Journal: Experimental & molecular medicine

Article Title: NR4A1 suppresses breast cancer growth by repressing c-Fos-mediated lipid and redox dyshomeostasis.

doi: 10.1038/s12276-025-01430-3

Figure Lengend Snippet: Fig. 1 NR4A1 has a tumor-suppressive role in BC progression. a Analysis of the TCGA database shows that the NR4A1 mRNA level is decreased in all four types of BC samples compared with normal breast tissues. Unpaired Student’s t-test, ***P < 0.001. b Representative IHC staining of NR4A1 in cohort 1 TMA. Scale bars, 200 μm. c Quantitative IRS of NR4A1 as in b. Paired Student’s t-tests, ***P < 0.001. d Stratification of patients in cohort 2 with NR4A1 high expression (with IRS 6–12) or low expression (with IRS 0–4) at the protein level in TMA and its association with clinicopathological factors. Chi-square test. e IHC staining of NR4A1 in different AJCC TNM stages of BC. Scale bar, 200 μm. f, g Kaplan–Meier survival plots of patients with BC based on NR4A1 expression in TCGA cohort (f) and cohort 2 (g). OS, overall survival; HR, hazard ratio. h, i Log-rank tests. Univariate (h) and multivariate (i) analysis was performed in cohort 2. The bars correspond to 95% confidence interval (CI).

Article Snippet: The sections were then exposed to antigen and incubated with a specific primary antibody against NR4A1 (Proteintech, cat. no. 12235-1-AP; 1:100 dilution), c-Fos (Abcam, cat. no. ab208942; 1:200 dilution) or PRDX6 (Abcam, cat. no. ab133348; 1:100 dilution) at 4 °C overnight, followed by incubating with the corresponding secondary antibodies at 37 °C for 1 h. Representative images were taken using an Olympus light microscope.

Techniques: Immunohistochemistry, Expressing

Journal: Experimental & molecular medicine

Article Title: NR4A1 suppresses breast cancer growth by repressing c-Fos-mediated lipid and redox dyshomeostasis.

doi: 10.1038/s12276-025-01430-3

Figure Lengend Snippet: Fig. 2 NR4A1 deficiency promotes the proliferation of BC cells. a Immunoblotting analysis of NR4A1 knockout efficiency in MCF7 and T47D cells. b Cell proliferation assay was performed by CCK-8 assay in NR4A1-knockout and parental control MCF7 cells or T47D cells. c Colony formation assay was performed in NR4A1-knockout and parental control MCF7 cells or T47D cells. d, e Flow cytometry analysis (left) and statistical quantification of MFI (right) for Edu incorporation in NR4A1-knockout and parental control MCF7 cells (d) or T47D cells (e). MFI, mean fluorescence intensity. f–h NR4A1-knockout and parental control MCF7 cells were injected into the flank of female athymic nude mice (n = 6 per group). Tumor volumes were measured every 3 days. Tumor images (f), growth curves (g) and tumor weight (h) were obtained at day 21 after dissection. i Representative IHC staining of Ki67 and the statistical analysis of Ki67-positive percentages in tumors from f. Scale bar, 100 μm. Unpaired Student’s t-tests were used in c–e, h and i, and one-way ANOVA was used in b and g, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The sections were then exposed to antigen and incubated with a specific primary antibody against NR4A1 (Proteintech, cat. no. 12235-1-AP; 1:100 dilution), c-Fos (Abcam, cat. no. ab208942; 1:200 dilution) or PRDX6 (Abcam, cat. no. ab133348; 1:100 dilution) at 4 °C overnight, followed by incubating with the corresponding secondary antibodies at 37 °C for 1 h. Representative images were taken using an Olympus light microscope.

Techniques: Western Blot, Knock-Out, Proliferation Assay, CCK-8 Assay, Control, Colony Assay, Flow Cytometry, Injection, Dissection, Immunohistochemistry

Journal: Experimental & molecular medicine

Article Title: NR4A1 suppresses breast cancer growth by repressing c-Fos-mediated lipid and redox dyshomeostasis.

doi: 10.1038/s12276-025-01430-3

Figure Lengend Snippet: Fig. 3 NR4A1 deficiency regulates the lipid remodeling and phospholipid accumulation. a Rank-ordered depiction of fold changes for all analyzed genes quantified by RNA-seq with the significantly changed genes of 73 increased and 28 decreased (fold change >1.5 and FDR <0.05 difference) in NR4A1-knockout MCF7 cells compared with parental MCF7 cells. b GSEA analysis was conducted to identify the different gene profiles between NR4A1-knockout and parental MCF7 cells. c Single-sample GSEA analysis was performed to show the pathways closely correlated with NR4A1 expression in MCF7 cells. d Volcano plots of metabolites detected by UHPLC–QTOF–MS-based nontargeted metabolomics analysis in NR4A1-knockout and parental MCF7 cells. Red represents lipids and lipid-like metabolites (n = 61). e Heat map showed the classification of the significantly changed metabolites of 91 increased (fold change >1.5, FDR <0.05) in NR4A1-knockout MCF7 cells compared with parental MCF7 cells. f Enriched metabolic signaling pathways based on significantly changed metabolites (n = 199) cluster identified by pathway analysis (https://www.metaboanalyst.ca/). g, h Flow cytometry analysis (left) and statistical quantification of MFI (right) for BODIPY FL C16 to compare fatty acid uptake ability in NR4A1-knockout and parental control MCF7 or T47D cells. i, j RT-qPCR (i) and immunoblotting analysis (j) of CD36 in NR4A1-knockout and parental MCF7 or T47D cells. Unpaired Student’s t-tests were used in g–i, **P < 0.01, ***P < 0.001.

Article Snippet: The sections were then exposed to antigen and incubated with a specific primary antibody against NR4A1 (Proteintech, cat. no. 12235-1-AP; 1:100 dilution), c-Fos (Abcam, cat. no. ab208942; 1:200 dilution) or PRDX6 (Abcam, cat. no. ab133348; 1:100 dilution) at 4 °C overnight, followed by incubating with the corresponding secondary antibodies at 37 °C for 1 h. Representative images were taken using an Olympus light microscope.

Techniques: RNA Sequencing, Knock-Out, Expressing, Protein-Protein interactions, Flow Cytometry, Control, Quantitative RT-PCR, Western Blot

Journal: Experimental & molecular medicine

Article Title: NR4A1 suppresses breast cancer growth by repressing c-Fos-mediated lipid and redox dyshomeostasis.

doi: 10.1038/s12276-025-01430-3

Figure Lengend Snippet: Fig. 4 Suppression of NR4A1 exacerbates the redox balance disruption. a Seahorse extracellular flux analyzer measurement of ECAR metabolic profile in NR4A1-knockout and parental MCF7 or T47D cells. b Assessments of ATP production ability in NR4A1-knockout and parental MCF7 or T47D cells. c Seahorse extracellular flux analyzer measurement of OCR metabolic profile in NR4A1-knockout and parental MCF7 or T47D cells. d Intracellular GSH level in MCF7 and T47D cells with or without NR4A1 expression. e, f Flow cytometry analysis of intracellular ROS levels by DCFH-DA staining in NR4A1-knockout and parental MCF7 (e) or T47D (f) cells. ROS levels were quantified by MFI. g, h Lipid peroxidation measured by flow cytometry using the lipid peroxidation reagent in NR4A1-knockout and parental MCF7 (g) or T47D (h) cells. Lipid peroxidation was quantified by the ratio of red (PE)/green (FITC) fluorescence intensities, and the decreased ratio was correlated with higher lipid peroxidation. Unpaired Student’s t-tests were used in b and d–h, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The sections were then exposed to antigen and incubated with a specific primary antibody against NR4A1 (Proteintech, cat. no. 12235-1-AP; 1:100 dilution), c-Fos (Abcam, cat. no. ab208942; 1:200 dilution) or PRDX6 (Abcam, cat. no. ab133348; 1:100 dilution) at 4 °C overnight, followed by incubating with the corresponding secondary antibodies at 37 °C for 1 h. Representative images were taken using an Olympus light microscope.

Techniques: Disruption, Knock-Out, Expressing, Flow Cytometry, Staining, Cytometry

Journal: Experimental & molecular medicine

Article Title: NR4A1 suppresses breast cancer growth by repressing c-Fos-mediated lipid and redox dyshomeostasis.

doi: 10.1038/s12276-025-01430-3

Figure Lengend Snippet: Fig. 5 NR4A1–c-Fos interaction affects the transcriptional activity of c-Fos. a, b Prediction of transcription factors regulating the significantly upregulated genes in NR4A1-knockout MCF7 cells by using TFEA method in ChEA3 databases. Statistical results (a) and co- regulatory network for the functional interaction (b) for the top six predicted transcription factors. c Enrichment plot of the c-Fos dataset in GSEA analysis. Heat map of the top 20 genes upregulated in NR4A1-knockout cells from the c-Fos dataset. d Cell proliferation assay was performed by CCK-8 assay in ectopic c-Fos- or empty vector (EV)-overexpressed MCF7 cells or T47D cells. One-way ANOVA, ***P < 0.001. e, f Co-IP experiments to detect the interaction between NR4A1 and c-Fos. HEK293T cells were transfected for 24 h with plasmids encoding either Flag-NR4A1 or HA-c-Fos alone or in combination. Cell lysates were immunoprecipitated with Flag (e) or HA (f) antibodies. g c-Fos domain structure and deletion mutants used for Co-IP experiments. h Co-IP experiments were used to identify the interaction domain for c-Fos binding to NR4A1.

Article Snippet: The sections were then exposed to antigen and incubated with a specific primary antibody against NR4A1 (Proteintech, cat. no. 12235-1-AP; 1:100 dilution), c-Fos (Abcam, cat. no. ab208942; 1:200 dilution) or PRDX6 (Abcam, cat. no. ab133348; 1:100 dilution) at 4 °C overnight, followed by incubating with the corresponding secondary antibodies at 37 °C for 1 h. Representative images were taken using an Olympus light microscope.

Techniques: Activity Assay, Knock-Out, Functional Assay, Proliferation Assay, CCK-8 Assay, Plasmid Preparation, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Binding Assay

Journal: Experimental & molecular medicine

Article Title: NR4A1 suppresses breast cancer growth by repressing c-Fos-mediated lipid and redox dyshomeostasis.

doi: 10.1038/s12276-025-01430-3

Figure Lengend Snippet: Fig. 6 NR4A1 competitively inhibits c-Fos binding to targeted genes in BC cells. a Genome-wide profiles of c-Fos in NR4A1-knockout and control MCF7 cells. b Heat maps of c-Fos ChIP–seq signals sorted on the basis of increased c-Fos peaks between NR4A1-knockout and control MCF7 cells. c Genomic annotations of the increased c-Fos peaks in NR4A1-knockout cells by chromosome location. d GO analysis and KEGG analysis of NR4A1-competitive c-Fos occupied genes. e Motif sequences (left) and matched transcription factors (middle) with corresponding P values (right) from de novo motif analysis of NR4A1-competitive c-Fos peaks. f c-Fos ChIP–seq tracks at PRDX6 gene locus. g RT-qPCR analysis of PRDX6 mRNA levels in NR4A1-knockout and control cells. h Immunoblotting analysis of PRDX6 in BC cells with or without NR4A1. i Schematic diagram of PRDX6 promoter showing c-Fos and NR4A1 binding motifs in the regulator region. pGL3-NBREwt and pGL3-NBREdel stand for the PRDX6 promoter region with NBRE-like elements or NBRE-like elements deleted sequences, which were cloned upstream of the firefly luciferase gene in the pGL3-basic vector. j ChIP–qPCR analysis of c-Fos and NR4A1 enrichment around the c-Fos motif and NBRE-like elements on PRDX6 promoter in NR4A1 knockout and control MCF7 cells. k PRDX6 promoter constructs were co-transfected with NR4A1, c-Fos or empty vector (EV) to detect luciferase activity in HEK293T cells. pRL-TK was transfected for normalization, and luciferase activity was measured by using a dual luciferase reporter assay system. Unpaired Student’s t-tests were used in g, j and k, **P < 0.01, ***P < 0.001, NS nonsignificant.

Article Snippet: The sections were then exposed to antigen and incubated with a specific primary antibody against NR4A1 (Proteintech, cat. no. 12235-1-AP; 1:100 dilution), c-Fos (Abcam, cat. no. ab208942; 1:200 dilution) or PRDX6 (Abcam, cat. no. ab133348; 1:100 dilution) at 4 °C overnight, followed by incubating with the corresponding secondary antibodies at 37 °C for 1 h. Representative images were taken using an Olympus light microscope.

Techniques: Binding Assay, Genome Wide, Knock-Out, Control, ChIP-sequencing, Quantitative RT-PCR, Western Blot, Clone Assay, Luciferase, Plasmid Preparation, ChIP-qPCR, Construct, Transfection, Activity Assay, Reporter Assay

Journal: Experimental & molecular medicine

Article Title: NR4A1 suppresses breast cancer growth by repressing c-Fos-mediated lipid and redox dyshomeostasis.

doi: 10.1038/s12276-025-01430-3

Figure Lengend Snippet: Fig. 7 NR4A1 agonist represses the transcriptional activity of c-Fos. a Immunoblotting analysis of NR4A1 in MCF7 cells treated by different concentrations of Csn-B or vehicle. b Cell proliferation assay was performed by CCK-8 assay in MCF7 or T47D cells treated by different concentrations of Csn-B or vehicle. c Comparison of cell growth between Csn-B (10 μM) and vehicle treatment in NR4A1-knockout MCF7 cells or T47D cells. d–f, Csn-B inhibited BC growth in xenografts. Female athymic nude mice bearing wild-type MCF7 tumor were treated daily with vehicle or Csn-B. Tumor images (d), growth curves (e) and tumor weight (f) were obtained at the end of treatment. n = 10 per group. g Representative IHC staining of Ki67 and the statistical analysis of Ki67-positive percentages in tumors d. Scale bar, 100 μm. h Co-IP experiments to detect interactions between NR4A1 and c-Fos in MCF7 cells treated by 10 μM Csn-B or vehicle for 24 h. i ChIP assays of the c-Fos enrichment on PRDX6 promoter in MCF7 cells treated by 10 μM Csn-B or vehicle for 24 h. j, k RT-qPCR (j) and immunoblotting analysis (k) of PRDX6 in MCF7 or T47D cells treated by 10 μM Csn-B or vehicle for 24 h. l Comparison of PRDX6 mRNA levels in NR4A1-knockout and parental MCF7 cells treated by 10 μM Csn-B or vehicle for 24 h. m Cell proliferation assay was performed by CCK-8 assay in ectopic c-Fos- or EV- overexpressed MCF7 cells treated by 10 μM Csn-B or vehicle. Unpaired Student’s t-tests were used in f, g, i, j and l, and one-way ANOVA was used in b, c, e and m, *P < 0.05, **P < 0.01, ***P < 0.001, NS nonsignificant.

Article Snippet: The sections were then exposed to antigen and incubated with a specific primary antibody against NR4A1 (Proteintech, cat. no. 12235-1-AP; 1:100 dilution), c-Fos (Abcam, cat. no. ab208942; 1:200 dilution) or PRDX6 (Abcam, cat. no. ab133348; 1:100 dilution) at 4 °C overnight, followed by incubating with the corresponding secondary antibodies at 37 °C for 1 h. Representative images were taken using an Olympus light microscope.

Techniques: Activity Assay, Western Blot, Proliferation Assay, CCK-8 Assay, Comparison, Knock-Out, Immunohistochemistry, Co-Immunoprecipitation Assay, Quantitative RT-PCR

Journal: Experimental & molecular medicine

Article Title: NR4A1 suppresses breast cancer growth by repressing c-Fos-mediated lipid and redox dyshomeostasis.

doi: 10.1038/s12276-025-01430-3

Figure Lengend Snippet: Fig. 8 The impact of NR4A1–c-Fos–PRDX6 axis on BC prognosis. a Representative IHC staining of NR4A1, c-Fos and PRDX6 in cohort 2 TMA. Scale bars, 200 μm. b Pearson correlation analysis between NR4A1, c-Fos and PRDX6 protein expression based on IRS in cohort 2 TMA. R represents the Pearson correlation coefficient. c Kaplan–Meier survival plots of patients with BC in cohort 2 TMA based on c-Fos, or PRDX6 expression. d Kaplan–Meier survival plots of patients with BC in cohort 2 TMA based on the protein levels of NR4A1 co-expressed with PRDX6, or c-Fos co-expressed with PRDX6. e Kaplan–Meier survival plots of patients with BC in cohort 2 TMA based on the protein levels of NR4A1 co- expressed with c-Fos, or NR4A1 co-expressed with c-Fos and PRDX6. Log-rank tests were used in c–e. f Schematic diagram of the proposed mechanism.

Article Snippet: The sections were then exposed to antigen and incubated with a specific primary antibody against NR4A1 (Proteintech, cat. no. 12235-1-AP; 1:100 dilution), c-Fos (Abcam, cat. no. ab208942; 1:200 dilution) or PRDX6 (Abcam, cat. no. ab133348; 1:100 dilution) at 4 °C overnight, followed by incubating with the corresponding secondary antibodies at 37 °C for 1 h. Representative images were taken using an Olympus light microscope.

Techniques: Immunohistochemistry, Expressing

Journal: Experimental & molecular medicine

Article Title: NR4A1 suppresses breast cancer growth by repressing c-Fos-mediated lipid and redox dyshomeostasis.

doi: 10.1038/s12276-025-01430-3

Figure Lengend Snippet: Fig. 1 NR4A1 has a tumor-suppressive role in BC progression. a Analysis of the TCGA database shows that the NR4A1 mRNA level is decreased in all four types of BC samples compared with normal breast tissues. Unpaired Student’s t-test, ***P < 0.001. b Representative IHC staining of NR4A1 in cohort 1 TMA. Scale bars, 200 μm. c Quantitative IRS of NR4A1 as in b. Paired Student’s t-tests, ***P < 0.001. d Stratification of patients in cohort 2 with NR4A1 high expression (with IRS 6–12) or low expression (with IRS 0–4) at the protein level in TMA and its association with clinicopathological factors. Chi-square test. e IHC staining of NR4A1 in different AJCC TNM stages of BC. Scale bar, 200 μm. f, g Kaplan–Meier survival plots of patients with BC based on NR4A1 expression in TCGA cohort (f) and cohort 2 (g). OS, overall survival; HR, hazard ratio. h, i Log-rank tests. Univariate (h) and multivariate (i) analysis was performed in cohort 2. The bars correspond to 95% confidence interval (CI).

Article Snippet: All cell lines were obtained from the American Type Culture Collection, cultured at 37 °C in a humidified atmosphere with 5% CO2 and ensured to be mycoplasmanegative cultures by monthly mycoplasma tests. sgRNA-mediated knockout cell generation For NR4A1 stable knockout using CRISPR single guide RNAs (sgRNAs), sgRNAs were first cloned into LentiCRISPR-V2 vector (Addgene, cat. no. 52961) using BsmBI sites.

Techniques: Immunohistochemistry, Expressing

Journal: Experimental & molecular medicine

Article Title: NR4A1 suppresses breast cancer growth by repressing c-Fos-mediated lipid and redox dyshomeostasis.

doi: 10.1038/s12276-025-01430-3

Figure Lengend Snippet: Fig. 2 NR4A1 deficiency promotes the proliferation of BC cells. a Immunoblotting analysis of NR4A1 knockout efficiency in MCF7 and T47D cells. b Cell proliferation assay was performed by CCK-8 assay in NR4A1-knockout and parental control MCF7 cells or T47D cells. c Colony formation assay was performed in NR4A1-knockout and parental control MCF7 cells or T47D cells. d, e Flow cytometry analysis (left) and statistical quantification of MFI (right) for Edu incorporation in NR4A1-knockout and parental control MCF7 cells (d) or T47D cells (e). MFI, mean fluorescence intensity. f–h NR4A1-knockout and parental control MCF7 cells were injected into the flank of female athymic nude mice (n = 6 per group). Tumor volumes were measured every 3 days. Tumor images (f), growth curves (g) and tumor weight (h) were obtained at day 21 after dissection. i Representative IHC staining of Ki67 and the statistical analysis of Ki67-positive percentages in tumors from f. Scale bar, 100 μm. Unpaired Student’s t-tests were used in c–e, h and i, and one-way ANOVA was used in b and g, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: All cell lines were obtained from the American Type Culture Collection, cultured at 37 °C in a humidified atmosphere with 5% CO2 and ensured to be mycoplasmanegative cultures by monthly mycoplasma tests. sgRNA-mediated knockout cell generation For NR4A1 stable knockout using CRISPR single guide RNAs (sgRNAs), sgRNAs were first cloned into LentiCRISPR-V2 vector (Addgene, cat. no. 52961) using BsmBI sites.

Techniques: Western Blot, Knock-Out, Proliferation Assay, CCK-8 Assay, Control, Colony Assay, Flow Cytometry, Injection, Dissection, Immunohistochemistry

Journal: Experimental & molecular medicine

Article Title: NR4A1 suppresses breast cancer growth by repressing c-Fos-mediated lipid and redox dyshomeostasis.

doi: 10.1038/s12276-025-01430-3

Figure Lengend Snippet: Fig. 3 NR4A1 deficiency regulates the lipid remodeling and phospholipid accumulation. a Rank-ordered depiction of fold changes for all analyzed genes quantified by RNA-seq with the significantly changed genes of 73 increased and 28 decreased (fold change >1.5 and FDR <0.05 difference) in NR4A1-knockout MCF7 cells compared with parental MCF7 cells. b GSEA analysis was conducted to identify the different gene profiles between NR4A1-knockout and parental MCF7 cells. c Single-sample GSEA analysis was performed to show the pathways closely correlated with NR4A1 expression in MCF7 cells. d Volcano plots of metabolites detected by UHPLC–QTOF–MS-based nontargeted metabolomics analysis in NR4A1-knockout and parental MCF7 cells. Red represents lipids and lipid-like metabolites (n = 61). e Heat map showed the classification of the significantly changed metabolites of 91 increased (fold change >1.5, FDR <0.05) in NR4A1-knockout MCF7 cells compared with parental MCF7 cells. f Enriched metabolic signaling pathways based on significantly changed metabolites (n = 199) cluster identified by pathway analysis (https://www.metaboanalyst.ca/). g, h Flow cytometry analysis (left) and statistical quantification of MFI (right) for BODIPY FL C16 to compare fatty acid uptake ability in NR4A1-knockout and parental control MCF7 or T47D cells. i, j RT-qPCR (i) and immunoblotting analysis (j) of CD36 in NR4A1-knockout and parental MCF7 or T47D cells. Unpaired Student’s t-tests were used in g–i, **P < 0.01, ***P < 0.001.

Article Snippet: All cell lines were obtained from the American Type Culture Collection, cultured at 37 °C in a humidified atmosphere with 5% CO2 and ensured to be mycoplasmanegative cultures by monthly mycoplasma tests. sgRNA-mediated knockout cell generation For NR4A1 stable knockout using CRISPR single guide RNAs (sgRNAs), sgRNAs were first cloned into LentiCRISPR-V2 vector (Addgene, cat. no. 52961) using BsmBI sites.

Techniques: RNA Sequencing, Knock-Out, Expressing, Protein-Protein interactions, Flow Cytometry, Control, Quantitative RT-PCR, Western Blot

Journal: Experimental & molecular medicine

Article Title: NR4A1 suppresses breast cancer growth by repressing c-Fos-mediated lipid and redox dyshomeostasis.

doi: 10.1038/s12276-025-01430-3

Figure Lengend Snippet: Fig. 4 Suppression of NR4A1 exacerbates the redox balance disruption. a Seahorse extracellular flux analyzer measurement of ECAR metabolic profile in NR4A1-knockout and parental MCF7 or T47D cells. b Assessments of ATP production ability in NR4A1-knockout and parental MCF7 or T47D cells. c Seahorse extracellular flux analyzer measurement of OCR metabolic profile in NR4A1-knockout and parental MCF7 or T47D cells. d Intracellular GSH level in MCF7 and T47D cells with or without NR4A1 expression. e, f Flow cytometry analysis of intracellular ROS levels by DCFH-DA staining in NR4A1-knockout and parental MCF7 (e) or T47D (f) cells. ROS levels were quantified by MFI. g, h Lipid peroxidation measured by flow cytometry using the lipid peroxidation reagent in NR4A1-knockout and parental MCF7 (g) or T47D (h) cells. Lipid peroxidation was quantified by the ratio of red (PE)/green (FITC) fluorescence intensities, and the decreased ratio was correlated with higher lipid peroxidation. Unpaired Student’s t-tests were used in b and d–h, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: All cell lines were obtained from the American Type Culture Collection, cultured at 37 °C in a humidified atmosphere with 5% CO2 and ensured to be mycoplasmanegative cultures by monthly mycoplasma tests. sgRNA-mediated knockout cell generation For NR4A1 stable knockout using CRISPR single guide RNAs (sgRNAs), sgRNAs were first cloned into LentiCRISPR-V2 vector (Addgene, cat. no. 52961) using BsmBI sites.

Techniques: Disruption, Knock-Out, Expressing, Flow Cytometry, Staining, Cytometry

Journal: Experimental & molecular medicine

Article Title: NR4A1 suppresses breast cancer growth by repressing c-Fos-mediated lipid and redox dyshomeostasis.

doi: 10.1038/s12276-025-01430-3

Figure Lengend Snippet: Fig. 5 NR4A1–c-Fos interaction affects the transcriptional activity of c-Fos. a, b Prediction of transcription factors regulating the significantly upregulated genes in NR4A1-knockout MCF7 cells by using TFEA method in ChEA3 databases. Statistical results (a) and co- regulatory network for the functional interaction (b) for the top six predicted transcription factors. c Enrichment plot of the c-Fos dataset in GSEA analysis. Heat map of the top 20 genes upregulated in NR4A1-knockout cells from the c-Fos dataset. d Cell proliferation assay was performed by CCK-8 assay in ectopic c-Fos- or empty vector (EV)-overexpressed MCF7 cells or T47D cells. One-way ANOVA, ***P < 0.001. e, f Co-IP experiments to detect the interaction between NR4A1 and c-Fos. HEK293T cells were transfected for 24 h with plasmids encoding either Flag-NR4A1 or HA-c-Fos alone or in combination. Cell lysates were immunoprecipitated with Flag (e) or HA (f) antibodies. g c-Fos domain structure and deletion mutants used for Co-IP experiments. h Co-IP experiments were used to identify the interaction domain for c-Fos binding to NR4A1.

Article Snippet: All cell lines were obtained from the American Type Culture Collection, cultured at 37 °C in a humidified atmosphere with 5% CO2 and ensured to be mycoplasmanegative cultures by monthly mycoplasma tests. sgRNA-mediated knockout cell generation For NR4A1 stable knockout using CRISPR single guide RNAs (sgRNAs), sgRNAs were first cloned into LentiCRISPR-V2 vector (Addgene, cat. no. 52961) using BsmBI sites.

Techniques: Activity Assay, Knock-Out, Functional Assay, Proliferation Assay, CCK-8 Assay, Plasmid Preparation, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Binding Assay

Journal: Experimental & molecular medicine

Article Title: NR4A1 suppresses breast cancer growth by repressing c-Fos-mediated lipid and redox dyshomeostasis.

doi: 10.1038/s12276-025-01430-3

Figure Lengend Snippet: Fig. 6 NR4A1 competitively inhibits c-Fos binding to targeted genes in BC cells. a Genome-wide profiles of c-Fos in NR4A1-knockout and control MCF7 cells. b Heat maps of c-Fos ChIP–seq signals sorted on the basis of increased c-Fos peaks between NR4A1-knockout and control MCF7 cells. c Genomic annotations of the increased c-Fos peaks in NR4A1-knockout cells by chromosome location. d GO analysis and KEGG analysis of NR4A1-competitive c-Fos occupied genes. e Motif sequences (left) and matched transcription factors (middle) with corresponding P values (right) from de novo motif analysis of NR4A1-competitive c-Fos peaks. f c-Fos ChIP–seq tracks at PRDX6 gene locus. g RT-qPCR analysis of PRDX6 mRNA levels in NR4A1-knockout and control cells. h Immunoblotting analysis of PRDX6 in BC cells with or without NR4A1. i Schematic diagram of PRDX6 promoter showing c-Fos and NR4A1 binding motifs in the regulator region. pGL3-NBREwt and pGL3-NBREdel stand for the PRDX6 promoter region with NBRE-like elements or NBRE-like elements deleted sequences, which were cloned upstream of the firefly luciferase gene in the pGL3-basic vector. j ChIP–qPCR analysis of c-Fos and NR4A1 enrichment around the c-Fos motif and NBRE-like elements on PRDX6 promoter in NR4A1 knockout and control MCF7 cells. k PRDX6 promoter constructs were co-transfected with NR4A1, c-Fos or empty vector (EV) to detect luciferase activity in HEK293T cells. pRL-TK was transfected for normalization, and luciferase activity was measured by using a dual luciferase reporter assay system. Unpaired Student’s t-tests were used in g, j and k, **P < 0.01, ***P < 0.001, NS nonsignificant.

Article Snippet: All cell lines were obtained from the American Type Culture Collection, cultured at 37 °C in a humidified atmosphere with 5% CO2 and ensured to be mycoplasmanegative cultures by monthly mycoplasma tests. sgRNA-mediated knockout cell generation For NR4A1 stable knockout using CRISPR single guide RNAs (sgRNAs), sgRNAs were first cloned into LentiCRISPR-V2 vector (Addgene, cat. no. 52961) using BsmBI sites.

Techniques: Binding Assay, Genome Wide, Knock-Out, Control, ChIP-sequencing, Quantitative RT-PCR, Western Blot, Clone Assay, Luciferase, Plasmid Preparation, ChIP-qPCR, Construct, Transfection, Activity Assay, Reporter Assay

Journal: Experimental & molecular medicine

Article Title: NR4A1 suppresses breast cancer growth by repressing c-Fos-mediated lipid and redox dyshomeostasis.

doi: 10.1038/s12276-025-01430-3

Figure Lengend Snippet: Fig. 7 NR4A1 agonist represses the transcriptional activity of c-Fos. a Immunoblotting analysis of NR4A1 in MCF7 cells treated by different concentrations of Csn-B or vehicle. b Cell proliferation assay was performed by CCK-8 assay in MCF7 or T47D cells treated by different concentrations of Csn-B or vehicle. c Comparison of cell growth between Csn-B (10 μM) and vehicle treatment in NR4A1-knockout MCF7 cells or T47D cells. d–f, Csn-B inhibited BC growth in xenografts. Female athymic nude mice bearing wild-type MCF7 tumor were treated daily with vehicle or Csn-B. Tumor images (d), growth curves (e) and tumor weight (f) were obtained at the end of treatment. n = 10 per group. g Representative IHC staining of Ki67 and the statistical analysis of Ki67-positive percentages in tumors d. Scale bar, 100 μm. h Co-IP experiments to detect interactions between NR4A1 and c-Fos in MCF7 cells treated by 10 μM Csn-B or vehicle for 24 h. i ChIP assays of the c-Fos enrichment on PRDX6 promoter in MCF7 cells treated by 10 μM Csn-B or vehicle for 24 h. j, k RT-qPCR (j) and immunoblotting analysis (k) of PRDX6 in MCF7 or T47D cells treated by 10 μM Csn-B or vehicle for 24 h. l Comparison of PRDX6 mRNA levels in NR4A1-knockout and parental MCF7 cells treated by 10 μM Csn-B or vehicle for 24 h. m Cell proliferation assay was performed by CCK-8 assay in ectopic c-Fos- or EV- overexpressed MCF7 cells treated by 10 μM Csn-B or vehicle. Unpaired Student’s t-tests were used in f, g, i, j and l, and one-way ANOVA was used in b, c, e and m, *P < 0.05, **P < 0.01, ***P < 0.001, NS nonsignificant.

Article Snippet: All cell lines were obtained from the American Type Culture Collection, cultured at 37 °C in a humidified atmosphere with 5% CO2 and ensured to be mycoplasmanegative cultures by monthly mycoplasma tests. sgRNA-mediated knockout cell generation For NR4A1 stable knockout using CRISPR single guide RNAs (sgRNAs), sgRNAs were first cloned into LentiCRISPR-V2 vector (Addgene, cat. no. 52961) using BsmBI sites.

Techniques: Activity Assay, Western Blot, Proliferation Assay, CCK-8 Assay, Comparison, Knock-Out, Immunohistochemistry, Co-Immunoprecipitation Assay, Quantitative RT-PCR

Journal: Experimental & molecular medicine

Article Title: NR4A1 suppresses breast cancer growth by repressing c-Fos-mediated lipid and redox dyshomeostasis.

doi: 10.1038/s12276-025-01430-3

Figure Lengend Snippet: Fig. 8 The impact of NR4A1–c-Fos–PRDX6 axis on BC prognosis. a Representative IHC staining of NR4A1, c-Fos and PRDX6 in cohort 2 TMA. Scale bars, 200 μm. b Pearson correlation analysis between NR4A1, c-Fos and PRDX6 protein expression based on IRS in cohort 2 TMA. R represents the Pearson correlation coefficient. c Kaplan–Meier survival plots of patients with BC in cohort 2 TMA based on c-Fos, or PRDX6 expression. d Kaplan–Meier survival plots of patients with BC in cohort 2 TMA based on the protein levels of NR4A1 co-expressed with PRDX6, or c-Fos co-expressed with PRDX6. e Kaplan–Meier survival plots of patients with BC in cohort 2 TMA based on the protein levels of NR4A1 co- expressed with c-Fos, or NR4A1 co-expressed with c-Fos and PRDX6. Log-rank tests were used in c–e. f Schematic diagram of the proposed mechanism.

Article Snippet: All cell lines were obtained from the American Type Culture Collection, cultured at 37 °C in a humidified atmosphere with 5% CO2 and ensured to be mycoplasmanegative cultures by monthly mycoplasma tests. sgRNA-mediated knockout cell generation For NR4A1 stable knockout using CRISPR single guide RNAs (sgRNAs), sgRNAs were first cloned into LentiCRISPR-V2 vector (Addgene, cat. no. 52961) using BsmBI sites.

Techniques: Immunohistochemistry, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Expression of Prooncogenic Nuclear Receptor 4A (NR4A)-Regulated Genes β1-Integrin and G9a Inhibited by Dual NR4A1/2 Ligands

doi: 10.3390/ijms26083909

Figure Lengend Snippet: DIM-3,5 analogs inhibit cancer cell growth and modulate expression of NR4A1-regulated gene products. RKO ( A ), SW480 ( B ) and HCT116 ( C ) cells were treated with 2.5–20 µmol/L DIM-3,5-CI 2 , DIM-3-CI-5-CF 3 and DIM-3,5-Br 2 for 24 h and effects on cell proliferation were determined as outlined in . RKO and SW480 cells were treated with DIM-3,5 analogs for 24 h and whole-cell lysates were analyzed by Western blots (in triplicate), G9a, β1-integrin and apoptosis ( D , E ) and Sp/NR4A gene products ( F , G ). Results are expressed as means ± SE for at least 3 determinations for each treatment group and significant ( p < 0.05) inhibition/induction compared to DMSO (control) is indicated (*) ( A – C ); quantitative results for ( D – G ) are summarized in ( H , I ) within the figure.

Article Snippet: The following antibodies were used for ChIP assays: NR4A1 (sc-365113X), NR4A2 (sc-376984X), Sp1 (sc-17824X), Sp4 (sc-390124X) and IgG (sc-515946), purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Western Blot, Inhibition, Control

Journal: International Journal of Molecular Sciences

Article Title: Expression of Prooncogenic Nuclear Receptor 4A (NR4A)-Regulated Genes β1-Integrin and G9a Inhibited by Dual NR4A1/2 Ligands

doi: 10.3390/ijms26083909

Figure Lengend Snippet: NR4A1 and NR4A2 knockdown on colon cancer cell growth and expression of selected genes. RKO, SW480 and HCT116 cells were transfected with oligonucleotides targeting NR4A1 (siNR4A1) and NR4A2 (siNR4A2) ( A – C ) and cell viability was determined. Multiple oligonucleotides targeting NR4A1 and NR4A2 and their combination were transfected into RKO or SW480 cells and whole-cell lysates were analyzed by Western blots knockdown to determine efficiency ( D ) and effects on selected gene products ( E ) as outlined in . Significant ( p < 0.05) inhibition is indicated (*) in ( A – C ).

Article Snippet: The following antibodies were used for ChIP assays: NR4A1 (sc-365113X), NR4A2 (sc-376984X), Sp1 (sc-17824X), Sp4 (sc-390124X) and IgG (sc-515946), purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Knockdown, Expressing, Transfection, Western Blot, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Expression of Prooncogenic Nuclear Receptor 4A (NR4A)-Regulated Genes β1-Integrin and G9a Inhibited by Dual NR4A1/2 Ligands

doi: 10.3390/ijms26083909

Figure Lengend Snippet: Effects of DIM-3,5 analogs on receptor-dependent transactivation. RKO ( A ), SW480 ( B ) and HCT116 ( C ) cells were transfected with GAL4-NR4A1 or GAL4-NR4A2 and a UAS-luc reporter gene and treated with DIM-3,5 analogs and luciferase activity was determined as outlined in . Results are expressed as means ± SE for at least 3 replicate determinations per treatment group, and significant ( p < 0.05) induction/inhibition is indicated (*).

Article Snippet: The following antibodies were used for ChIP assays: NR4A1 (sc-365113X), NR4A2 (sc-376984X), Sp1 (sc-17824X), Sp4 (sc-390124X) and IgG (sc-515946), purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Transfection, Luciferase, Activity Assay, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Expression of Prooncogenic Nuclear Receptor 4A (NR4A)-Regulated Genes β1-Integrin and G9a Inhibited by Dual NR4A1/2 Ligands

doi: 10.3390/ijms26083909

Figure Lengend Snippet: NR4A1 and NR4A2 knockdown and DIM-3,5 analogs inhibit invasion and induce TUNEL staining. SW480 cells were transfected with siNR4A1 and siNR4A2 or treated with DIM-3,5 analogs, and migration ( A ) and TUNEL ( B ) staining were determined as outlined in the Results are expressed as means ± SE for at least 3 determinations per treatment group, and significant ( p < 0.05) differences from the control group are indicated (*).

Article Snippet: The following antibodies were used for ChIP assays: NR4A1 (sc-365113X), NR4A2 (sc-376984X), Sp1 (sc-17824X), Sp4 (sc-390124X) and IgG (sc-515946), purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Knockdown, TUNEL Assay, Staining, Transfection, Migration, Control

Journal: International Journal of Molecular Sciences

Article Title: Expression of Prooncogenic Nuclear Receptor 4A (NR4A)-Regulated Genes β1-Integrin and G9a Inhibited by Dual NR4A1/2 Ligands

doi: 10.3390/ijms26083909

Figure Lengend Snippet: Effects of Sp1/Sp4 downregulation and the ChIP assay on the G9a and β1-integrin promoters in ( A ). ( A ) RKO and SW480 cells were transfected with oligonucleotides targeting Sp1 (siSp1) or Sp4 (siSp4) and whole-cell lysates were analyzed by Western blots as outlined in . RKO cells were treated with DIM-3,5-CI 2 for 24 h and a ChIP assay was determined using primers for the β1-integrin ( B , C ) and G9a ( D , E ) promoters and interactions of NR4A1, NR4A2, Sp1 and Sp4 with the gene promoters was determined as outlined in the . Results ( B , D ) are means ± SE from 3 replicate experiments, and significantly ( p < 0.05) decreased mRNA levels are indicated (*).

Article Snippet: The following antibodies were used for ChIP assays: NR4A1 (sc-365113X), NR4A2 (sc-376984X), Sp1 (sc-17824X), Sp4 (sc-390124X) and IgG (sc-515946), purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Transfection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Expression of Prooncogenic Nuclear Receptor 4A (NR4A)-Regulated Genes β1-Integrin and G9a Inhibited by Dual NR4A1/2 Ligands

doi: 10.3390/ijms26083909

Figure Lengend Snippet: Results of scRNAseq analysis. Effects of knockdown ( A ) and DIM-3,5-CI 2 treatment ( B ) on various mRNA levels by RT-PCR. ( C ) UMAP plots of the transcriptional landscape across individual cells. ( D , E ) Comparison of SW480 single-cell expression profiles, e.g., NR4A1, NR4A2, c-parp (PARP1), β1-integrin (ITGB1), G9a (EHMT2), Sp1 and Sp4 in control, Ctrl (DMSO) versus the DIM-3,5-CI 2 treatment group. The small decrease in β1-integrin mRNA levels may be due, in part, to the single 24 h timepoint, since the decrease in gene expression varies over time. Significant ( p < 0.05) effects are indicated (*) from triplicate determinations.

Article Snippet: The following antibodies were used for ChIP assays: NR4A1 (sc-365113X), NR4A2 (sc-376984X), Sp1 (sc-17824X), Sp4 (sc-390124X) and IgG (sc-515946), purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Comparison, Expressing, Control, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Expression of Prooncogenic Nuclear Receptor 4A (NR4A)-Regulated Genes β1-Integrin and G9a Inhibited by Dual NR4A1/2 Ligands

doi: 10.3390/ijms26083909

Figure Lengend Snippet: DE genes and pathways and their overlap. The overlap of DE genes identified when comparing the SW480 control versus transfected siRNAs individually targeting NR4A1 or NR4A2, versus treatment with DIM-3,5-CI 2 ( A ), and combined NR4A1 and NR4A2 siRNAs ( B ). The top 500 up- or downregulated DE genes from each pair comparison are included in the Venn diagrams. ( C ) Consensus gene ontology terms and pathways enriched in DE genes. DE analysis was conducted between control, transfected or treated groups, i.e., control versus DIM-3,5-CI 2 , control versus siNR4A1, control versus siNR4A2 and control versus siNR4A1 and siNR4A2 (combined). DE genes identified between each of these pairs were subjected to functional enrichment analysis. Consensus gene ontology terms and pathways are those commonly identified.

Article Snippet: The following antibodies were used for ChIP assays: NR4A1 (sc-365113X), NR4A2 (sc-376984X), Sp1 (sc-17824X), Sp4 (sc-390124X) and IgG (sc-515946), purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Control, Transfection, Comparison, Functional Assay

Journal: Biological Research

Article Title: Daphnetin alleviates unexplained recurrent spontaneous abortion by regulating the NR4A1/BACH2 axis in mice

doi: 10.1186/s40659-025-00658-7

Figure Lengend Snippet: Daphnetin upregulates the protein expression of NR4A1. A Chemical structure of daphnetin from PubChem Substance. B The differentially expressed genes between decidual tissues from URSA patients and controls with induced abortions in the GSE113790 dataset. C Intersection of differentially expressed genes in the GSE113790 dataset and daphnetin downstream targets. D Detection of NR4A1 expression in mouse decidual tissue by RT-qPCR. E Positive cells of NR4A1 in mouse decidual tissue were detected by immunohistochemistry. F The molecular interaction between daphnetin and NR4A1 protein. G The residual intracellular NR4A1 protein expression in CD4 + T cells with 20 μM daphnetin and 10 μg/mL CHX for varying durations was measured using Western blot analysis. H NR4A1 expression in the nuclei and cytoplasm of CD4 + T cells treated with 20 μM daphnetin for 48 h was measured using Western blot analysis. Data is presented as mean ± SEM (n = 10 or 3). One-way ANOVA for DE, and two-way ANOVA for G

Article Snippet: Antibodies against NR4A1 (1/500, NB100-56745SS, Novus Biological Inc., Littleton, CO, USA) were used to precipitate the NR4A1-bound chromatin, using rabbit IgG as a control.

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Western Blot

Journal: Biological Research

Article Title: Daphnetin alleviates unexplained recurrent spontaneous abortion by regulating the NR4A1/BACH2 axis in mice

doi: 10.1186/s40659-025-00658-7

Figure Lengend Snippet: Knockdown of NR4A1 abates the mitigating effects of daphnetin on URSA in mice. A Knockdown efficiency of NR4A1 in the decidual tissue of URSA mice detected by RT-qPCR. B Embryo resorption rate in URSA mice after knockdown of NR4A1. C Morphologic changes (the black arrow indicates the site of hemorrhage, while the green arrow indicates the area of necrosis) in decidual tissues of URSA mice after the knockdown of NR4A1 were assessed by HE staining. D The protein expression of NR4A1, IGFBP-1, and PRL in decidual tissues of mice after the knockdown of NR4A1 was detected by western blot analysis. E Treg and Th17 cell populations in the spleens of mice were analyzed using flow cytometry. F The number of Tregs (CD25 + FoxP3 + ) and Th17 cells (CD4 + IL-17A + ) infiltrating the decidual tissues of mice was assessed by immunofluorescence staining. Data is presented as mean ± SEM (n = 10). Unpaired t-test for ABDEF

Article Snippet: Antibodies against NR4A1 (1/500, NB100-56745SS, Novus Biological Inc., Littleton, CO, USA) were used to precipitate the NR4A1-bound chromatin, using rabbit IgG as a control.

Techniques: Knockdown, Quantitative RT-PCR, Staining, Expressing, Western Blot, Flow Cytometry, Immunofluorescence

Journal: Biological Research

Article Title: Daphnetin alleviates unexplained recurrent spontaneous abortion by regulating the NR4A1/BACH2 axis in mice

doi: 10.1186/s40659-025-00658-7

Figure Lengend Snippet: NR4A1 induces the transcription of BACH2 in CD4 + T cells. A The intersection of the first 500 downstream targets of NR4A1 downloaded from hTFtarget with differentially expressed genes in the GSE113790 dataset. B Binding sites for NR4A1 present in the promoter region of BACH2 (chr6: 90296739–90297088) were analyzed by JASPAR. C BACH2 was identified as a downstream target of NR4A1 in the hTFtarget database. D Detection of NR4A1 and BACH2 mRNA expression in CD4 + T cells by RT-qPCR. E Detection of NR4A1 and BACH2 protein expression in CD4 + T cells by western blot analysis. Expression of NR4A1 and BACH2 in CD4 + T cells after knockdown of NR4A1 was detected by RT-qPCR F and western blot G . H Enrichment of the BACH2 promoter immunoprecipitated by anti-NR4A1 in CD4 + T cells was analyzed using ChIP-qPCR. I Luciferase activity of the BACH2 promoter luciferase reporter plasmid in CD4 + T cells following NR4A1 knockdown was detected by dual-luciferase assays. Data is presented as mean ± SEM of three independent experiments. Unpaired t-test for FGHI, and one-way ANOVA for DE

Article Snippet: Antibodies against NR4A1 (1/500, NB100-56745SS, Novus Biological Inc., Littleton, CO, USA) were used to precipitate the NR4A1-bound chromatin, using rabbit IgG as a control.

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Knockdown, Immunoprecipitation, ChIP-qPCR, Luciferase, Activity Assay, Plasmid Preparation

Journal: Biological Research

Article Title: Daphnetin alleviates unexplained recurrent spontaneous abortion by regulating the NR4A1/BACH2 axis in mice

doi: 10.1186/s40659-025-00658-7

Figure Lengend Snippet: Overexpression of BACH2 is beneficial in maintaining Th17/Treg cell homeostasis in CD4 + T cells in the presence of sh-NR4A1. A Overexpression efficiency of BACH2 in CD4 + T cells detected by RT-qPCR. B Percentage of Treg and Th17 cells in CD4 + T cells analyzed by flow cytometry. C Detection of mRNA expression of FoxP3 and RORγt in CD4 + T cells by RT-qPCR. D Concentrations of Treg cell-associated cytokines (TGF-β, IL-10) and Th17 cell-associated cytokines (IL-17, IL-23) in the supernatant of CD4 + T cells were determined by ELISA. E The levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β in CD4 + T cells were measured by ELISA. Data is presented as mean ± SEM of three independent experiments. Unpaired t-test for A, one-way ANOVA for BCDE

Article Snippet: Antibodies against NR4A1 (1/500, NB100-56745SS, Novus Biological Inc., Littleton, CO, USA) were used to precipitate the NR4A1-bound chromatin, using rabbit IgG as a control.

Techniques: Over Expression, Quantitative RT-PCR, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Biological Research

Article Title: Daphnetin alleviates unexplained recurrent spontaneous abortion by regulating the NR4A1/BACH2 axis in mice

doi: 10.1186/s40659-025-00658-7

Figure Lengend Snippet: Overexpression of BACH2 overturns the accentuating effects of sh-NR4A1 on URSA in mice. A Overexpression efficiency of BACH2 in the decidual tissue of URSA mice detected by immunohistochemistry. B Embryo resorption rate in URSA mice after knockdown of NR4A1 and overexpression of BACH2. C Morphologic changes (the black arrow indicates the site of hemorrhage, while the green arrow indicates the area of necrosis) in decidual tissues of URSA mice after the knockdown of NR4A1 and overexpression of BACH2 were assessed by HE staining. D The protein expression of IGFBP-1 and PRL in decidual tissues of mice after the knockdown of NR4A1 and overexpression of BACH2 was detected by western blot analysis. E Treg and Th17 cell populations in the spleens of mice were analyzed using flow cytometry. F The number of Tregs (CD25 + FoxP3 +) and Th17 cells (CD4 + IL-17A + ) infiltrating the decidual tissues of mice was assessed by immunofluorescence staining. G mRNA expression of FoxP3 and RORγt in decidual tissue of URSA mice by RT-qPCR. Data is presented as mean ± SEM (n = 10). Unpaired t-test for ABDEF, and one-way ANOVA for G

Article Snippet: Antibodies against NR4A1 (1/500, NB100-56745SS, Novus Biological Inc., Littleton, CO, USA) were used to precipitate the NR4A1-bound chromatin, using rabbit IgG as a control.

Techniques: Over Expression, Immunohistochemistry, Knockdown, Staining, Expressing, Western Blot, Flow Cytometry, Immunofluorescence, Quantitative RT-PCR

Journal: Biological Research

Article Title: Daphnetin alleviates unexplained recurrent spontaneous abortion by regulating the NR4A1/BACH2 axis in mice

doi: 10.1186/s40659-025-00658-7

Figure Lengend Snippet: The protective effect of daphnetin on URSA and the underlying mechanisms. CD4 + T cells in URSA are polarized to Th17 differentiation, while Treg differentiation is reduced. Daphnetin ameliorated the imbalance of the Treg/Th17 ratio by promoting NR4A1-mediated BACH2 transcriptional expression, thereby inhibiting URSA progression

Article Snippet: Antibodies against NR4A1 (1/500, NB100-56745SS, Novus Biological Inc., Littleton, CO, USA) were used to precipitate the NR4A1-bound chromatin, using rabbit IgG as a control.

Techniques: Expressing