, CD86, and CD163 expression in mouse tumors. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Cultured cells were treated with IFN-β (TMPY-03145, TargetMol, Massachusetts, USA), poly(I:C) ( T12516 , TargetMol) and BMS-986165 ( T14687 , TargetMol).

Techniques: CCK-8 Assay, In Vivo, Expressing

Journal: Cell Death & Disease

Article Title: The TRIM3/TLR3 axis overrides IFN-β feedback inhibition to suppress NSCLC progression

doi: 10.1038/s41419-025-08265-w

Figure Lengend Snippet: A – H Bioinformatic analysis was conducted to evaluate the correlations between TLR3 expression and the immune infiltration levels of CD4⁺ T cells, M1 macrophages, M2 macrophages and NK cells in LUAD and LUSC. I Kaplan–Meier survival curves were plotted to correlate the combined TRIM3 and TLR3 mRNA expression with the overall survival of patients with NSCLC. The log-rank test was used for statistical analysis. J Schematic diagram depicting the mechanism by which the TRIM3/TLR3 axis overrides IFN-β feedback inhibition to suppress NSCLC progression.

Article Snippet: Cultured cells were treated with IFN-β (TMPY-03145, TargetMol, Massachusetts, USA), poly(I:C) ( T12516 , TargetMol) and BMS-986165 ( T14687 , TargetMol).

Techniques: Expressing, Inhibition

Journal: Nature communications

Article Title: Primate-Specific miR-576-3p Sets Host Defense Signaling Threshold

doi: 10.1038/ncomms5963

Figure Lengend Snippet: a , Schematic showing the region of the 3’ UTR of STING that is recognized by the miR-576-3p seed sequence. b , HBEC were transfected with control or miR-576-3p (M-576-3p) mimic for 72 h. Cells were then mock infected or infected with VSV-GFP at an MOI 3 for 3 h. Total RNA was harvested from cells and STING mRNA levels were determined by qPCR and normalized to levels of β-actin. c , HBEC were transfected with miRNA mimic (M-576-3p) or inhibitor (I-576-3p) as in b and mock-infected or infected with VSV-GFP at MOI 0.1 for 18 h. Cell lysates were harvested and subjected to western blot analysis with anti-STING antibodies. β-actin serves as loading control. d,e , HBEC were transfected with 1 µg/ml of poly (I:C) for 6 h ( d ) or treated with 100 U/ml of IFNβ for 18 h ( e ) and levels of pri-mir-576 and SEC24B mRNA were analyzed by qPCR. f,g , HBEC were transfected with poly (I:C) as in d after siRNA knockdown of NFκB (P65) or IRF3. Relative pri-mir-576 ( f ) or SEC24B mRNA ( g ) levels were measured by qPCR. h , HBEC expressing control or miR-576-3p mimic (M-576-3p) or an siRNA targeting STING were transfected with poly (I:C) and levels of IFNβ mRNA were measured by qPCR. i , HBEC were transfected with control or miR-576-3p mimic (M-576-3p) and pre-treated with 100 U/ml of IFNβ prior to VSV infection. Cell viability was determined by measuring ATP levels of mock or infected cells. j , HBEC expressing control or miR-576-3p inhibitor (I-576-3p) were transfected with poly (I:C) and levels of IFNβ were measured by qPCR. Data are representative of three independent experiments. Unpaired two tailed t-test was used and error bars represent SD. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Recombinant human IFNβ was obtained from R&D systems.

Techniques: Sequencing, Transfection, Control, Infection, Western Blot, Knockdown, Expressing, Two Tailed Test

Journal: Nature communications

Article Title: Primate-Specific miR-576-3p Sets Host Defense Signaling Threshold

doi: 10.1038/ncomms5963

Figure Lengend Snippet: a , HBEC were transfected with 1 µg/ml of poly (I:C) for 6 h or treated with 100 U/ml of IFNβ for 18 h and levels of mature miR-576-3p were determined by qPCR. b , HBEC were mock infected or infected with HSV-1 at MOI 10 for 3 h and levels of miR-576-3p were measured by qPCR. Unpaired two tailed t-test was used and error bars represent SD. *p<0.05, **p<0.01. ns, non significant with respect to control.

Article Snippet: Recombinant human IFNβ was obtained from R&D systems.

Techniques: Transfection, Infection, Two Tailed Test, Control

Journal: Nature communications

Article Title: Primate-Specific miR-576-3p Sets Host Defense Signaling Threshold

doi: 10.1038/ncomms5963

Figure Lengend Snippet: a , Model illustrates regulation of IFN expression by miR-576-3p as a feedback mechanism. Targets of miR-576-3p are indicated by inhibitory red lines. b , Public available microRNA array datasets (GSE37425 and GSE37426) of synovial or renal tissues from RA or SLE patients, respectively, along with controls were obtained from the GEO database . Normalized expression values of miR-576-3p from each sample were used to calculate the relative levels and p-values of miR-576-3p for SLE or RA patients as compared to controls. Unpaired two tailed t-test was used and error bars represent SD. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Recombinant human IFNβ was obtained from R&D systems.

Techniques: Expressing, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: HELZ2: a new, interferon-regulated, human 3′-5′ exoribonuclease of the RNB family is expressed from a non-canonical initiation codon

doi: 10.1093/nar/gkad673

Figure Lengend Snippet: Mass spectrometry analysis of protein extracts from mock- or IFNβ- treated HeLa cells. ( A ) Volcano-plot showing the log2 fold change versus -log 10 ( P -value) between mock- or interferon-treated HeLa cells. Analyses were performed in triplicate and a total of 4828 proteins were quantified by mass spectrometry. Filled or open squares indicate human proteins whose expression at mRNA or protein levels have been reported to be induced at least 3 fold by interferon in the Interferome Database . The names of some of these factors are indicated. Open square and triangle represent measurement for peptides corresponding to the HELZ2β isoform and the HELZ2 extension, respectively. All other proteins are indicated by grey dots. Dashed lines delimit regions of p-value higher or lower than 0.01 and up- or down-regulation of at least 2-fold. ( B ) Graphical representation of the number of PSM per 100 amino acids for the N-terminal extension sequence of HELZ2 and the HELZ2β isoform. ( C ) Representative microscopic image of HeLa cells transfected with a plasmid expressing GFP-HELZ2 to monitor its localization. Green: GFP signals, blue: DAPI signal, and merged. White scale bar: 15 μm.

Article Snippet: For interferon β stimulation, cells were seeded in 6 well plates and then mock-treated or treated with 5000 U/mL of human recombinant IFNβ (R&D systems, 8499-IF/CF) for 24 h.

Techniques: Mass Spectrometry, Expressing, Sequencing, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Human interferon-ϵ and interferon-κ exhibit low potency and low affinity for cell-surface IFNAR and the poxvirus antagonist B18R

doi: 10.1074/jbc.RA118.003617

Figure Lengend Snippet: Surface plasmon resonance-derived binding constants and ISFG3 assay EC 50 values —, not determined.

Article Snippet: Reagents used in the assays included anti-IFNα (MMHA-2, PBL Assay Science), anti-IFNAR1 (ab10739, Abcam), and anti-IFNAR2 (MMHAR-2, PBL Assay Science) neutralizing antibodies, soluble receptors IFNAR1-FC (245-AB-050, R&D Systems) and IFNAR2-FC (4015-AB-050, R&D Systems), and viral type-I antagonist B18R (34-8185-85, eBioscience).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Human interferon-ϵ and interferon-κ exhibit low potency and low affinity for cell-surface IFNAR and the poxvirus antagonist B18R

doi: 10.1074/jbc.RA118.003617

Figure Lengend Snippet: SPR analysis of IFNϵ, IFNκ, IFNα2, and IFNω binding to IFNAR1-FC, IFNAR2-FC, and the IFNAR1/IFNAR2-FC heterodimer. SPR sensorgrams for each IFN-IFNAR interaction are shown in black. The calculated sensorgrams, derived from fitting the data to a 1:1 binding model, are shown in black (IFNα2), red (IFNκ), blue (IFNϵ), and green (IFNω). Kinetic and equilibrium constants derived from the data are shown in Table 2.

Article Snippet: Reagents used in the assays included anti-IFNα (MMHA-2, PBL Assay Science), anti-IFNAR1 (ab10739, Abcam), and anti-IFNAR2 (MMHAR-2, PBL Assay Science) neutralizing antibodies, soluble receptors IFNAR1-FC (245-AB-050, R&D Systems) and IFNAR2-FC (4015-AB-050, R&D Systems), and viral type-I antagonist B18R (34-8185-85, eBioscience).

Techniques: Binding Assay, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Human interferon-ϵ and interferon-κ exhibit low potency and low affinity for cell-surface IFNAR and the poxvirus antagonist B18R

doi: 10.1074/jbc.RA118.003617

Figure Lengend Snippet: Molecular model of unique IFNϵ/κ and IFNα1 residues that modulate IFNAR2 and B18R binding. A and B, schematic models and structures of the (A) IFNα2/IFNAR1/IFNAR2 complex (PDB 3SE3) and the (B) IFNα2/B18R complex (IFNα2 from PDB 3S9D, and B18R derived from the C12R structure, PDB 3OQ3). C, ribbon diagram of the IFNα2 backbone with key residues that regulate IFNAR2 and B18R binding affinity, as discussed in the text, shown in yellow, orange, and magenta. D, superposition of IFNs in IFN/IFNAR2 and IFN/B18R structures with B18R D3 domain in red and the IFNAR2 D1 domain cyan. Three negatively charged residues conserved in IFNAR2 (green) and B18R (yellow) are positioned by IFNα arginines: Arg-35, Arg-56, and Arg=172 (magenta). E, amino acids within the AB loop region of IFNs that distinguish strong and weak IFNAR2 and B18R binders. Amino acid substitutions made to produce IFNα1 and IFNκ mutants are shown in bold.

Article Snippet: Reagents used in the assays included anti-IFNα (MMHA-2, PBL Assay Science), anti-IFNAR1 (ab10739, Abcam), and anti-IFNAR2 (MMHAR-2, PBL Assay Science) neutralizing antibodies, soluble receptors IFNAR1-FC (245-AB-050, R&D Systems) and IFNAR2-FC (4015-AB-050, R&D Systems), and viral type-I antagonist B18R (34-8185-85, eBioscience).

Techniques: Binding Assay, Derivative Assay

Journal: Cell host & microbe

Article Title: Gestational stage and IFN-λ signaling regulate ZIKV infection in utero

doi: 10.1016/j.chom.2017.08.012

Figure Lengend Snippet: A. Schematic of the intrauterine compartment during human pregnancy denoting chorionic villi, fetal membranes (amnion and chorion), and decidua basalis. B. Confocal micrographs of human chorionic villi, fetal membrane, or decidua stained for cytokeratin-19 (green, left panel; red, middle panel), HLA-G (green, right panel), or β-actin (red, left of right panels; green, middle panel). DAPI-stained nuclei are shown in blue. Scale bar, 50 μm. C. IFN-λ induction of OAS1 in midgestation chorionic villi (**, P < 0.01; n = 6), fetal membranes (P = 0.07; n = 6), or decidua (**, P < 0.01; n = 3) after treatment with 100 ng/mL of IFN-λ (λ1 or λ3) for 16 h as assessed by RT-qPCR. Experiments with recombinant IFN-β (100 ng/ml) were performed in parallel (***, P < 0.001). Data are normalized to mock-treated controls and are shown as mean ± standard deviation. D. Infectious ZIKV-Brazil titers from midgestation chorionic villi (n = 7), fetal membranes (n = 6), or decidua (n = 3) preparations pre-treated with medium (Mock) or 100 ng/ml of IFN-β, IFN-λ1, or IFN-λ3 overnight (~16 h) and then infected with ZIKV-Brazil for 72 h. Data are shown as mean ± standard deviations. Statistical analyses in C and D were peformed using a Kruskal-Wallis ANOVA (*, P < 0.05). See also Figures S3, S4, and S5.

Article Snippet: Human placental explants For infections, placental, decidual, or fetal membrane explants were infected immediately with 5 x 10 5 FFU/ml of ZIKV for 72 h following isolation and treatment (~16 h) with 100 ng/mL of recombinant IFN-β, IFN-λ1 or IFN-λ3 (R&D Systems; 1598-IL-025, 5259-IL-025, 8499-IF-010).

Techniques: Membrane, Staining, Quantitative RT-PCR, Recombinant, Standard Deviation, Infection