Journal: Mechanisms of ageing and development

Article Title: Long-term caloric restriction ameliorates T cell immunosenescence in mice.

doi: 10.1016/j.mad.2022.111710

Figure Lengend Snippet: Fig. 7. Effects of caloric restriction on expression of T cell exhaustion-associated transcription factors in the spleen. Spleen cells were isolated from groups of mice fed and sacrificed according to the protocols described in Fig. 1. Cells were stained with antibodies against CD4, CD8, NR4A1, and TOX and the proportions of NR4A1+ (A) and TOX+ (B) T cells were analyzed by flow cytometry. Data are the means ± SE of n = 7 (Young, CR, and sCR) or n = 6 (CON) mice per group. * P < 0.05 vs CON by ANOVA followed by Dunnett’s test.

Article Snippet: For the detection of intracellular NR4A1 and TOX, splenocytes were incubated with FITC-anti-CD4 or APC-anti-CD8 mAbs, fixed and permeabilized as described above, and incubated with PE-antiNR4A1 (REA704; Miltenyi Biotec, San Diego, CA, USA) or PE-antiTOX (TXRX10, Invitrogen) mAbs.

Techniques: Expressing, Isolation, Staining, Flow Cytometry

Journal: Cell death discovery

Article Title: Nur77-IRF1 axis inhibits esophageal squamous cell carcinoma growth and improves anti-PD-1 treatment efficacy.

doi: 10.1038/s41420-024-02019-x

Figure Lengend Snippet: Fig. 1 Nur77 inhibited ESCC cell proliferation in vitro and tumorigenesis in vivo. A, B qRT-PCR and western blot analysis of Nur77 expression in NEEC and ESCC cell lines (n = 3). C Overexpression of Nur77 in Kyse520 and TE-1 cell lines was analyzed by western blot. GAPDH was used as a loading control (n = 3). D The viability of ESCC cells overexpressing Nur77 was inhibited, as determined by the CCK-8 assay (n = 3). E The colony formation ability of ESCC cells overexpressing Nur77 was reduced (n = 3). F The percentage of Kyse520 and TE-1 cells that underwent Nur77 overexpression was increased (n = 3). G Western blotting was performed to investigate the expression levels of Bcl-2, Bax, cleaved caspase-3, and cleaved PARP in Kyse520 and TE-1 cells overexpressing Nur77. GAPDH was used as a loading control (n = 3). Representative tumor images (H), tumor volumes (I) and tumor weights (J) were collected from nude mice with tumor xenografts derived from TE-1 cells stably overexpressing Nur77 (n = 5). Nur77 protein expression in tissue samples from the xenografts was detected via immunohistochemistry (K) and western blotting (L) (n = 3). M Expression of cleaved caspase-3, Ki67, and PCNA in tumor xenografts was determined via immunohistochemistry (n = 3). Scale bars: 20 μm. All in vitro experiments were performed with three independent experiments. An unpaired Student’s t tests were used for statistical analysis, and the error bars indicate the means ± S.D. **P < 0.01 indicates a significant difference from the vector group.

Article Snippet: Using protein A/G magnetic beads (Cat# 88802, Thermo Fisher Scientific) and antibodies against Nur77 (1:200, Cat# NB10056745, Novus) or control rabbit IgG (1:100, Cat# 2729, Cell Signaling Technology), the nuclear lysate was incubated at 4 °C overnight on a rotator.

Techniques: In Vitro, In Vivo, Quantitative RT-PCR, Western Blot, Expressing, Over Expression, Control, CCK-8 Assay, Derivative Assay, Stable Transfection, Immunohistochemistry, Plasmid Preparation

Journal: Cell death discovery

Article Title: Nur77-IRF1 axis inhibits esophageal squamous cell carcinoma growth and improves anti-PD-1 treatment efficacy.

doi: 10.1038/s41420-024-02019-x

Figure Lengend Snippet: Fig. 2 Silencing of Nur77 promoted tumor cell proliferation in vitro and in vivo. A Verification of Nur77 knockdown in Eca109 and Kyse510 cells by western blot analysis. GAPDH was used as a loading control (n = 3). Nur77 knockdown increased cell proliferation (B) and colony formation ability (C) (n = 3). D The percentage of Eca109 and Kyse510 cells undergoing apoptosis following Nur77 knockdown was decreased (n = 3). E Nur77 knockdown increased the expression of Bcl-2 and decreased the expression of Bax, cleaved caspase-3, and cleaved PARP. Cell lysates were assessed by western blotting (n = 3). F Images of tumor tissues collected from nude mice with stable Nur77 knockdown xenograft tumors derived from TE-1 cells (n = 5). Xenograft tumor growth was monitored (G) and weighed (H) (n = 5). Nur77 knockdown in tissue samples from the xenografts was evaluated via immunohistochemistry (I) and western blotting (J) (n = 3). K The expression of cleaved caspase-3, Ki67, and PCNA in tumor xenografts was determined by immunohistochemical analysis (n = 3). Scale bars: 20 μm. All in vitro experiments were performed with three independent experiments. An unpaired Student’s t-test was used for statistical analysis, and the error bars indicate the means ± S.D. **P < 0.01 indicates a significant difference from the shNC group.

Article Snippet: Using protein A/G magnetic beads (Cat# 88802, Thermo Fisher Scientific) and antibodies against Nur77 (1:200, Cat# NB10056745, Novus) or control rabbit IgG (1:100, Cat# 2729, Cell Signaling Technology), the nuclear lysate was incubated at 4 °C overnight on a rotator.

Techniques: In Vitro, In Vivo, Knockdown, Western Blot, Control, Expressing, Derivative Assay, Immunohistochemistry, Immunohistochemical staining

Journal: Cell death discovery

Article Title: Nur77-IRF1 axis inhibits esophageal squamous cell carcinoma growth and improves anti-PD-1 treatment efficacy.

doi: 10.1038/s41420-024-02019-x

Figure Lengend Snippet: Fig. 4 IRF1 knockdown suppressed ESCC cell proliferation, and upregulating IRF1 in ESCC cells overexpressing Nur77 rescued the suppressive effects of Nur77 in vitro. Confirmation of IRF1 knockdown in Kyse520 and TE-1 cells by western blot (A, B) and qRT‒PCR (C). GAPDH was used as a loading control (n = 3). IRF1 knockdown decreased cell proliferation (D) and colony formation ability (E) (n = 3). F The percentage of Kyse520 and TE-1 cells undergoing apoptosis following IRF1 knockdown was increased (n = 3). G Western blot analysis showed that IRF1 knockdown decreased the expression of Bcl-2 and increased the expression of Bax, cleaved caspase-3, and cleaved PARP (n = 3). H Western blot of IRF1 protein expression in Nur77-overexpressing ESCC cells overexpressing IRF1. GAPDH was used as a loading control (n = 3). The influence of IRF1 overexpression on the inhibitory effects of Nur77 on cell growth (I) and colony formation (J, K) in vitro was detected by CCK-8 and colony formation assays (n = 3). The data are shown as the mean ± S.D. from experiments with three replicates. An unpaired Student’s t test was used for statistical analysis. **P < 0.01 indicates a significant difference compared with the vehicle group.

Article Snippet: Using protein A/G magnetic beads (Cat# 88802, Thermo Fisher Scientific) and antibodies against Nur77 (1:200, Cat# NB10056745, Novus) or control rabbit IgG (1:100, Cat# 2729, Cell Signaling Technology), the nuclear lysate was incubated at 4 °C overnight on a rotator.

Techniques: Knockdown, In Vitro, Western Blot, Control, Expressing, Over Expression, CCK-8 Assay

Journal: Cell death discovery

Article Title: Nur77-IRF1 axis inhibits esophageal squamous cell carcinoma growth and improves anti-PD-1 treatment efficacy.

doi: 10.1038/s41420-024-02019-x

Figure Lengend Snippet: Fig. 7 Nur77 expression was negatively correlated with IRF1 expression in primary ESCC tissues. The mRNA expression of Nur77 (A) and IRF1 (B) in 72 paired ESCC and nontumor tissues was detected via qRT‒PCR. Nur77 (C) and IRF1 (D) expression levels in ESCC and nontumor tissues in the GSE38129, GSE45670, and GSE53625 datasets. E The correlation between Nur77 and IRF1 mRNA expression in 72 paired cancerous/noncancerous esophageal tissues from primary ESCC patients. The Pearson correlation coefficients (r) and p values are indicated. F The protein expression levels of Nur77, IRF1, and PD-L1 in eight paired ESCC and nontumor tissues were detected via western blotting (n = 3). G Representative images of IHC staining for the Nur77, IRF1, and PD-L1 proteins in cancerous and noncancerous esophageal tissues. Scale bars: 20 μm. H Kaplan‒Meier analysis of overall survival stratified by Nur77 and IRF1 expression in 72 ESCC patients. The log-rank test was used to determine statistical significance. The data are shown as the mean ± S.D. from experiments with three replicates. Paired Student’s t tests were used for statistical analysis. *P < 0.05, **P < 0.01 indicate significant differences from the normal group.

Article Snippet: Using protein A/G magnetic beads (Cat# 88802, Thermo Fisher Scientific) and antibodies against Nur77 (1:200, Cat# NB10056745, Novus) or control rabbit IgG (1:100, Cat# 2729, Cell Signaling Technology), the nuclear lysate was incubated at 4 °C overnight on a rotator.

Techniques: Expressing, Western Blot, Immunohistochemistry

Journal: Theranostics

Article Title: Single-cell RNA-Seq analysis of molecular changes during radiation-induced skin injury: the involvement of Nur77

doi: 10.7150/thno.100417

Figure Lengend Snippet: Changes in the transcriptional profiles of skin fibroblast during radiation-induced skin injury. (A) Bubble plot of TFs alterations in rat and human skin cells with or without radiation. (B) Venn plots showing the number of shared upregulated (upper) and downregulated (lower) DEGs between different groups of human and rat skin samples. Violin plots showing the expression levels of 5 common DEGs in human and rat skin. (C) The t-SNE plot displays rat (left) and human (right) fibroblast. Bar plots showing the cell number of each cell subtypes contributed. (D) Violin plot showing Nur77 gene expression changes across different fibroblast subcluster in rat (left) and human (right). (E) Pseudotime ordering on rat fibroblasts and human fibroblasts (F) arranged them into a major trajectory, with two minor bifurcations. Each dot represents a single cell. The black arrow indicates the start and direction of the trajectory. Feature plots of expression distribution for Nur77 across pseudotime. (G) Heatmap showing the top 5 markers for fibroblast subcluster from the rat (left) and human (right) skin.

Article Snippet: Cells were blocked with blocking buffer (phosphate buffered saline, 1% Triton X-100, and 5% BSA) and incubated at 4 °C with Nur77 (#NBP2-66980, Novus) antibody (1: 200) overnight, FITC-conjugated goat anti-mouse antibody (1:300) was added for 2 h at room temperature, and cells were dyed with 200 nM MitoTracker Red CMXRos (#C1035, Beyotime) at 37 °C for 30 min in darkness.

Techniques: Expressing, Gene Expression

Journal: Theranostics

Article Title: Single-cell RNA-Seq analysis of molecular changes during radiation-induced skin injury: the involvement of Nur77

doi: 10.7150/thno.100417

Figure Lengend Snippet: Nur77 is involved in the irradiation process of skin cells. (A) qRT-PCR analysis of Nur77 mRNA expression in response to radiation in WS1 and HaCaT cells. (B) Western blotting analysis showing Nur77 expression in WS1 and HaCaT cells after different time post irradiation or different dose of irradiation. (C) and (D) Detection of radiation-induced nuclear-cytoplasmic distribution of Nur77 determined by preforming immunofluorescence analysis separating nuclear and cytoplasmic fraction and separating nuclear and cytoplasmic fractions. (E) Expression of Nur77 in normal and irradiated human skin tissues. (F) Western blot analysis showing the distribution of Nur77 in different organs and changes over time from 3 to 6 days after irradiation. (G) The effect of C-DIM8 on ROS production after different dose of irradiation as determined by DCFH-DA staining in WS1 and HaCaT cells. (H) The effect of C-DIM8 on cell apoptosis determined by AV/PI staining in WS1 and HaCaT cells. (I) The effect of Nur77 inhibitor C-DIM8 on radiosensitivity as determined by a colony formation assay following different doses of radiation. (J) Western blotting analysis showing cell death-related biomarker expression in irradiated WS1 cells treated with C-DIM8. * P < 0.05 and ** P < 0.01, compared with the control group. Scale bar = 200 μm.

Article Snippet: Cells were blocked with blocking buffer (phosphate buffered saline, 1% Triton X-100, and 5% BSA) and incubated at 4 °C with Nur77 (#NBP2-66980, Novus) antibody (1: 200) overnight, FITC-conjugated goat anti-mouse antibody (1:300) was added for 2 h at room temperature, and cells were dyed with 200 nM MitoTracker Red CMXRos (#C1035, Beyotime) at 37 °C for 30 min in darkness.

Techniques: Irradiation, Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining, Colony Assay, Biomarker Discovery, Control

Journal: Theranostics

Article Title: Single-cell RNA-Seq analysis of molecular changes during radiation-induced skin injury: the involvement of Nur77

doi: 10.7150/thno.100417

Figure Lengend Snippet: Loss of Nur77 aggravates radiation-induced skin injury in mouse models. (A) The phenotypes and genotypes of wild-type ( Nur77 +/+ ) and Nur77 knockout ( Nur77 -/- ) mice. (B) Schematic of the workflow showing the establishment of the three mouse models with radiation-induced skin injury. 1) Acute radiation-induced skin injury; 2) Radiation fractionation; 3) A mouse model of full-thickness skin wounds combined with 4 Gy total-body irradiation. Loss of Nur77 aggravates radiation-induced skin injury in three mouse models. (C) Pictures showing the weight change and the scoring curves of the whole course of radiogenic injury in mice with different Nur77 genotypes. (D) Wound healing, body weight, and wound score results of radiation fractionation model in wild-type and Nur77 knockout mice. (E) Wound healing, body weight, and wound healing score results of the radiation combined injury model in mice. * P < 0.05 and ** P < 0.01, compared with the control group.

Article Snippet: Cells were blocked with blocking buffer (phosphate buffered saline, 1% Triton X-100, and 5% BSA) and incubated at 4 °C with Nur77 (#NBP2-66980, Novus) antibody (1: 200) overnight, FITC-conjugated goat anti-mouse antibody (1:300) was added for 2 h at room temperature, and cells were dyed with 200 nM MitoTracker Red CMXRos (#C1035, Beyotime) at 37 °C for 30 min in darkness.

Techniques: Knock-Out, Fractionation, Irradiation, Control

Journal: Theranostics

Article Title: Single-cell RNA-Seq analysis of molecular changes during radiation-induced skin injury: the involvement of Nur77

doi: 10.7150/thno.100417

Figure Lengend Snippet: scRNA-Seq reveals the complex mechanism by which Nur77 mediates radiation-induced skin injury. (A) Diagram displaying the process of sequencing single cells from radiation-induced skin injury samples obtained from wild-type ( Nur77 +/+ ) and Nur77 knockout ( Nur77 -/- ) mice. (B) The t-SNE plot displays main cell types in wild-type and Nur77 knockout mice. Each dot represents only one cell. (C) Dot plot showing the expression of representative genes for each cell type. (D) The U-MAP plot displays cell types mouse skin with or without radiation. Each dot represents only one cell. (E) Bar plots show the proportions that each group contributes to each cluster. (F) The Venn diagram shows the number of up-regulated DEpcGs and down-regulated DEpcGs in different cell types. (G) Significant signaling pathways were ranked based on differences in the overall information flow within the inferred networks between Nur77 -/- and Nur77 +/+ mouse skin. The overall information flow of a signaling network is calculated by summarizing all communication probabilities in that network. An overview of cell-cell interactions. Arrow and edge color indicate direction. Bar plots showing overall information flow of each signaling pathway. (H) Heatmap shows outgoing signaling patterns of Nur77 -/- and Nur77 +/+ mouse skin. (I) Comparison of the significant ligand-receptor pairs between Nur77 -/- and Nur77 +/+ mouse skin, which contribute to the signaling from fibroblast to other cells.

Article Snippet: Cells were blocked with blocking buffer (phosphate buffered saline, 1% Triton X-100, and 5% BSA) and incubated at 4 °C with Nur77 (#NBP2-66980, Novus) antibody (1: 200) overnight, FITC-conjugated goat anti-mouse antibody (1:300) was added for 2 h at room temperature, and cells were dyed with 200 nM MitoTracker Red CMXRos (#C1035, Beyotime) at 37 °C for 30 min in darkness.

Techniques: Sequencing, Knock-Out, Expressing, Protein-Protein interactions, Comparison

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

Journal: Cancer Research

Article Title: Modulation of Orphan Nuclear Receptor Nur77-Mediated Apoptotic Pathway by Acetylshikonin and Analogues

doi: 10.1158/0008-5472.can-08-1972

Figure Lengend Snippet: Figure 2. Induction of apoptosis by SK07 and the role of Nur77. A, DAPI staining. NIH-H460 cells cultured in serum-free medium were treated with SK03, SK06, SK07 (5 Amol/L), or vehicle for 24 h and subjected to DAPI staining. Apoptotic cells were compared between different treatments. *, P < 0.05 (vs. control); **, P < 0.01 (vs. control); ##, P < 0.01 (vs. SK03 or SK06). B, PARP cleavage and caspase-3 activation. NIH-H460 or HeLa cells were treated with 5 Amol/L of SK07 in serum-free medium for the indicated times. Total cell lysates were subjected to Western blotting assay for PARP cleavage using anti-PARP antibody (top). For the analysis of caspase-3 activation (bottom), NIH-H460 cells were treated with shikonins as described above and stained with an antibody recognizing the cleaved caspase-3. Nuclei were visualized by costaining with DAPI. C, transfection of Nur77 mediates the apoptotic effect of SK07. HEK293T cells were transfected with full-length Nur77, N-Nur77 (A/B domain), or C-Nur77 (E/F domain) and subjected to SK07 treatment (5 Amol/L) or vehicle in serum-free medium for 8 h. Apoptotic cells examined by DAPI staining were compared between transfected cells carrying different mutants. **, P < 0.01 (vs. control). D, the apoptotic effect of SK07 is impaired in Nur77 knockout MEFs. MEF cells or MEF Nur77/ cells were treated with vehicle or the indicated concentrations of SK07 or SK03 for 24 h and stained with PI/Annexin V. Apoptosis was analyzed by fluorescence-activated cell sorting analysis.

Article Snippet: © 2008 American Association for Cancercancerres.aacrjournals.org Downloaded from anti-rabbit and anti-mouse secondary antibody conjugated to horseradish peroxidase from Thermo Fisher Scientific, Inc., anti-mouse IgG conjugated with Cy3 from Chemicon International, polyclonal anti-Nur77 (sc-5569), anti-Hsp60 (sc-7150), anti–Bcl-2 (sc-509), anti-Bax (sc-493), anti-Bax (6A7; sc-23959), FITC-labeled anti-rabbit IgG from Santa Cruz Biotechnology, anti–poly(ADP-ribose) polymerase (PARP; 556494), and anti-cytochrome c (556433) from BD Biosciences, monoclonal anti–h-actin antibody from Sigma, monoclonal anti-Nur77 antibody from R&D Systems, anti–Bcl-2 BH3 (AP1303a) from Abgent, anti–cleaved caspase-3 (Asp175) from Cell Signaling Technology, polyvinylidene difluoride membranes from Millipore, and a cocktail of proteinase inhibitors (80-6501-23) from Amersham were used in this study.

Techniques: Staining, Cell Culture, Control, Activation Assay, Western Blot, Transfection, Knock-Out, Fluorescence, FACS

Journal: Cancer Research

Article Title: Modulation of Orphan Nuclear Receptor Nur77-Mediated Apoptotic Pathway by Acetylshikonin and Analogues

doi: 10.1158/0008-5472.can-08-1972

Figure Lengend Snippet: Figure 5. SK07 induces Bcl-2 conformational change and Bax activation. A, colocalization of Nur77 with Bcl-2. NIH-H460 cells treated with vehicle or 5 Amol/L of SK07 in the presence or absence of LMB (10 ng/mL) for 24 h were subjected to costaining with anti-Nur77 and anti–Bcl-2 antibodies. Fluorescent microscopy was used to analyze the subcellular localization of Nur77 and Bcl-2. B, Bcl-2 conformational change and Bax activation. NIH-H460 cells treated with 5 Amol/L of SK07 were examined for Bcl-2 conformational change by anti–Bcl-2(BH3) antibody. Its association with Bax activation was analyzed by costaining with anti-Bax(6A7) antibody. Cells were also stained with DAPI to visualize the nucleus. C, translocation of Bax to mitochondria. NIH-H460 cells treated with 5 Amol/L of SK07 were examined for Bax activation by antibody against Bax(6A7). Cells were costained with anti-Hsp60 antibody to reveal Bax colocalization with mitochondria. DAPI staining was used to visualize the nuclei. The colocalization of active Bax with mitochondria and their association with apoptotic nuclear morphology were compared. D, Bax activation and cytochrome c release. NIH-H460 cells treated with 5 Amol/L of SK07 were stained with anti-Bax(6A7) and anti–cytochrome c (Cyt c) antibodies. Bax activation was compared with its induction of cytochrome c release as indicated by diffusive cytochrome c staining. LMB was used to determine its effect on both Bax activation and cytochrome c release.

Article Snippet: © 2008 American Association for Cancercancerres.aacrjournals.org Downloaded from anti-rabbit and anti-mouse secondary antibody conjugated to horseradish peroxidase from Thermo Fisher Scientific, Inc., anti-mouse IgG conjugated with Cy3 from Chemicon International, polyclonal anti-Nur77 (sc-5569), anti-Hsp60 (sc-7150), anti–Bcl-2 (sc-509), anti-Bax (sc-493), anti-Bax (6A7; sc-23959), FITC-labeled anti-rabbit IgG from Santa Cruz Biotechnology, anti–poly(ADP-ribose) polymerase (PARP; 556494), and anti-cytochrome c (556433) from BD Biosciences, monoclonal anti–h-actin antibody from Sigma, monoclonal anti-Nur77 antibody from R&D Systems, anti–Bcl-2 BH3 (AP1303a) from Abgent, anti–cleaved caspase-3 (Asp175) from Cell Signaling Technology, polyvinylidene difluoride membranes from Millipore, and a cocktail of proteinase inhibitors (80-6501-23) from Amersham were used in this study.

Techniques: Activation Assay, Microscopy, Staining, Translocation Assay

Journal: Molecular and Cellular Biology

Article Title: NR4A1 Antagonists Inhibit β1-Integrin-Dependent Breast Cancer Cell Migration

doi: 10.1128/mcb.00912-15

Figure Lengend Snippet: Fig. 1. NR4A1 regulates β1-integrin expression in breast cancer cells and tumors. (A) Breast 598

Article Snippet: NR4A1 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Expressing

Journal: Molecular and Cellular Biology

Article Title: NR4A1 Antagonists Inhibit β1-Integrin-Dependent Breast Cancer Cell Migration

doi: 10.1128/mcb.00912-15

Figure Lengend Snippet: Fig. 3. Role of NR4A1/p300/Sp1 in regulation of β1- and β3-integrin. (A) Analysis of polII, 620

Article Snippet: NR4A1 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: NR4A1 Antagonists Inhibit β1-Integrin-Dependent Breast Cancer Cell Migration

doi: 10.1128/mcb.00912-15

Figure Lengend Snippet: Fig. 5. Role of NR4A1 on TGFβ-induced migration of MDA-MB-231 cells. (A) MDA-MB-231 647

Article Snippet: NR4A1 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Migration

Journal: International Journal of Molecular Sciences

Article Title: NR4A1 Mediates Bronchopulmonary Dysplasia-Like Lung Injury Induced by Intrauterine Inflammation in Mouse Offspring

doi: 10.3390/ijms26146931

Figure Lengend Snippet: Increased NR4A1 expression was associated with impaired lung development induced by IUI. ( a ) Representative image of hematoxylin- and eosin-stained lung tissues from C57BL/6 neonatal mice exposed to IUI and control mice on postnatal day 1. Scale bars, 100 μm ( n = 6–7). ( b ) Quantification of alveolar size for ( a ). ( c ) Quantification of alveolar number for ( a ). ( d ) Western Blot analysis of NR4A1 expression in lung tissues from C57BL/6 neonatal mice exposed to IUI and control mice on postnatal day 1. ( e ) The relative protein level of NR4A1 was quantified for ( d ), with GAPDH used as the loading control ( n = 7). ( f ) Immunohistochemistry analysis of NR4A1 expression in lung tissues from C57BL/6 neonatal mice exposed to IUI and control mice on postnatal day 1. Scale bars, 100 μm ( n = 6–7). ( g ) Quantification of NR4A1-positive areas for ( f ). ( h ) Representative image of hematoxylin and eosin-stained lung tissues from C57BL/6 neonatal mice exposed to IUI and control mice at 3 months postnatal. Scale bars, 100 μm ( n = 11–12). ( i ) Quantification of alveolar size for ( h ). ( j ) Quantification of alveolar number for ( h ). All data are presented as the mean ± SD. Statistical analysis was performed using unpaired Student’s t tests. * p < 0.05, ** p < 0.01 as indicated. Arrows in subfigure (f) indicate NR4A1-positive cells.

Article Snippet: The sections were probed with rabbit anti-NR4A1 antibodies (1:350; Novus, St. Louis, MI, USA) in the incubator overnight at 4 °C.

Techniques: Expressing, Staining, Control, Western Blot, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: NR4A1 Mediates Bronchopulmonary Dysplasia-Like Lung Injury Induced by Intrauterine Inflammation in Mouse Offspring

doi: 10.3390/ijms26146931

Figure Lengend Snippet: Involvement of NR4A1 in Lung Injury Induced by IUI. The efficiency of NR4A1 RNA interference was evaluated by RT-qPCR ( a ) and Western blot ( b ). Representative image of lung tissues from C57BL/6 neonatal mice exposed to IUI on postnatal day 7, with NR4A1 siRNA administered intranasally from postnatal day 2 to 5, stained with hematoxylin and eosin. Scale bars, 200 μm ( n = 11–20) ( c ). ( d ) Quantification of alveolar size for ( c ). ( e ) Quantification of alveoli number for ( c ). All data are presented as the mean ± SD. Statistical analysis was performed using unpaired Student’s t tests. * p < 0.05, ** p < 0.01 as indicated.

Article Snippet: The sections were probed with rabbit anti-NR4A1 antibodies (1:350; Novus, St. Louis, MI, USA) in the incubator overnight at 4 °C.

Techniques: Quantitative RT-PCR, Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: NR4A1 Mediates Bronchopulmonary Dysplasia-Like Lung Injury Induced by Intrauterine Inflammation in Mouse Offspring

doi: 10.3390/ijms26146931

Figure Lengend Snippet: Differential transcriptome and proliferation caused by overexpression of NR4A1 in MLE-12 cells. The efficiency of NR4A1 overexpression was evaluated by Western blot ( a ). ( b ) Representative images of EdU-positive cells in NR4A1-overexpressing and control MLE-12 cells ( n = 6). ( c ) The quantification of EdU-positive cells for ( b ). ( d ) A volcano plot of DEGs in MLE-12 cells overexpressing NR4A1. ( e ) The top 15 DEGs associated with lung injury induced by NR4A1 overexpression. ( f , g ) The DEGs significantly regulated by NR4A1 overexpression are enriched in the top 30 KEGG and GO pathways. ( h ) Western Blot analysis of whole-cell lysates prepared from NR4A1-overexpressing and control MLE-12 cells after transfection with the vectors for 48 h. The relative protein level of pERK1/2/tERK1/2 ( i ) and pAKT/tAKT ( j ) were quantified for ( h ), with GAPDH used as the loading control (n = 9). All data are presented as the mean ± SD. Statistical analysis was performed using unpaired Student’s t test. * p < 0.05, ** p < 0.01, and *** p < 0.001 as indicated. DAPI: 4′,6-diamidino-2-phenylindole; EdU: 5-ethynyl-2-deoxyuridine; DEGs: differentially expressed genes.

Article Snippet: The sections were probed with rabbit anti-NR4A1 antibodies (1:350; Novus, St. Louis, MI, USA) in the incubator overnight at 4 °C.

Techniques: Over Expression, Western Blot, Control, Transfection

Journal: International Journal of Molecular Sciences

Article Title: NR4A1 Mediates Bronchopulmonary Dysplasia-Like Lung Injury Induced by Intrauterine Inflammation in Mouse Offspring

doi: 10.3390/ijms26146931

Figure Lengend Snippet: EREG is a key downstream target of NR4A1. ( a ) Verification of DEGs associated with lung injury in MLE-12 cells after transfection with the vectors for 48 h by RT-qPCR (n = 6–8). ( b ) Western Blot analysis of whole-cell lysates prepared from NR4A1-overexpressing and control MLE-12 cells after transfection with the vectors for 48 h. ( c ) The relative protein level of EREG in ( b ) (n = 5). ( d ) ELISA analysis of the protein levels of EREG in the lung tissues of neonatal mice with IUI on postnatal day 1 (n = 11). ( e ) Schematic illustration of predicted NR4A1 binding elements (S1–S3) in the total 2000 bp sequence of the promoter and 5′UTR of the murine Ereg gene. ( f ) Effect of S1–3 fragments EREG transcription activity mediated by NR4A1 (n = 6). All data are presented as the mean ± SD. The data were analyzed using unpaired t -tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, # p < 0.05; ## p < 0.01, and #### p < 0.0001 as indicated.

Article Snippet: The sections were probed with rabbit anti-NR4A1 antibodies (1:350; Novus, St. Louis, MI, USA) in the incubator overnight at 4 °C.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Binding Assay, Sequencing, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: NR4A1 Mediates Bronchopulmonary Dysplasia-Like Lung Injury Induced by Intrauterine Inflammation in Mouse Offspring

doi: 10.3390/ijms26146931

Figure Lengend Snippet: NR4A1-EREG-EGFR signaling pathway involved in development of pulmonary fibrosis induced by IUI. ( a ) Representative image of collagen fiber staining using Masson’s trichrome staining in the lungs of neonatal mice with IUI on postnatal day 1 ( n = 7–9). ( b ) Quantification of Masson-positive areas for ( b ). ( c ) Representative images of collagen fiber staining using Masson’s trichrome staining in the lungs of neonatal mice with IUI at 6 months postnatal ( n = 4–5). ( d ) Quantification of Masson-positive areas for ( c ). EMT-related gene expression was analyzed by RT-qPCR in MLE-12 cells ( e ) and A549 cells ( j ) after 72h transfection with NR4A1-overexpressing vectors (n = 4–5). EMT-related gene expression was analyzed in L929 cells ( f ) by RT-qPCR following 24 h treatment with conditioned media derived from NR4A1-overexpressing MLE-12 cells cultured for 48 h ( n = 3–4). EMT-related gene expression was analyzed using RT-qPCR in MLE-12 cells ( g ) and A549 cells ( k ) pretreated with gefitinib (10 μmoL) for 1 h, followed by EREG (50 ng/mL) for 12 h ( n = 3–5). ( h ) Western Blot analysis of whole-cell lysates prepared from NR4A1-overexpressing and control MLE-12 cells treated with DMSO or gefitinib (10 μmoL), respectively, after transfection with the vectors for 48 h. ( i ) The relative protein levels of pERK1/2/tERK1/2 and pAKT/tAKT were quantified for ( h ), with GAPDH used as the loading control ( n = 3). All data are presented as the mean ± SD. The data were analyzed using unpaired t -tests. * p < 0.05, ** p <0.01, *** p <0.001 as indicated.

Article Snippet: The sections were probed with rabbit anti-NR4A1 antibodies (1:350; Novus, St. Louis, MI, USA) in the incubator overnight at 4 °C.

Techniques: Staining, Gene Expression, Quantitative RT-PCR, Transfection, Derivative Assay, Cell Culture, Western Blot, Control