Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: TRAF3 is positively correlated with favorable prognosis in breast cancer. ( a ) High TRAF3 mRNA expression levels are associated with better OS (Living vs. Diseased, Mann–Whitney U test), lower disease stage (Bonferroni correction), lower lymph node stage (N) (N0 vs. N1: p = 0.004, Bonferroni correction) and lower tumor stage (T) (T1 vs. T3: p = 0.019, Bonferroni correction) in the TCGA-BRCA cohort. ( b ) High TRAF3 mRNA expression presents with a statistically significant better OS ( p = 0.00405) and DMFS ( p = 0.00729) in the ER-negative breast cancer cohort employed by GOBO, with ER-positive disease presenting a similar association despite not reaching statistical significance.

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: Forced TRAF3 expression in breast cancer cell lines induces partial EMT and affects cell proliferation. ( a ) Invasion, migration and colony formation assays depicting an opposing phenotype between migratory and proliferative states of MCF7-TRAF3 cells. ( b ) Western blot analyses for the indicated proteins in MDA-MB-231 and MCF-7 cells (control and TRAF3 expressing). ( c ) ICC for the indicated proteins in MCF-7 cells, indicating significant downregulation of key molecules upon TRAF3 expression (ns: no significance; *** p < 0.001 Student’s t -test).

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, Migration, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: Identification of interactors, pathways and processes of TRAF3 in breast cancer. ( a ) Volcano plot of significant TRAF3 interactions in MCF-7 cells (FDR < 0.05). ( b ) Top 20 enriched pathways (Metascape) among proteins that interact with TRAF3 in MCF-7 cells with −log10(Padj) > 10 −20 . ( c ) Significantly enriched pathways among genes co-expressed with TRAF3 in the TCGA BRCA cohort. ( d ) Representative BRCA cases from the TCGA cohort presenting with High and Low TILs (upper panel). High TRAF3 mRNA expression is correlated ( p = 0.02, Mann–Whitney U Test) with High stromal TILs in the TCGA BRCA cohort ( n = 200).

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: TRAF3 expression across cell populations in the scRNA human breast cancer dataset. ( a ) UMAP visualization of 81,389 quality-filtered single cells derived from the Breast Cancer Atlas, colored by cell type annotation. ( b ) Feature plot showing log-normalized TRAF3 expression projected onto the UMAP embedding. ( c ) Violin plots depicting log-normalized TRAF3 expression across each of the cell types. Statistical comparisons were performed using Wilcoxon rank-sum tests, comparing each cell type against all remaining cells, followed by Benjamini–Hochberg correction for multiple testing. Asterisks (*) indicate adj p -values < 0.05. ( d ) Volcano Plot of Differential expression of TRAF3 -positive ( TRAF3 +) vs. negative ( TRAF3 -) Cancer Epithelial (CE) cells. The x-axis represents the log 2 fold change of expression in TRAF3 -positive versus TRAF3 -negative cells, and the y-axis shows the −log 10 adjusted p -value (FDR). Points are colored according to FDR significance, while labels highlight specific immunologically relevant genes, colored according to the following categories: (i) Immunogenicity—Immunogenicity/Antigen Presentation; (ii) MHC-I—MHC class I pathway (CD8 + T-cell recognition); (iii) MHC-II—MHC class II (tumor-intrinsic or antigen-presenting cell mediated); (iv) Checkpoint—Checkpoint blockade/Immune Modulation; (v) Infiltration—Increase immune infiltration into tumors; and (vi) Non-self—Promote tumor cell recognition as “non-self”. Selected genes of interest not in the above categories are colored black (‘Other’ category). ( e ) Gene Ontology (GO) Enrichment Analysis of the filtered top DE genes (FDR < 0.05 & |log2FC| > 0.1) identified via differential expression analysis between TRAF3 + and TRAF3 -cancer epithelial (CE) cells. X-axis represents the Fold Enrichment, and y-axis represents the immune-related Biological Process and Molecular Function GO terms, grouped into clusters based on functional similarity (for the full GO term graph with all the immune and non-immune related GO terms, see ). Dot size is analogous to the number of specific genes associated with each GO term, while their color gradient corresponds to the FDR-adjusted p -value (Q value). Abbreviations used include the following: CE (Cancer Epithelial cells), NE (Normal Epithelial cells), PVL (PeriVascular-Like cells), CAFs (Cancer-Associated Fibroblasts), PR (Positive Regulation), R (Regulation), prd (production), MM (Molecular Mediator), MBP (Macromolecule Biosynthetic Process), MMP (Macromolecule Metabolic Process), CR (Cellular Response), env/tal (environmental), RSP (receptor signaling pathway), SP (signaling pathway), resp. (response), ext. (external), and If-M (interferon-mediated).

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, Derivative Assay, Quantitative Proteomics, Immunopeptidomics, Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: Dual Role of Cancer Epithelial-Specific TRAF3 in Regulating Breast Cancer Cell Survival and Lymphocyte Activity

doi: 10.3390/ijms27104414

Figure Lengend Snippet: TRAF3 expression in cancer cells affects PBMC subpopulations and cytokine expression. ( a ) FACs analysis of PBMCs co-cultured with MCF7-TRAF3 cells indicates the downregulation of the CD25+CD127low (Tregs) subpopulation of CD4+ T cells. ( b ) FACS analysis of PBMCs co-cultured with MCF7-TRAF3 cells indicates the upregulation of the CD56+CD16- subpopulation of NK-cells. ( c ) Diagrams depicting absolute quantification of IFN-γ, TNF-α and IL-10 in the supernatants of co-cultured PBMCs/MCF7-TRAF3 cells. ( d ) FACs analysis for live/dead MCF-7 breast cancer cells co-cultured with PBMCs depicting a shift from alive to dead cells in the MCF7-TRAF3 cell population in comparison to MCF7-control cells. ( e ) IHC stain for PD-L1 (CD274) on MCF7-control and MCF7-TRAF3. Arrowheads depict PD-L1 expression only on MCF7-control cells. ( f ) Schematic illustration of a proposed model of TRAF3 action in breast cancer epithelial cells and on the surrounding tumor microenvironmental cells.

Article Snippet: The TRAF3 coding sequence was amplified from a pCMV6-TRAF3-GFP (RG210417, Origene, Rockville, MD, USA) vector and cloned into pLenti-EF1a-GFP-2A-Puro (LV067, ABM Inc, Richmond, BC, Canada).

Techniques: Expressing, Cell Culture, Quantitative Proteomics, Comparison, Control, Staining

Journal: Biochemistry and Biophysics Reports

Article Title: The miR-2110/ TRAF3 axis is associated with endothelial dysfunction and atherosclerosis in coronary heart disease

doi: 10.1016/j.bbrep.2026.102508

Figure Lengend Snippet: Validation of TRAF3 as a direct target of miR-2110 by RT-qPCR, luciferase reporter assay, and Western blot analysis ( A ) RT-qPCR analysis of TRAF3 mRNA levels in OE2110 and OENC cells, calculated using the 2 −ΔΔCt method, normalized to GAPDH, and expressed relative to the control group (set to 1). ( B ) TRAF3 mRNA levels in peripheral blood samples from healthy controls (n = 30) and CHD patients (n = 34), normalized to GAPDH. ( C ) Schematic representation of the predicted binding sites between miR-2110 and the wild-type (WT) 3′-UTR of TRAF3 , and the corresponding mutated (MT) sequence used for luciferase assays. ( D ) Dual-luciferase reporter assay showing decreased luciferase activity in cells co-transfected with miR-2110 mimics and TRAF3 -WT but not TRAF3 -MT. ( E, F ) Western blot analysis and quantification of TRAF3 protein expression in OE2110 and OENC, with GAPDH as internal reference. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ns: not significant. Panels A, D, and F: Data are presented as mean ± SD from three independent biological experiments (N = 3) using EA.hy926 cells. Panel E: Representative blots are shown. Statistical comparisons were performed using unpaired two-tailed Student's t-tests. Panel B (patient samples): Data were presented as median (interquartile range) and were analyzed using the Mann–Whitney U test.

Article Snippet: After blocking with 5% skimmilk in PBS, the membranes were immunoblotted overnight at 4 °C with antibodies against TRAF3 (1:2000, Proteintech, USA, 18099-1-AP) and GAPDH (1:50,000, Proteintech, USA, 10494-1-AP), followed by HRP-conjugated goat Anti-rabbit IgG (H + L) (1:10000, Servicebio, China, GB23303).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Luciferase, Reporter Assay, Western Blot, Control, Binding Assay, Sequencing, Activity Assay, Transfection, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Biochemistry and Biophysics Reports

Article Title: The miR-2110/ TRAF3 axis is associated with endothelial dysfunction and atherosclerosis in coronary heart disease

doi: 10.1016/j.bbrep.2026.102508

Figure Lengend Snippet: Increased TRAF3 expression in cardiac and aortic tissues of ApoE −/− mice fed a high-fat diet ( A ) Relative Traf3 mRNA expression in the aortic root and adjacent heart tissue quantified by RT-qPCR, calculated using the 2 −ΔΔCt method, normalized to GAPDH, and expressed relative to the control group (set to 1). ( B, C ) Western blot analysis and quantification of TRAF3 protein expression in heart tissues. ( D, E ) Western blot analysis and quantification of TRAF3 protein expression in aortic tissues. ( F ) Representative immunohistochemical staining of TRAF3 in aortic root sections from control and ApoE −/− HFD mice, showing predominant localization in the tunica intima, including the endothelial layer and subendothelial regions containing vascular smooth muscle cells and infiltrating leukocytes (200 × ). Red boxes highlight regions with enhanced staining in HFD mice. (G) Quantification of TRAF3-positive area in the tunica intima. ∗ P < 0.05. Data represent mean ± SD; n = 6 mice per group. Statistical comparisons were performed using unpaired two-tailed Student's t-tests.

Article Snippet: After blocking with 5% skimmilk in PBS, the membranes were immunoblotted overnight at 4 °C with antibodies against TRAF3 (1:2000, Proteintech, USA, 18099-1-AP) and GAPDH (1:50,000, Proteintech, USA, 10494-1-AP), followed by HRP-conjugated goat Anti-rabbit IgG (H + L) (1:10000, Servicebio, China, GB23303).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV VP2 suppresses IFN-I production by targeting the upstream of TBK1. ( A–D ) HEK293T cells were transfected with an IFN-β-luc reporter plasmid, a control plasmid, and plasmids expressing ALSV VP2 protein, along with MAVS ( A ), TRAF3 ( B ), TBK1 ( C ), and IRF3 ( D ) expressing plasmids to induce IFN-I production. At 24 hpt, cells were subjected to the luciferase activity assay and immunoblotting analysis using the phosphorylation and total antibodies of IRF3 and TBK1, along with the tag antibodies. Gray-scale statistical analysis of phosphorylation relative to total protein is conducted. ( E and F ) HEK293T cells were transfected with plasmids expressing ALSV VP2 protein, along with the indicated signaling molecules. At 48 hpt, cells were subjected to immunoblotting analysis. The relative levels of the indicated proteins normalized to β-Actin are shown. Data from independent experiments ( n ≥ 3) were statistically analyzed using two-way ANOVA with multiple comparison correction (*** P < 0.001 and **** P < 0.0001).

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Luciferase, Activity Assay, Western Blot, Phospho-proteomics, Comparison

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV VP2 interacts with RIG-I to suppress its activity. ( A ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, or an empty vector. At 48 hpt, cells were subjected to anti-HA immunoprecipitates and analyzed by immunoblotting. ( B ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Myc immunoprecipitates and analyzed by immunoblotting. ( C ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, MDA5, or an empty vector. At 48 hpt, anti-HA immunoprecipitates were analyzed by immunoblotting. ( D ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( E ) HEK293T cells were transfected with Flag-VP2, along with HA-RIG-I, HA-TBK1, HA-TRAF3, or Myc-IRF3. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( F ) HEK293T cells were transfected with Flag-VP2 or an empty vector. At 48 hpt, anti-Flag immunoprecipitates were analyzed by immunoblotting with the indicated endogenous antibodies.

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Western Blot

Journal: Biochemistry and Biophysics Reports

Article Title: The miR-2110/ TRAF3 axis is associated with endothelial dysfunction and atherosclerosis in coronary heart disease

doi: 10.1016/j.bbrep.2026.102508

Figure Lengend Snippet: Validation of TRAF3 as a direct target of miR-2110 by RT-qPCR, luciferase reporter assay, and Western blot analysis ( A ) RT-qPCR analysis of TRAF3 mRNA levels in OE2110 and OENC cells, calculated using the 2 −ΔΔCt method, normalized to GAPDH, and expressed relative to the control group (set to 1). ( B ) TRAF3 mRNA levels in peripheral blood samples from healthy controls (n = 30) and CHD patients (n = 34), normalized to GAPDH. ( C ) Schematic representation of the predicted binding sites between miR-2110 and the wild-type (WT) 3′-UTR of TRAF3 , and the corresponding mutated (MT) sequence used for luciferase assays. ( D ) Dual-luciferase reporter assay showing decreased luciferase activity in cells co-transfected with miR-2110 mimics and TRAF3 -WT but not TRAF3 -MT. ( E, F ) Western blot analysis and quantification of TRAF3 protein expression in OE2110 and OENC, with GAPDH as internal reference. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ns: not significant. Panels A, D, and F: Data are presented as mean ± SD from three independent biological experiments (N = 3) using EA.hy926 cells. Panel E: Representative blots are shown. Statistical comparisons were performed using unpaired two-tailed Student's t-tests. Panel B (patient samples): Data were presented as median (interquartile range) and were analyzed using the Mann–Whitney U test.

Article Snippet: Endogenous peroxidase activity was quenched with 3% H 2 O 2 for 10 min at room temperature, followed by blocking with 5% normal goat serum (Solarbio, China, SL038) in PBS for 1 h. Sections were incubated overnight at 4 °C with rabbit anti-TRAF3 monoclonal antibody (1:200, HUABIO, China, PSH01-30), then probed with HRP-conjugated goat anti-rabbit IgG (1:500, Servicebio, China, GB23303) for 1 h at room temperature.

Techniques: Biomarker Discovery, Quantitative RT-PCR, Luciferase, Reporter Assay, Western Blot, Control, Binding Assay, Sequencing, Activity Assay, Transfection, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Biochemistry and Biophysics Reports

Article Title: The miR-2110/ TRAF3 axis is associated with endothelial dysfunction and atherosclerosis in coronary heart disease

doi: 10.1016/j.bbrep.2026.102508

Figure Lengend Snippet: Increased TRAF3 expression in cardiac and aortic tissues of ApoE −/− mice fed a high-fat diet ( A ) Relative Traf3 mRNA expression in the aortic root and adjacent heart tissue quantified by RT-qPCR, calculated using the 2 −ΔΔCt method, normalized to GAPDH, and expressed relative to the control group (set to 1). ( B, C ) Western blot analysis and quantification of TRAF3 protein expression in heart tissues. ( D, E ) Western blot analysis and quantification of TRAF3 protein expression in aortic tissues. ( F ) Representative immunohistochemical staining of TRAF3 in aortic root sections from control and ApoE −/− HFD mice, showing predominant localization in the tunica intima, including the endothelial layer and subendothelial regions containing vascular smooth muscle cells and infiltrating leukocytes (200 × ). Red boxes highlight regions with enhanced staining in HFD mice. (G) Quantification of TRAF3-positive area in the tunica intima. ∗ P < 0.05. Data represent mean ± SD; n = 6 mice per group. Statistical comparisons were performed using unpaired two-tailed Student's t-tests.

Article Snippet: Endogenous peroxidase activity was quenched with 3% H 2 O 2 for 10 min at room temperature, followed by blocking with 5% normal goat serum (Solarbio, China, SL038) in PBS for 1 h. Sections were incubated overnight at 4 °C with rabbit anti-TRAF3 monoclonal antibody (1:200, HUABIO, China, PSH01-30), then probed with HRP-conjugated goat anti-rabbit IgG (1:500, Servicebio, China, GB23303) for 1 h at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Immunohistochemical staining, Staining, Two Tailed Test