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: 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: 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: 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: 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, Fluorescence, 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: 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 i n 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: 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: 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: 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: Knock-Out, Expressing, Flow Cytometry, Staining, Fluorescence

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: 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: 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: 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: 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: 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: Genome Wide, Knock-Out, Control, ChIP-sequencing, Quantitative RT-PCR, Western Blot, Binding Assay, 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: 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: 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, 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: 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: 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