Journal: Journal of Translational Medicine

Article Title: Network medicine modeling of the m⁶A regulatory landscape identifies a KLF6–WTAP axis as a therapeutic target in pulmonary fibrosis

doi: 10.1186/s12967-026-07876-x

Figure Lengend Snippet: WTAP promotes fibroblast proliferation and migration in pulmonary fibrosis through m⁶A methylation. ( A ) Representative immunofluorescence images of the merged photos in normal and IPF samples. ( B ) Representative western blot images of WTAP. ( C ) qRT-PCR result of the transfection efficiency. ( D ) Knockdown of WTAP significantly reduced the expression of α-SMA and Collagen III in TGF-β–stimulated fibroblasts and reversed their upregulation induced by TGF-β. ( E ) Representative immunofluorescence images to detect WTAP, Ki-67 and α-SMA expression in TGF-β-induced groups at 48 h. Scale bars, 20 μm. ( F ) Cell proliferation was measured by EdU staining. ( G - H ) Fibroblasts migration ability was measured by the wound healing and transwell assay. ( I ) Dot blot assay using an anti-m⁶A antibody in TGF-β-induced fibroblasts, shWTAP and NC groups. MB staining was included as a loading control. P < 0.05 was considered significant. ( J ) Western blot images of IGFBP5, KLF6, NR4A3 and MYC levels. ( K ) MeRIP-qPCR analysis of MYC, IGFBP5, KLF6 and NR4A3 in TGF-β-induced fibroblasts. ( L ) Putative KLF6 binding site on the promoter region of WTAP. ( M ) KLF6 binds to the promoter region of WTAP in TGF-β induced fibroblasts. Chromatin-IP was performed using KLF6 antibody or control IgG. Values are percentage of input. ( O ) KLF6 activates WTAP promoter activity in dual-luciferase reporter assay. ( O ) Western blotting analysis and correlation analyses of WTAP expression with expression of KLF6 in 8 freshly collected human IPF samples. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to the corresponding groups

Article Snippet: The membranes were blocked with 5% bovine serum albumin (BSA) and incubated overnight at 4 °C with primary antibodies against WTAP (Proteintech, Cat No. 60188-1-Ig), Collagen III(Proteintech, Cat No. 22734-1-AP), KLF6 (Proteintech, Cat No. 14716-1-AP), NR4A3 (Proteintech, Cat No. 55405-1-AP), MYC(Proteintech, Cat No. 10828-1-AP), GAPDH (Proteintech, Cat No. 60004-1-Ig), α-SMA (Proteintech, Cat No. 14395-1-AP).

Techniques: Migration, Methylation, Immunofluorescence, Western Blot, Quantitative RT-PCR, Transfection, Knockdown, Expressing, Staining, Transwell Assay, Dot Blot, Control, Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Luciferase, Reporter Assay

Journal: Journal of Atherosclerosis and Thrombosis

Article Title: GLP-1 Receptor Agonist Exendin-4 Attenuates NR4A Orphan Nuclear Receptor NOR1 Expression in Vascular Smooth Muscle Cells

doi: 10.5551/jat.43414

Figure Lengend Snippet: Ex-4 suppresses cell growth after vascular injury and expression of NOR1 and PCNA in VSMCs. (A) Sections were subjected to immunohistochemistry for αSMA (red) and counterstained with DAPI to visualize nuclei (blue). Magnification, ×630. (B) Total cell count of the intima and media. One-way ANOVA was performed to calculate statistical significance. Data are mean ± SEM. * P < 0.05 vs. control (C) Representative immunofluorescence image of injured femoral arteries showing NOR1 (cyan) and PCNA (green). Magnification, ×630. (D) (E) NOR1- and PCNA-positive cells were quantified by analyzing the fraction of stained cells to the total number of nuclei (Control, n = 7; Ex-4 LD, n = 8; and Ex-4 HD, n = 6). Values are expressed as the percentage of positive cells. One-way ANOVA was performed to calculate statistical significance. * P < 0.05 vs. control, ** P < 0.01 vs. control.

Article Snippet: The following primary antibodies were used: NOR1 (TA804872, ORIGENE), phosphop44/42 MAPK (Thr202/Tyr204) (#9101; Cell Signaling), p44/42 MAPK (#9102; Cell Signaling), phospho-CREB (Ser133) (#9198; Cell Signaling), CREB (#9197; Cell Signaling), phospho-Akt (Ser473) (#4058; Cell Signaling), Protein kinase B (Akt) (#9272; Cell Signaling), phospho-mTOR (Ser2448) (#2971; Cell Signaling), mTOR (#2983; Cell Signaling), p27 Kip1 (#3686; Cell Signaling), and GAPDH (sc-20357; Santa Cruz).

Techniques: Expressing, Immunohistochemistry, Cell Counting, Control, Immunofluorescence, Staining

Journal: Journal of Atherosclerosis and Thrombosis

Article Title: GLP-1 Receptor Agonist Exendin-4 Attenuates NR4A Orphan Nuclear Receptor NOR1 Expression in Vascular Smooth Muscle Cells

doi: 10.5551/jat.43414

Figure Lengend Snippet: FBS-induced NOR1 expression is suppressed by Ex-4 pretreatment in vitro . (A) Serum-deprived human aortic SMCs were stimulated with 10% FBS with 0–10 nM Ex-4 pretreatment. NOR1 protein expression was analyzed at 6 h after FBS stimulation by western blotting. (B) FBS-induced NOR1 mRNA expression was analyzed at 2 h after FBS stimulation with 0–10 nM Ex-4 pretreatment by qRT-PCR. One-way ANOVA was performed to calculate statistical significance. Data are mean ± SEM. * P < 0.05 vs. FBS(+) 0 nM Ex-4, ** P < 0.01 vs. FBS(+) 0 nM Ex-4. (C) Rat aortic SMCs were transiently transfected with 1.7 kb NOR1 promoter constructs. Serum-deprived cells were pretreated with or without 10 nM Ex-4 and then stimulated with 10% FBS for 12 h. Following stimulation, cells were harvested and luciferase activities were analyzed. Statistical significance was calculated using unpaired t -tests. Data are mean ± SEM. * P < 0.05 vs. Control FBS(+). (D) Serum-deprived SMCs were treated with 10 nM Ex-4 or PBS and pretreated for 30 min with or without 100 nM Exendin (9–39) or 10 µM PKI and subsequently stimulated with FBS at a final concentration of 10%. After 2 h of stimulation, NOR1 mRNA expression was determined. One-way ANOVA was performed to calculate statistical significance. Data are mean ± SEM. * P < 0.05 vs. FBS(+) control, # P < 0.05 vs. FBS(+) Exendin (9–39).

Article Snippet: The following primary antibodies were used: NOR1 (TA804872, ORIGENE), phosphop44/42 MAPK (Thr202/Tyr204) (#9101; Cell Signaling), p44/42 MAPK (#9102; Cell Signaling), phospho-CREB (Ser133) (#9198; Cell Signaling), CREB (#9197; Cell Signaling), phospho-Akt (Ser473) (#4058; Cell Signaling), Protein kinase B (Akt) (#9272; Cell Signaling), phospho-mTOR (Ser2448) (#2971; Cell Signaling), mTOR (#2983; Cell Signaling), p27 Kip1 (#3686; Cell Signaling), and GAPDH (sc-20357; Santa Cruz).

Techniques: Expressing, In Vitro, Western Blot, Quantitative RT-PCR, Transfection, Construct, Luciferase, Control, Concentration Assay

Journal: Journal of Atherosclerosis and Thrombosis

Article Title: GLP-1 Receptor Agonist Exendin-4 Attenuates NR4A Orphan Nuclear Receptor NOR1 Expression in Vascular Smooth Muscle Cells

doi: 10.5551/jat.43414

Figure Lengend Snippet: Ex-4 suppresses cell growth after vascular injury and expression of NOR1 and PCNA in VSMCs under high-fat diet feeding. (A) Sections were subjected to immunohistochemistry for α SMA (red) and counterstained with DAPI to visualize nuclei (blue). Magnification, ×630. (B) Total cell count of the intima and media. One-way ANOVA was performed to calculate statistical significance. Data are mean ± SEM. (C) Representative immunofluorescence image of injured femoral arteries showing NOR1 (cyan) and PCNA (green). Magnification, ×630. (D) (E) NOR1- and PCNA-positive cells were quantified by analyzing the fraction of stained cells to the total number of nuclei (Control, n = 5; Ex-4 LD, n = 5; and Ex-4 HD, n = 5). Values are expressed as the percentage of positive cells. One-way ANOVA was performed to calculate statistical significance. * P < 0.05 vs. control, ** P < 0.01 vs. control.

Article Snippet: The following primary antibodies were used: NOR1 (TA804872, ORIGENE), phosphop44/42 MAPK (Thr202/Tyr204) (#9101; Cell Signaling), p44/42 MAPK (#9102; Cell Signaling), phospho-CREB (Ser133) (#9198; Cell Signaling), CREB (#9197; Cell Signaling), phospho-Akt (Ser473) (#4058; Cell Signaling), Protein kinase B (Akt) (#9272; Cell Signaling), phospho-mTOR (Ser2448) (#2971; Cell Signaling), mTOR (#2983; Cell Signaling), p27 Kip1 (#3686; Cell Signaling), and GAPDH (sc-20357; Santa Cruz).

Techniques: Expressing, Immunohistochemistry, Cell Counting, Immunofluorescence, Staining, Control

Journal: American journal of clinical pathology

Article Title: NR4A3 Immunostain Is a Highly Sensitive and Specific Marker for Acinic Cell Carcinoma in Cytologic and Surgical Specimens.

doi: 10.1093/ajcp/aqab099

Figure Lengend Snippet: FIGURE 1 Nuclear receptor subfamily 4 group A member 3 (NR4A3) immunohistochemistry in acinic cell carcinoma and secretory carcinoma in cytology specimens (×100). Cell block preparations from fine-needle aspirates from acinic cell carcinoma (A, B) and secretory carcinoma (C, D), a key entity on the differential diagnosis, were stained with H&E or immunostained with NR4A3. Nuclear NR4A3 was seen only in acinic cell carcinoma cases but not in secretory carcinoma cases.

Article Snippet: Representative examples of a low-grade acinic cell carcinoma surgical resection negative with the Origene NR4A3 antibody clone (A, B; ×40) and a falsepositive high-grade mucoepidermoid carcinoma (C, D; ×100).

Techniques: Immunohistochemistry, Blocking Assay, Biomarker Discovery, Staining

Journal: American journal of clinical pathology

Article Title: NR4A3 Immunostain Is a Highly Sensitive and Specific Marker for Acinic Cell Carcinoma in Cytologic and Surgical Specimens.

doi: 10.1093/ajcp/aqab099

Figure Lengend Snippet: FIGURE 2 Nuclear receptor subfamily 4 group A member 3 (NR4A3) immunohistochemistry in other primary oncocytic salivary gland neoplasms in cytology specimens (×100). Cell block preparations from fine-needle aspirates from oncocytoma (A, B), low-grade mucoepidermoid carcinoma (C, D), and Warthin tumor (E, F) were stained with H&E or immunostained with NR4A3. Nuclear NR4A3 was not identifiable in any of these oncocytic/oncocyotid salivary gland neoplasms. Nonspecific cytoplasmic granular NR4A3 staining was seen in the case of low-grade mucoepidermoid carcinoma.

Article Snippet: Representative examples of a low-grade acinic cell carcinoma surgical resection negative with the Origene NR4A3 antibody clone (A, B; ×40) and a falsepositive high-grade mucoepidermoid carcinoma (C, D; ×100).

Techniques: Immunohistochemistry, Blocking Assay, Staining

Journal: American journal of clinical pathology

Article Title: NR4A3 Immunostain Is a Highly Sensitive and Specific Marker for Acinic Cell Carcinoma in Cytologic and Surgical Specimens.

doi: 10.1093/ajcp/aqab099

Figure Lengend Snippet: FIGURE 4 Nuclear receptor subfamily 4 group A member 3 (NR4A3) immunohistochemistry in acinic cell carcinoma and secretory carcinoma in surgical resections (×100). Representative sections from surgical resection specimens of acinic cell carcinoma (A, B) and secretory carcinoma (C, D) were stained with H&E or immunostained with NR4A3. Similar to cytologic preparations, nuclear NR4A3 was seen only in acinic cell carcinoma but not in secretory carcinoma.

Article Snippet: Representative examples of a low-grade acinic cell carcinoma surgical resection negative with the Origene NR4A3 antibody clone (A, B; ×40) and a falsepositive high-grade mucoepidermoid carcinoma (C, D; ×100).

Techniques: Immunohistochemistry, Staining

Journal: American journal of clinical pathology

Article Title: NR4A3 Immunostain Is a Highly Sensitive and Specific Marker for Acinic Cell Carcinoma in Cytologic and Surgical Specimens.

doi: 10.1093/ajcp/aqab099

Figure Lengend Snippet: FIGURE 5 Nuclear receptor subfamily 4 group A member 3 (NR4A3) immunohistochemistry in other primary oncocytic salivary gland neoplasms in surgical resections (×100). Representative sections from surgical resection specimens of oncocytoma (A, B), oncocytic mucoepidermoid carcinoma (C, D), and Warthin tumor (E, F) were stained with H&E or immunostained with NR4A3. Similar to cytologic preparations, nuclear NR4A3 was not seen in other oncocytic salivary gland neoplasms, a few of which showed nonspecific granular cytoplasmic staining.

Article Snippet: Representative examples of a low-grade acinic cell carcinoma surgical resection negative with the Origene NR4A3 antibody clone (A, B; ×40) and a falsepositive high-grade mucoepidermoid carcinoma (C, D; ×100).

Techniques: Immunohistochemistry, Staining

Journal: American journal of clinical pathology

Article Title: NR4A3 Immunostain Is a Highly Sensitive and Specific Marker for Acinic Cell Carcinoma in Cytologic and Surgical Specimens.

doi: 10.1093/ajcp/aqab099

Figure Lengend Snippet: FIGURE 6 Nuclear receptor subfamily 4 group A member 3 (NR4A3) expression is retained in acinic cell carcinoma with high-grade transformation. Representative sections from two different resections of acinic cell carcinoma with high-grade transformation (A-D) were stained with H&E or immunostained with NR4A3 (×200).

Article Snippet: Representative examples of a low-grade acinic cell carcinoma surgical resection negative with the Origene NR4A3 antibody clone (A, B; ×40) and a falsepositive high-grade mucoepidermoid carcinoma (C, D; ×100).

Techniques: Expressing, Transformation Assay, Staining

Journal: American journal of clinical pathology

Article Title: NR4A3 Immunostain Is a Highly Sensitive and Specific Marker for Acinic Cell Carcinoma in Cytologic and Surgical Specimens.

doi: 10.1093/ajcp/aqab099

Figure Lengend Snippet: FIGURE 7 Examples of false-positive and a single negative case of nuclear receptor subfamily 4 group A member 3 (NR4A3) immunohistochemistry. Representative examples of a low-grade acinic cell carcinoma surgical resection negative with the Origene NR4A3 antibody clone (A, B; ×40) and a false- positive high-grade mucoepidermoid carcinoma (C, D; ×100).

Article Snippet: Representative examples of a low-grade acinic cell carcinoma surgical resection negative with the Origene NR4A3 antibody clone (A, B; ×40) and a falsepositive high-grade mucoepidermoid carcinoma (C, D; ×100).

Techniques: Immunohistochemistry

Journal: NPJ Breast Cancer

Article Title: NR4A3 potentials M1-like macrophage polarization to facilitate anti-tumor immune responses in breast cancer

doi: 10.1038/s41523-025-00785-0

Figure Lengend Snippet: A , B Kaplan–Meier curves showing the OS ( A ) and DFS ( B ) with high NR4A3 expression versus low NR4A3 expression in the TCGA-BRCA cohort. C Pearson correlation analysis of scores of ESTIMATE, immune cell, and stroma, and expression of NR4A subfamily ( NR4A1, NR4A2, and NR4A3 ) in the TCGA-BRCA cohort. D Violin plot showing NR4A3 expression among different immune subtypes in the TCGA-BRCA cohort. The C2 subtype has the highest M1/M2-like macrophage polarization and the highest lymphocyte infiltration with the greatest TCR diversity. The C3 subtype is defined by elevated Th17- and Th1-related genes and low to moderate tumor cell proliferation. The C4 and C6 subtypes, characterized by Th1 suppression and high M2 response, confer the worst prognosis. E Differences in IPS between the high NR4A3 and low NR4A3 group for CTLA4 negative /PD-1 negative , CTLA4 negtive /PD-1 positive , CTLA4 positive /PD-1 negative , CTLA4 positive /PD-1 positive in the TCGA-BRCA cohort. p values were calculated by Pearson correlation analysis ( C ), Kruskal–Wallis ( D ), and Wilcoxon test ( E ).

Article Snippet: Slides were stained with CD4 (Abcam, ab133616), CD8 (Abcam, ab101500), IFN-γ (Abcam, ab231036), CD68 (Abcam, ab233172), NR4A3 (Atlas Antibodies, HPA043360), iNOS (Abcam, ab283655), CD68 (Abcam, ab283654), CD86 (Invitrogen, 14-0862-85), phospho-NF-κB p65 (Cell Signaling Technology, 3033), pan-Cytokeratin (Abcam, ab7753), caspase 3 (Abcam, ab4051), and DAPI in proper order.

Techniques: Expressing

Journal: NPJ Breast Cancer

Article Title: NR4A3 potentials M1-like macrophage polarization to facilitate anti-tumor immune responses in breast cancer

doi: 10.1038/s41523-025-00785-0

Figure Lengend Snippet: A The CIBERSORT algorithm was used to assess the abundance of 22 immune cells between high NR4A3 and low NR4A3 groups in the TCGA-BRCA cohort. B UMAP plot showing the cell types in the microenvironment of the BC single-cell database ( GSE176078 ), colored by cell types. C Boxplot showing the percentage of indicated cell types in the high NR4A3 and low NR4A3 samples ( GSE176078 ). D Representative images of IHC staining of NR4A3 (left panel) and mIHC staining of CD4, CD8, pan-cytokeratin (PanCK), and DAPI (right panel) in high NR4A3 and low NR4A3 expression slides of BC (scale bar, 20 μm). The IHC staining was independently assessed by two pathologists without patient data, and the statistical analysis of NR4A3 was based on the IHC score (values, 0–12). Patients were divided into high- and low-expression groups according to IHC scores. E The number of CD4 + or CD8 + T cells in high NR4A3 and low NR4A3 expression slides of BC tissues within a 20 × field ( n = 32). The mean number of CD4 + or CD8 + cells was quantified from four random fields of each section. p values were calculated by the Wilcoxon test ( A , C ) and Student’s t -test ( E ). ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Slides were stained with CD4 (Abcam, ab133616), CD8 (Abcam, ab101500), IFN-γ (Abcam, ab231036), CD68 (Abcam, ab233172), NR4A3 (Atlas Antibodies, HPA043360), iNOS (Abcam, ab283655), CD68 (Abcam, ab283654), CD86 (Invitrogen, 14-0862-85), phospho-NF-κB p65 (Cell Signaling Technology, 3033), pan-Cytokeratin (Abcam, ab7753), caspase 3 (Abcam, ab4051), and DAPI in proper order.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: NPJ Breast Cancer

Article Title: NR4A3 potentials M1-like macrophage polarization to facilitate anti-tumor immune responses in breast cancer

doi: 10.1038/s41523-025-00785-0

Figure Lengend Snippet: A , B The correlation between the level of NR4A3 and MHC molecules ( A ) and immune stimulatory molecules ( B ) in the TCGA-BRCA cohort. C Volcano plot of DEGs between high NR4A3 and low NR4A3 samples in a subpopulation of T cells from GSE176078 . D The DEGs were related to TNF-α signaling via NF-κB, interferon-gamma response, regulation of CD4 positive alpha/beta T-cell activation, and T-cell differentiation involved in immune response between high NR4A3 and low NR4A3 samples by GSEA ( GSE176078 ). E Representative images of IHC staining of NR4A3 (left panels) and mIHC staining of CD4, CD8, IFN-γ, pan-cytokeratin (PanCK), caspase 3 and DAPI (right panels) in high NR4A3 and low NR4A3 expression slides of BC (scale bar, 20 μm). The IHC staining was independently assessed by two pathologists without patient data, and the statistical analysis of NR4A3 levels was based on the IHC score (values, 0–12). Patients were divided into high- and low-expression groups according to IHC scores. F The numbers of IFN-γ-producing CD4 + or CD8 + T cells and the proportion of caspase 3 + tumor cells in high NR4A3 and low NR4A3 expression slides of BC tissues within a 20 × field ( n = 32). The mean number of IFN-γ-producing CD4 + or CD8 + T cells and the proportion of caspase 3 + tumor cells were quantified from four random fields of each section. p values were calculated by Pearson correlation analysis ( A , B ) and Student’s t -test ( F ). * p < 0.05.

Article Snippet: Slides were stained with CD4 (Abcam, ab133616), CD8 (Abcam, ab101500), IFN-γ (Abcam, ab231036), CD68 (Abcam, ab233172), NR4A3 (Atlas Antibodies, HPA043360), iNOS (Abcam, ab283655), CD68 (Abcam, ab283654), CD86 (Invitrogen, 14-0862-85), phospho-NF-κB p65 (Cell Signaling Technology, 3033), pan-Cytokeratin (Abcam, ab7753), caspase 3 (Abcam, ab4051), and DAPI in proper order.

Techniques: Activation Assay, Cell Differentiation, Immunohistochemistry, Staining, Expressing

Journal: NPJ Breast Cancer

Article Title: NR4A3 potentials M1-like macrophage polarization to facilitate anti-tumor immune responses in breast cancer

doi: 10.1038/s41523-025-00785-0

Figure Lengend Snippet: A Violin plot showing the NR4A3 expression levels in different cell types ( GSE176078 ). B Heatmap showing the NR4A3 expression levels in different cell types ( GSE176078 ). C UMAP plot showing the CD68 and NR4A3 double-positive cells in the BC single-cell database ( GSE176078 ). D The mRNA expression of CD68 and NR4A3 in MCF-7, T74D, HCC1954, BT549, MDA-MB-231, THP-1-monocytes, and THP-1-macrophages was determined by RT-qPCR (normalized with GAPDH mRNA). E The correlation between NR4A3 expression level and M1-like scores in a subpopulation of myeloid cells from GSE176078 . F The correlation between NR4A3 expression level and M2-like scores in a subpopulation of myeloid cells from GSE176078 . G CIBERSORT algorithm to assess the percentages of M0-like macrophages, M1-like macrophages, and M2-like macrophages in high NR4A3 and low NR4A3 groups in the TCGA-BRCA cohort. H Heatmap showing the expression of markers of M1-like macrophages in THP-1-derived macrophages after treatment with IL-4 or IFN-γ/LPS for 24 h ( GSE140235 ). I The mRNA expression of iNOS, TNFA, CD206 , and NR4A3 in THP-1-derived macrophages after treatment with IL-4 or IFN-γ/LPS for 24 h was determined by RT-qPCR (normalized with GAPDH mRNA). J Representative images of mIHC staining of NR4A3, CD68, and iNOS within BC tissue slides (scale bar, 20 μm). p values were calculated by Student’s t -test ( I ), Pearson correlation analysis ( E , F ), and Wilcoxon test ( G ). ns p > 0.05, *** p < 0.001.

Article Snippet: Slides were stained with CD4 (Abcam, ab133616), CD8 (Abcam, ab101500), IFN-γ (Abcam, ab231036), CD68 (Abcam, ab233172), NR4A3 (Atlas Antibodies, HPA043360), iNOS (Abcam, ab283655), CD68 (Abcam, ab283654), CD86 (Invitrogen, 14-0862-85), phospho-NF-κB p65 (Cell Signaling Technology, 3033), pan-Cytokeratin (Abcam, ab7753), caspase 3 (Abcam, ab4051), and DAPI in proper order.

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Staining

Journal: NPJ Breast Cancer

Article Title: NR4A3 potentials M1-like macrophage polarization to facilitate anti-tumor immune responses in breast cancer

doi: 10.1038/s41523-025-00785-0

Figure Lengend Snippet: A Knocking down NR4A3 expression in THP-1 cells with siRNA, the production of CD86 + and CD80 + in CD68 + cells in THP-1-derived macrophages after treatment with IFN-γ/LPS for 24 h was determined by flow cytometry. B The production of CD86 + and CD80 + in CD68 + cells in THP-1-derived macrophages with or without NR4A3 -overexpressing ( NR4A3 -OE) after treatment with IFN-γ/LPS for 24 h was determined by flow cytometry. C Knocking down NR4A3 expression in THP-1 cells with siRNA, the mRNA expression of NR4A3, TNFA, CD86, iNOS, IL1B , and IL6 in THP-1-derived macrophages after treatment with IFN-γ/LPS for 24 h was determined by RT-qPCR (normalized with GAPDH mRNA). D The mRNA expression of NR4A3, TNFA, CD86, iNOS, IL1B , and IL6 in THP-1-derived macrophages with or without NR4A3 -OE after treatment with IFN-γ/LPS for 24 h was determined by RT-qPCR (normalized with GAPDH mRNA). E Dot plot of the HALLMARK enrichment analysis. The size of the dot represents the gene count, and the color of the dot represents the p- value ( GSE176078 ). F The protein levels of NR4A3, p65, p-p65, and β-Tubulin in THP-1-derived macrophages with or without NR4A3 -OE were measured after treatment with IFN-γ/LPS for 24 h. G The mRNA expression of iNOS, TNFA, CD86, IL1B , and IL6 in NR4A3 -OE M1-like macrophage with NF-κB inhibitor BAY 11-7082 (5 μM, 24 h) treatment was determined by RT-qPCR (normalized with GAPDH mRNA). H Predicted protein–protein docking model between NR4A3 (slate cartoon) and NF-κB p65 (cyan cartoon). I , J Immunoprecipitation (IP) and western blot analysis of the NR4A3/p65 interaction in THP-1-derived macrophages after treatment with IFN-γ/LPS for 24 h. K The NR4A3/p65 interaction was analyzed using confocal microscopy in THP-1-derived macrophages after treatment with IFN-γ/LPS for 24 h (scale bar, 10 μm). p values were calculated by one-way ANOVA ( A , C ) and Student’s t -test ( B , D , G ). ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Slides were stained with CD4 (Abcam, ab133616), CD8 (Abcam, ab101500), IFN-γ (Abcam, ab231036), CD68 (Abcam, ab233172), NR4A3 (Atlas Antibodies, HPA043360), iNOS (Abcam, ab283655), CD68 (Abcam, ab283654), CD86 (Invitrogen, 14-0862-85), phospho-NF-κB p65 (Cell Signaling Technology, 3033), pan-Cytokeratin (Abcam, ab7753), caspase 3 (Abcam, ab4051), and DAPI in proper order.

Techniques: Expressing, Derivative Assay, Flow Cytometry, Quantitative RT-PCR, Immunoprecipitation, Western Blot, Confocal Microscopy

Journal: NPJ Breast Cancer

Article Title: NR4A3 potentials M1-like macrophage polarization to facilitate anti-tumor immune responses in breast cancer

doi: 10.1038/s41523-025-00785-0

Figure Lengend Snippet: A The schematic illustration of E0771 alone or co-injected with wild-type BMDMs or Nr4a3 -overexpressing ( Nr4a3 -OE)/ Nr4a3 -knockdown BMDMs model with C57BL/6 mice. B The efficacy of Nr4a3 overexpression in BMDMs was determined by RT-qPCR (normalized with Gapdh mRNA). C – E Immunocompetent C57BL/6 mice were transplanted with E0771 alone or co-injected with wild-type BMDMs or Nr4a3 -OE BMDMs for 4 weeks. The volumes of tumors. Mean values ± SEM ( C ), the images of tumors ( D ), and the weight of tumors ( E ) were quantified ( n = 5). F Representative images of mIHC staining of CD68, iNOS, CD86, p-p65, and DAPI in tumor tissues derived from the syngeneic mouse tumor model (scale bar, 20 μm). G – I Quantification of iNOS + CD68 + cells ( G ), CD86 + CD68 + cells ( H ), and the mean fluorescence intensity (MFI) of p-p65 of CD68 + cells ( I ), about two random fields (1 mm 2 per field) were selected under a microscope in each section. J The efficacy of Nr4a3 knockdown in BMDMs was determined by RT-qPCR (normalized with Gapdh mRNA). K – N The production of CD86 + cells ( K ), CD80 + cells ( L ), CD206 + cells ( M ), and CD163 + cells ( N ) among F4/80 + and CD11b + cells in E0771 tumor-bearing mice co-injected with Nr4a3 -knockdown BMDMs was examined by flow cytometry. p values were assessed by the Student’s t -test ( B ), one-way ANOVA ( E , G – I , K – N ), and two-way analysis of variance in C . ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Slides were stained with CD4 (Abcam, ab133616), CD8 (Abcam, ab101500), IFN-γ (Abcam, ab231036), CD68 (Abcam, ab233172), NR4A3 (Atlas Antibodies, HPA043360), iNOS (Abcam, ab283655), CD68 (Abcam, ab283654), CD86 (Invitrogen, 14-0862-85), phospho-NF-κB p65 (Cell Signaling Technology, 3033), pan-Cytokeratin (Abcam, ab7753), caspase 3 (Abcam, ab4051), and DAPI in proper order.

Techniques: Injection, Knockdown, Over Expression, Quantitative RT-PCR, Staining, Derivative Assay, Fluorescence, Microscopy, Flow Cytometry

Journal: NPJ Breast Cancer

Article Title: NR4A3 potentials M1-like macrophage polarization to facilitate anti-tumor immune responses in breast cancer

doi: 10.1038/s41523-025-00785-0

Figure Lengend Snippet: A , B Immunocompetent C57BL/6 mice were transplanted with E0771 alone or co-injected with wild-type BMDMs or Nr4a3 -overexpressing ( Nr4a3 -OE) BMDMs for 4 weeks. Representative images of CD4, CD8, Granzyme B, and IFN-γ IHC staining in tumor sections (scale bar, 20 μm) ( A ) and numbers of indicated immune cells per mm 2 ( B ), about two random fields (1 mm 2 per field) were selected under a microscope in each section. C , D Immunocompetent C57BL/6 mice were transplanted with E0771 alone or co-injected with wild-type BMDMs or Nr4a3 -knockdown BMDMs for 3 weeks. Representative images of CD4, CD8, Granzyme B, and IFN-γ B IHC staining in tumor sections (scale bar, 20 μm) ( C ) and numbers of indicated immune cells per mm 2 ( D ), about two random fields (1 mm 2 per field) were selected under a microscope in each section. E The schematic illustration of T cells co-culturing with THP-1 macrophages with or without NR4A3 -overexpressing ( NR4A3 -OE). F The production of IFN-γ and TNF-α in CD4 + T cells was examined by flow cytometry. G The production of IFN-γ and TNF-α in CD8 + T cells was examined by flow cytometry. p values were assessed by the one-way ANOVA ( B , D ), and Student’s t -test ( F , G ). ns p > 0.05, * p < 0.05, *** p < 0.001.

Article Snippet: Slides were stained with CD4 (Abcam, ab133616), CD8 (Abcam, ab101500), IFN-γ (Abcam, ab231036), CD68 (Abcam, ab233172), NR4A3 (Atlas Antibodies, HPA043360), iNOS (Abcam, ab283655), CD68 (Abcam, ab283654), CD86 (Invitrogen, 14-0862-85), phospho-NF-κB p65 (Cell Signaling Technology, 3033), pan-Cytokeratin (Abcam, ab7753), caspase 3 (Abcam, ab4051), and DAPI in proper order.

Techniques: Injection, Immunohistochemistry, Microscopy, Knockdown, Flow Cytometry

Journal: American journal of clinical pathology

Article Title: NR4A3 Immunostain Is a Highly Sensitive and Specific Marker for Acinic Cell Carcinoma in Cytologic and Surgical Specimens.

doi: 10.1093/ajcp/aqab099

Figure Lengend Snippet: FIGURE 1 Nuclear receptor subfamily 4 group A member 3 (NR4A3) immunohistochemistry in acinic cell carcinoma and secretory carcinoma in cytology specimens (×100). Cell block preparations from fine-needle aspirates from acinic cell carcinoma (A, B) and secretory carcinoma (C, D), a key entity on the differential diagnosis, were stained with H&E or immunostained with NR4A3. Nuclear NR4A3 was seen only in acinic cell carcinoma cases but not in secretory carcinoma cases.

Article Snippet: In SRs, 89% of AciCCs (8/9) showed strong diffuse NR4A3 staining when using the NR4A3 Origene antibody clone FIGURE 4 and FIGURE 5 .

Techniques: Immunohistochemistry, Blocking Assay, Biomarker Discovery, Staining

Journal: American journal of clinical pathology

Article Title: NR4A3 Immunostain Is a Highly Sensitive and Specific Marker for Acinic Cell Carcinoma in Cytologic and Surgical Specimens.

doi: 10.1093/ajcp/aqab099

Figure Lengend Snippet: FIGURE 2 Nuclear receptor subfamily 4 group A member 3 (NR4A3) immunohistochemistry in other primary oncocytic salivary gland neoplasms in cytology specimens (×100). Cell block preparations from fine-needle aspirates from oncocytoma (A, B), low-grade mucoepidermoid carcinoma (C, D), and Warthin tumor (E, F) were stained with H&E or immunostained with NR4A3. Nuclear NR4A3 was not identifiable in any of these oncocytic/oncocyotid salivary gland neoplasms. Nonspecific cytoplasmic granular NR4A3 staining was seen in the case of low-grade mucoepidermoid carcinoma.

Article Snippet: In SRs, 89% of AciCCs (8/9) showed strong diffuse NR4A3 staining when using the NR4A3 Origene antibody clone FIGURE 4 and FIGURE 5 .

Techniques: Immunohistochemistry, Blocking Assay, Staining

Journal: American journal of clinical pathology

Article Title: NR4A3 Immunostain Is a Highly Sensitive and Specific Marker for Acinic Cell Carcinoma in Cytologic and Surgical Specimens.

doi: 10.1093/ajcp/aqab099

Figure Lengend Snippet: FIGURE 4 Nuclear receptor subfamily 4 group A member 3 (NR4A3) immunohistochemistry in acinic cell carcinoma and secretory carcinoma in surgical resections (×100). Representative sections from surgical resection specimens of acinic cell carcinoma (A, B) and secretory carcinoma (C, D) were stained with H&E or immunostained with NR4A3. Similar to cytologic preparations, nuclear NR4A3 was seen only in acinic cell carcinoma but not in secretory carcinoma.

Article Snippet: In SRs, 89% of AciCCs (8/9) showed strong diffuse NR4A3 staining when using the NR4A3 Origene antibody clone FIGURE 4 and FIGURE 5 .

Techniques: Immunohistochemistry, Staining

Journal: American journal of clinical pathology

Article Title: NR4A3 Immunostain Is a Highly Sensitive and Specific Marker for Acinic Cell Carcinoma in Cytologic and Surgical Specimens.

doi: 10.1093/ajcp/aqab099

Figure Lengend Snippet: FIGURE 5 Nuclear receptor subfamily 4 group A member 3 (NR4A3) immunohistochemistry in other primary oncocytic salivary gland neoplasms in surgical resections (×100). Representative sections from surgical resection specimens of oncocytoma (A, B), oncocytic mucoepidermoid carcinoma (C, D), and Warthin tumor (E, F) were stained with H&E or immunostained with NR4A3. Similar to cytologic preparations, nuclear NR4A3 was not seen in other oncocytic salivary gland neoplasms, a few of which showed nonspecific granular cytoplasmic staining.

Article Snippet: In SRs, 89% of AciCCs (8/9) showed strong diffuse NR4A3 staining when using the NR4A3 Origene antibody clone FIGURE 4 and FIGURE 5 .

Techniques: Immunohistochemistry, Staining

Journal: American journal of clinical pathology

Article Title: NR4A3 Immunostain Is a Highly Sensitive and Specific Marker for Acinic Cell Carcinoma in Cytologic and Surgical Specimens.

doi: 10.1093/ajcp/aqab099

Figure Lengend Snippet: FIGURE 6 Nuclear receptor subfamily 4 group A member 3 (NR4A3) expression is retained in acinic cell carcinoma with high-grade transformation. Representative sections from two different resections of acinic cell carcinoma with high-grade transformation (A-D) were stained with H&E or immunostained with NR4A3 (×200).

Article Snippet: In SRs, 89% of AciCCs (8/9) showed strong diffuse NR4A3 staining when using the NR4A3 Origene antibody clone FIGURE 4 and FIGURE 5 .

Techniques: Expressing, Transformation Assay, Staining

Journal: American journal of clinical pathology

Article Title: NR4A3 Immunostain Is a Highly Sensitive and Specific Marker for Acinic Cell Carcinoma in Cytologic and Surgical Specimens.

doi: 10.1093/ajcp/aqab099

Figure Lengend Snippet: FIGURE 7 Examples of false-positive and a single negative case of nuclear receptor subfamily 4 group A member 3 (NR4A3) immunohistochemistry. Representative examples of a low-grade acinic cell carcinoma surgical resection negative with the Origene NR4A3 antibody clone (A, B; ×40) and a false- positive high-grade mucoepidermoid carcinoma (C, D; ×100).

Article Snippet: In SRs, 89% of AciCCs (8/9) showed strong diffuse NR4A3 staining when using the NR4A3 Origene antibody clone FIGURE 4 and FIGURE 5 .

Techniques: Immunohistochemistry