primary antibody against nr4a1  (Proteintech)

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: Molecular Medicine Reports

Article Title: Orphan nuclear receptor NR4A1 regulates both osteoblastogenesis and adipogenesis in human mesenchymal stem cells

doi: 10.3892/mmr.2024.13368

Figure Lengend Snippet: Effects of Nr4a1 or NR4A1 knockdown or overexpression in MC3T3-E1 cells and human BMD-MSCs. (A) Experimental design diagram. (B) Reverse transcription-quantitative PCR of Nr4a1 ( NR4A1 ) and (G) mRNA expression in MC3T3-E1 cells and BMD-MSCs. (C and H) Western blot analysis of NR4A1 expression in MC3T3-E1 cells and BMD-MSCs. (D) ALP staining performed on day 5 and (I) Alizarin red S staining performed on day 18 of culture. (E and J) ALP activity measured at 405 nm using alkaline phosphatase yellow liquid substrate system. (F and K) Alizarin red S-stained cells were extracted using cetylpyridinium chloride, and the mineralization level was quantified by measuring absorbance at 562 nm in control, siNr4a1 (siNR4A1) -treated, and Nr4a1 (NR4A1) -overexpressing cells. Data are presented as the mean ± standard errors of the mean of three biological replicates. *P<0.05, **P<0.005 vs. control. NR4A1, nuclear receptor subfamily 4 group A member 1; BMD-MSCs, bone marrow-derived mesenchymal stem cells; ALP, alkaline phosphatase; Ctrl-, Control; NC, negative control; si, small interfering; siNr4a1, siNr4a1 -treated MC3T3-E1 cells; siNR4A1, siNR4A1 -treated BMD-MSCs; Nr4a1, Nr4a1 -overexpressing MC3T3-E1 cells; NR4A1 NR4A1 -overexpressing BMD-MSCs.

Article Snippet: The membranes were blocked in 5% skim milk for 1 h at 20–22°C, then incubated overnight at 4°C with primary antibodies against NR4A1 (cat. no. MA5-32647, 1:500; Thermo Fisher Scientific, Inc.) or β-Actin (cat. no. A300-491A, 1:10,000; Bethyl Laboratories, Inc.).

Techniques: Knockdown, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Staining, Activity Assay, Control, Derivative Assay, Negative Control

Journal: Molecular Medicine Reports

Article Title: Orphan nuclear receptor NR4A1 regulates both osteoblastogenesis and adipogenesis in human mesenchymal stem cells

doi: 10.3892/mmr.2024.13368

Figure Lengend Snippet: Nr4a1 ( NR4A1 )-overexpression increases adipogenesis in 3T3-L1 cells and BMD-MSCs. (A) Experimental design diagram. (B) Reverse transcription-quantitative PCR of Nr4a1 mRNA expression in 3T3-L1 cells. (C) Western blot analysis of NR4A1 expression in 3T3-L1 cells. (D) Adipocyte differentiation and (F) quantification of Oil Red O staining in control, siNr4a1 (siNR4A1) -treated and Nr4a1 (NR4A1) -overexpressing 3T3-L1 cells. (E) Adipocyte differentiation and (G) quantification of Oil red O staining in control, siNr4a1 (siNR4A1) -treated, and Nr4a1 (NR4A1) -overexpressing BMD-MSCs. Data are presented as the mean ± standard errors of the mean of three biological replicates. *P<0.05, **P<0.005 vs. control. NR4A1, nuclear receptor subfamily 4 group A member 1; BMD-MSCs, bone marrow-derived mesenchymal stem cells; Ctrl-, Control; NC, negative control; si, small interfering; NR4A1 NR4A1 -overexpressing BMD-MSCs.

Article Snippet: The membranes were blocked in 5% skim milk for 1 h at 20–22°C, then incubated overnight at 4°C with primary antibodies against NR4A1 (cat. no. MA5-32647, 1:500; Thermo Fisher Scientific, Inc.) or β-Actin (cat. no. A300-491A, 1:10,000; Bethyl Laboratories, Inc.).

Techniques: Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Staining, Control, Derivative Assay, Negative Control

Journal: Molecular Medicine Reports

Article Title: Orphan nuclear receptor NR4A1 regulates both osteoblastogenesis and adipogenesis in human mesenchymal stem cells

doi: 10.3892/mmr.2024.13368

Figure Lengend Snippet: DIM-C-pPhOH and NR4A1 knockdown demonstrate similar effects on adipogenesis in BMD-MSCs. (A) Experimental design diagram. (B) ALP staining and Alizarin red S staining were performed on days 7 and 18 of culture, respectively. (C) ALP activity measured at 405 nm using alkaline phosphatase yellow liquid substrate system. (D) Alizarin red S-stained cells were extracted using cetylpyridinium chloride, and the mineralization level was quantified by measuring absorbance at 562 nm in control, DIM-C-pPhOH-treated, NR4A1 -overexpressing, and NR4A1 -overexpressing/DIM-C-pPhOH-treated BMD-MSCs. (E) Adipocyte differentiation and (F) quantification of Oil red O staining in human BMD-MSCs. *P<0.05, **P<0.005. DIM-C-pPhOH, 1,1-bis(3′-indolyl)-1-(p-hydroxyphenyl) methane; NR4A1, nuclear receptor subfamily 4 group A member 1; BMD-MSCs, bone marrow-derived mesenchymal stem cells; ALP, alkaline phosphatase; Ctrl-, Control; NC, negative control; si, small interfering; NR4A1 NR4A1 -overexpressing BMD-MSCs.

Article Snippet: The membranes were blocked in 5% skim milk for 1 h at 20–22°C, then incubated overnight at 4°C with primary antibodies against NR4A1 (cat. no. MA5-32647, 1:500; Thermo Fisher Scientific, Inc.) or β-Actin (cat. no. A300-491A, 1:10,000; Bethyl Laboratories, Inc.).

Techniques: Knockdown, Staining, Activity Assay, Control, Derivative Assay, Negative Control

Journal: Molecular Medicine Reports

Article Title: Orphan nuclear receptor NR4A1 regulates both osteoblastogenesis and adipogenesis in human mesenchymal stem cells

doi: 10.3892/mmr.2024.13368

Figure Lengend Snippet: Notch signaling mediates the effects of NR4A1 on osteoblastogenesis and adipogenesis in BMD-MSCs. (A) Experimental design diagram. (B) IPA revealed that the expression of the gene encoding the Notch signaling pathway component MAML3 was decreased, whereas those of JAG1, DTX4, NOTCH3, HES1 and PSEN2 were increased in siNR4A1 -treated cells. RT-qPCR data of the representative genes showing altered expression in IPA. Similar changes were observed in MAML3 mRNA expression. Data are presented as the mean ± standard errors of the mean of three biological replicates. *P<0.05, **P<0.005. NR4A1, nuclear receptor subfamily 4 group A member 1; BMD-MSCs, bone marrow-derived mesenchymal stem cells; IPA, ingenuity pathway analysis; MAML3, Mastermind-like transcriptional coactivator 3; RT-qPCR, reverse transcription-quantitative PCR; WB, western blot analysis; Ctrl-, Control; si, small interfering; siNr4a1, siNr4a1 -treated MC3T3-E1 cells; siNR4A1, siNR4A1 -treated BMD-MSCs; Nr4a1, Nr4a1 -overexpressing MC3T3-E1 cells.

Article Snippet: The membranes were blocked in 5% skim milk for 1 h at 20–22°C, then incubated overnight at 4°C with primary antibodies against NR4A1 (cat. no. MA5-32647, 1:500; Thermo Fisher Scientific, Inc.) or β-Actin (cat. no. A300-491A, 1:10,000; Bethyl Laboratories, Inc.).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Molecular Medicine Reports

Article Title: Orphan nuclear receptor NR4A1 regulates both osteoblastogenesis and adipogenesis in human mesenchymal stem cells

doi: 10.3892/mmr.2024.13368

Figure Lengend Snippet: Mechanisms by which NR4A1 regulates osteoporosis and adipogenesis via the Notch signaling pathway. The reduction in NR4A1 expression led to a decrease in CSL-MAML-dependent Notch1 signaling, consequently triggering the upregulation of the RUNX2 gene, either via direct or indirect pathways involving HES1. This cascade promotes osteoblastogenesis. Moreover, the reduction of HES1 may further inhibit adipogenesis. NR4A1, nuclear receptor subfamily 4 group A member 1; RUNX2, Runt-related transcription factor 2; HES1, Hes family bHLH transcription factor 1.

Article Snippet: The membranes were blocked in 5% skim milk for 1 h at 20–22°C, then incubated overnight at 4°C with primary antibodies against NR4A1 (cat. no. MA5-32647, 1:500; Thermo Fisher Scientific, Inc.) or β-Actin (cat. no. A300-491A, 1:10,000; Bethyl Laboratories, Inc.).

Techniques: Expressing

Journal: Bioengineered

Article Title: Nuclear receptor 4A1 (NR4A1) silencing protects hepatocyte against hypoxia-reperfusion injury in vitro by activating liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling

doi: 10.1080/21655979.2022.2053804

Figure Lengend Snippet: NR4A1 is significantly upregulated while proteins in LKB1/AMPK signaling is downregulated in H/R-treated BRL-3A cells. (a-b) Expression of NR4A1 in BRL-3A cells treated with H/R was detected using western blotting and RT-qPCR. (c) The expression or proteins in LKB1/AMPK signaling in BRL-3A cells treated with H/R were detected by western blotting. ***P < 0.001 vs. control.

Article Snippet: Prior to the overnight incubation with 1:1000 primary antibodies against NR4A1 (25,851-1-AP, Proteintech, Chicago, IL, USA), phospho (p)-LKB1 (3482S, Cell Signaling Technology, Boston, MA, USA), LKB1 (3047S, Cell Signaling Technology, Boston, MA, USA), p-AMPK (2535 T, Cell Signaling Technology, Boston, MA, USA), AMPK (5832 T, Cell Signaling Technology, Boston, MA, USA), p-acetyl-CoA carboxylase (p-ACC, ab68191, Abcam, Cambridge, UK), ACC (ab68191, Abcam, Cambridge, UK), B-cell lymphoma 2 (Bcl-2, ab196495, Abcam, Cambridge, UK), Bcl-2-associated X protein (Bax, 2772 T, Cell Signaling Technology, Boston, MA, USA), cleaved caspase-3 (9664 T, Cell Signaling Technology, Boston, MA, USA), caspase-3 (9662S, Cell Signaling Technology, Boston, MA, USA), Poly-ADP-Ribose polymerase (PARP, ab191217, Abcam, Cambridge, UK), cleaved PARP (94885S, Cell Signaling Technology, Boston, MA, USA), Beclin1 (3495 T, Cell Signaling Technology, Boston, MA, USA), autophagy-related protein 5 (ATG5, 12,994 T, Cell Signaling Technology, Boston, MA, USA), ATG7 (8558 T, Cell Signaling Technology, Boston, MA, USA), p62 (39749S, Cell Signaling Technology, Boston, MA, USA), and GAPDH (5174 T, Cell Signaling Technology, Boston, MA, USA) at 4°C, 5% nonfat milk was adopted to impede these membranes for 1 h. After that, secondary antibody conjugated to horseradish peroxidase-conjugated (HRP) were utilized to cultivate the membranes at room temperature for another 2 h. Immune bands were detected by enhanced chemiluminescence (ECL) chromogenic kit (Thermo Fisher Scientific, Inc.).

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

Journal: Bioengineered

Article Title: Nuclear receptor 4A1 (NR4A1) silencing protects hepatocyte against hypoxia-reperfusion injury in vitro by activating liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling

doi: 10.1080/21655979.2022.2053804

Figure Lengend Snippet: NR4A1 inhibits the activation of LKB1/AMPK pathway in BRL-3A cells treated with H/R. (a-b) siRNA NR4A1-mediated transfection efficiency was determined by western blotting and RT-qPCR. (c) Effect of NR4A1 knockdown on the phosphorylation levels of the LKB1/AMPK signaling in BRL-3A cells treated with H/R were detected by western blotting. ***P < 0.001 vs. control; ###P < 0.001 vs. H/R+ siRNA-NC. (d-e) Western blot and RT-qPCR were used for detecting the transfection efficiency of NR4A1 overexpressed plasmid. ***P < 0.001 vs. ov-NC. (f). Effect of NR4A1 overexpression on the phosphorylation levels of the LKB1/AMPK signaling pathway in BRL-3A cells treated with H/R were detected by western blotting. ***P < 0.001 vs. control; ### P < 0.001 vs. H/R+ Ov-NC.

Article Snippet: Prior to the overnight incubation with 1:1000 primary antibodies against NR4A1 (25,851-1-AP, Proteintech, Chicago, IL, USA), phospho (p)-LKB1 (3482S, Cell Signaling Technology, Boston, MA, USA), LKB1 (3047S, Cell Signaling Technology, Boston, MA, USA), p-AMPK (2535 T, Cell Signaling Technology, Boston, MA, USA), AMPK (5832 T, Cell Signaling Technology, Boston, MA, USA), p-acetyl-CoA carboxylase (p-ACC, ab68191, Abcam, Cambridge, UK), ACC (ab68191, Abcam, Cambridge, UK), B-cell lymphoma 2 (Bcl-2, ab196495, Abcam, Cambridge, UK), Bcl-2-associated X protein (Bax, 2772 T, Cell Signaling Technology, Boston, MA, USA), cleaved caspase-3 (9664 T, Cell Signaling Technology, Boston, MA, USA), caspase-3 (9662S, Cell Signaling Technology, Boston, MA, USA), Poly-ADP-Ribose polymerase (PARP, ab191217, Abcam, Cambridge, UK), cleaved PARP (94885S, Cell Signaling Technology, Boston, MA, USA), Beclin1 (3495 T, Cell Signaling Technology, Boston, MA, USA), autophagy-related protein 5 (ATG5, 12,994 T, Cell Signaling Technology, Boston, MA, USA), ATG7 (8558 T, Cell Signaling Technology, Boston, MA, USA), p62 (39749S, Cell Signaling Technology, Boston, MA, USA), and GAPDH (5174 T, Cell Signaling Technology, Boston, MA, USA) at 4°C, 5% nonfat milk was adopted to impede these membranes for 1 h. After that, secondary antibody conjugated to horseradish peroxidase-conjugated (HRP) were utilized to cultivate the membranes at room temperature for another 2 h. Immune bands were detected by enhanced chemiluminescence (ECL) chromogenic kit (Thermo Fisher Scientific, Inc.).

Techniques: Activation Assay, Transfection, Western Blot, Quantitative RT-PCR, Knockdown, Phospho-proteomics, Control, Plasmid Preparation, Over Expression

Journal: Bioengineered

Article Title: Nuclear receptor 4A1 (NR4A1) silencing protects hepatocyte against hypoxia-reperfusion injury in vitro by activating liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling

doi: 10.1080/21655979.2022.2053804

Figure Lengend Snippet: NR4A1-knockdown attenuates oxidative stress and inflammation of BRL-3A cells exposed to H/R through LKB1/AMPK pathway. (a) Cell viability was determined by CCK-8 assay. (b-c) Intracellular ROS level was examined by DCFH-DA staining. (d-e) Measurement of MDA and SOD by respective kits. (f-i) Expression levels of TNF-α, IL-1β, IL-6, and MCP-1 mRNA in BRL-3A cells treated with H/R was determined by RT-qPCR. ***P < 0.001 vs. control; ### P < 0.001 vs. H/R+ siRNA-NC; ΔΔ P<0.01, ΔΔΔ P<0.001 vs. H/R+ siRNA-NR4A1.

Article Snippet: Prior to the overnight incubation with 1:1000 primary antibodies against NR4A1 (25,851-1-AP, Proteintech, Chicago, IL, USA), phospho (p)-LKB1 (3482S, Cell Signaling Technology, Boston, MA, USA), LKB1 (3047S, Cell Signaling Technology, Boston, MA, USA), p-AMPK (2535 T, Cell Signaling Technology, Boston, MA, USA), AMPK (5832 T, Cell Signaling Technology, Boston, MA, USA), p-acetyl-CoA carboxylase (p-ACC, ab68191, Abcam, Cambridge, UK), ACC (ab68191, Abcam, Cambridge, UK), B-cell lymphoma 2 (Bcl-2, ab196495, Abcam, Cambridge, UK), Bcl-2-associated X protein (Bax, 2772 T, Cell Signaling Technology, Boston, MA, USA), cleaved caspase-3 (9664 T, Cell Signaling Technology, Boston, MA, USA), caspase-3 (9662S, Cell Signaling Technology, Boston, MA, USA), Poly-ADP-Ribose polymerase (PARP, ab191217, Abcam, Cambridge, UK), cleaved PARP (94885S, Cell Signaling Technology, Boston, MA, USA), Beclin1 (3495 T, Cell Signaling Technology, Boston, MA, USA), autophagy-related protein 5 (ATG5, 12,994 T, Cell Signaling Technology, Boston, MA, USA), ATG7 (8558 T, Cell Signaling Technology, Boston, MA, USA), p62 (39749S, Cell Signaling Technology, Boston, MA, USA), and GAPDH (5174 T, Cell Signaling Technology, Boston, MA, USA) at 4°C, 5% nonfat milk was adopted to impede these membranes for 1 h. After that, secondary antibody conjugated to horseradish peroxidase-conjugated (HRP) were utilized to cultivate the membranes at room temperature for another 2 h. Immune bands were detected by enhanced chemiluminescence (ECL) chromogenic kit (Thermo Fisher Scientific, Inc.).

Techniques: Knockdown, CCK-8 Assay, Staining, Expressing, Quantitative RT-PCR, Control

Journal: Bioengineered

Article Title: Nuclear receptor 4A1 (NR4A1) silencing protects hepatocyte against hypoxia-reperfusion injury in vitro by activating liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling

doi: 10.1080/21655979.2022.2053804

Figure Lengend Snippet: NR4A1-knockdown ameliorates BRL-3A cell apoptosis induced by H/R through LKB1/AMPK pathway. (a) Representative dot plots from flow cytometry analysis. (b) Cell apoptosis was determined by flow cytometry analysis. (c) Expression of apoptosis-related proteins (Bcl-2, Bax, caspase3, cleaved caspase3, PARP, and cleaved RAPR) were detected by western blotting. ***P < 0.001 vs. control; ### P < 0.001 vs. H/R+ siRNA-NC; Δ P<0.05, ΔΔ P<0.01, ΔΔΔ P<0.001 vs. H/R+ siRNA-NR4A1.

Article Snippet: Prior to the overnight incubation with 1:1000 primary antibodies against NR4A1 (25,851-1-AP, Proteintech, Chicago, IL, USA), phospho (p)-LKB1 (3482S, Cell Signaling Technology, Boston, MA, USA), LKB1 (3047S, Cell Signaling Technology, Boston, MA, USA), p-AMPK (2535 T, Cell Signaling Technology, Boston, MA, USA), AMPK (5832 T, Cell Signaling Technology, Boston, MA, USA), p-acetyl-CoA carboxylase (p-ACC, ab68191, Abcam, Cambridge, UK), ACC (ab68191, Abcam, Cambridge, UK), B-cell lymphoma 2 (Bcl-2, ab196495, Abcam, Cambridge, UK), Bcl-2-associated X protein (Bax, 2772 T, Cell Signaling Technology, Boston, MA, USA), cleaved caspase-3 (9664 T, Cell Signaling Technology, Boston, MA, USA), caspase-3 (9662S, Cell Signaling Technology, Boston, MA, USA), Poly-ADP-Ribose polymerase (PARP, ab191217, Abcam, Cambridge, UK), cleaved PARP (94885S, Cell Signaling Technology, Boston, MA, USA), Beclin1 (3495 T, Cell Signaling Technology, Boston, MA, USA), autophagy-related protein 5 (ATG5, 12,994 T, Cell Signaling Technology, Boston, MA, USA), ATG7 (8558 T, Cell Signaling Technology, Boston, MA, USA), p62 (39749S, Cell Signaling Technology, Boston, MA, USA), and GAPDH (5174 T, Cell Signaling Technology, Boston, MA, USA) at 4°C, 5% nonfat milk was adopted to impede these membranes for 1 h. After that, secondary antibody conjugated to horseradish peroxidase-conjugated (HRP) were utilized to cultivate the membranes at room temperature for another 2 h. Immune bands were detected by enhanced chemiluminescence (ECL) chromogenic kit (Thermo Fisher Scientific, Inc.).

Techniques: Knockdown, Flow Cytometry, Expressing, Western Blot, Control

Journal: Bioengineered

Article Title: Nuclear receptor 4A1 (NR4A1) silencing protects hepatocyte against hypoxia-reperfusion injury in vitro by activating liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling

doi: 10.1080/21655979.2022.2053804

Figure Lengend Snippet: NR4A1-knockdown promotes autophagy of BRL-3A cells treated with H/R through LKB1/AMPK pathway. Western blot was used to evaluate the expression of autophagy-related proteins. ***P < 0.001 vs. control; ### P < 0.001 vs. H/R+ siRNA-NC; ΔΔ P<0.01, ΔΔΔ P<0.001 vs. H/R+ siRNA-NR4A1.

Article Snippet: Prior to the overnight incubation with 1:1000 primary antibodies against NR4A1 (25,851-1-AP, Proteintech, Chicago, IL, USA), phospho (p)-LKB1 (3482S, Cell Signaling Technology, Boston, MA, USA), LKB1 (3047S, Cell Signaling Technology, Boston, MA, USA), p-AMPK (2535 T, Cell Signaling Technology, Boston, MA, USA), AMPK (5832 T, Cell Signaling Technology, Boston, MA, USA), p-acetyl-CoA carboxylase (p-ACC, ab68191, Abcam, Cambridge, UK), ACC (ab68191, Abcam, Cambridge, UK), B-cell lymphoma 2 (Bcl-2, ab196495, Abcam, Cambridge, UK), Bcl-2-associated X protein (Bax, 2772 T, Cell Signaling Technology, Boston, MA, USA), cleaved caspase-3 (9664 T, Cell Signaling Technology, Boston, MA, USA), caspase-3 (9662S, Cell Signaling Technology, Boston, MA, USA), Poly-ADP-Ribose polymerase (PARP, ab191217, Abcam, Cambridge, UK), cleaved PARP (94885S, Cell Signaling Technology, Boston, MA, USA), Beclin1 (3495 T, Cell Signaling Technology, Boston, MA, USA), autophagy-related protein 5 (ATG5, 12,994 T, Cell Signaling Technology, Boston, MA, USA), ATG7 (8558 T, Cell Signaling Technology, Boston, MA, USA), p62 (39749S, Cell Signaling Technology, Boston, MA, USA), and GAPDH (5174 T, Cell Signaling Technology, Boston, MA, USA) at 4°C, 5% nonfat milk was adopted to impede these membranes for 1 h. After that, secondary antibody conjugated to horseradish peroxidase-conjugated (HRP) were utilized to cultivate the membranes at room temperature for another 2 h. Immune bands were detected by enhanced chemiluminescence (ECL) chromogenic kit (Thermo Fisher Scientific, Inc.).

Techniques: Knockdown, Western Blot, Expressing, Control