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: Journal of Anesthesia

Article Title: Effects of sevoflurane exposure during different stages of pregnancy on the brain development of rat offspring

doi: 10.1007/s00540-021-02972-2

Figure Lengend Snippet: Effect of sevoflurane anesthesia during pregnancy on proteins related to the NR4A1/NF-κB pathway in the hippocampi of offspring rats. A Western blot analysis of NR4A1, p-P65, P65, p-IκBα, and IκBα protein; B Relative expression of NR4A1; C Relative expression of p-P65/P66; D Relative expression of p-IκBα/IκBα. There were 6 rats in each group, and the data are represented as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{X}$$\end{document} X ¯ ± SD. * p < 0.05 compared to the control group

Article Snippet: Next, the following polyclonal primary antibodies were applied: rabbit anti-human NR4A1 antibody (1:1000, orb127604, Biorbyt, Beijing, China), rabbit anti-human NF-κB p-p65 antibody (1:100, orb501839, Biorbyt, Beijing, China), rabbit anti-human NF-κB p65 antibody (1:100, orb344389, Biorbyt), rabbit anti-human IκBα antibody (1:1000, 4812, Cell Signaling Technology, Shanghai, China), rabbit anti-human p-IκBα antibody (1:800, 2859, Cell Signaling Technology, Shanghai, China), and rabbit anti-human GAPDH antibody (1:1000, 5174, Cell Signaling Technology, Shanghai, China).

Techniques: Western Blot, Expressing, Control

Journal: Advanced Science

Article Title: CD27 and ICOS as Targets of PD‐1/PD‐L1 Signaling to Regulate Resident Memory CD8 + T‐Cell‐Mediated Pulmonary Protection and Pathology

doi: 10.1002/advs.202512452

Figure Lengend Snippet: NR4A1 plays an intrinsic role in the regulation of NP 366–374 CD8 + T RM cells. A–J) Nur77–EGFP mice were infected with influenza PR8 and received either control IgG or the indicated antibodies from 21 to 25 d.p.i. Representative plots and frequencies of EGFP + NP 366–374 T RM cells are shown at 29 d.p.i. K–O) Bone marrow chimeric mice were infected with influenza PR8. K) Schematic of experimental design. L,M) Representative plots, frequency, and total cell numbers of Ki‐67 + NP 366–374 T RM cells at 28 d.p.i. N,O) Representative plots, frequencies, and total cell numbers of NP 366–374 T RM cells at 42 d.p.i. P) WT and NR4A1 −/− mice were infected with influenza PR8 and rechallenged with X31 (1×10 4 pfu) at 42 d.p.i. in the presence of FTY720. Percentages of original body weight following rechallenge were assessed daily. Q–V) WT and NR4A1 −/− mice were infected with influenza PR8 and received control IgG or α‐PD‐L1 from 21 to 25 d.p.i. or from 21 to 37 d.p.i. Q,R) Representative plots, frequency and total cell numbers of Ki‐67 + NP 366–374 T RM cells at 29 d.p.i. S,T) Representative plots, frequencies and total cell numbers of NP 366–374 T RM cells at 42 d.p.i. U) All mice were rechallenged with X31 (1×10 4 pfu) at 42 d.p.i. in the presence of FTY720. Percentages of original body weight following rechallenge were assessed daily. V) Lung pathology was evaluated at 60 d.p.i. Representative of three to four experiments ( n = 3–8). Data are mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, unpaired two‐tailed t ‐test or one‐way analysis of variance (ANOVA) with Tukey multiple comparison test.

Article Snippet: CD8α −/− and NR4A1 −/− mice were originally purchased from Cyagen Biosciences (Suzhou), Nur77–EGFP mice were originally purchased from the Jackson Laboratory and bred in house.

Techniques: Infection, Control, Two Tailed Test, Comparison

Journal: Advanced Science

Article Title: CD27 and ICOS as Targets of PD‐1/PD‐L1 Signaling to Regulate Resident Memory CD8 + T‐Cell‐Mediated Pulmonary Protection and Pathology

doi: 10.1002/advs.202512452

Figure Lengend Snippet: CD27 agonist provides better protection to hosts by maintaining NP 366–374 CD8 + T RM cells. A–H) WT C57BL/6 mice were infected with influenza PR8 and treated with either control IgG or CD27 agonist from 21 to 37 d.p.i. or from 21 to 57 d.p.i. A) Schematic of experimental design. B,C) Representative plots, frequencies, and total cell numbers of Ki‐67 + NP 366–374 T RM cells at 42 d.p.i. D,E) Representative plots, frequencies, and total cell numbers of NP 366–374 T RM cells at 60 d.p.i. F) CD69 expression level [mean florescence intensity (MFI)] on lung NP 366–374 T RM cells at 60 d.p.i. G) All mice were rechallenged with X31 (2×10 4 pfu) at 60 d.p.i. in the presence of FTY720. Percentages of original body weight after rechallenge were assessed daily. H) Lung pathology was evaluated at 60 d.p.i. I,J) Nur77–EGFP mice were infected with influenza PR8 and received either control IgG or CD27 agonist from 21 to 37 d.p.i. Representative plots and frequencies of EGFP + NP 366–374 T RM cells at 42 d.p.i. are shown. K–P) WT and NR4A1 −/− mice were infected with influenza PR8 and treated with either control IgG or CD27 agonist from 21 to 37 d.p.i. or from 21 to 57 d.p.i. K) Schematic of experimental design. L,M) Representative plots, frequencies and total cell numbers of NP 366–374 T RM cells at 60 d.p.i. N,O) Representative plots, frequencies, and total cell numbers of Ki‐67 + NP 366–374 T RM cells at 42 d.p.i. P) All mice were rechallenged with X31 (2×10 4 pfu) at 60 d.p.i. in the presence of FTY720. Percentages of original body weight following rechallenge were assessed daily. Representative of three to four experiments ( n = 4–9). Data are mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001, unpaired two‐tailed t ‐test or one‐way analysis of variance (ANOVA) with Tukey multiple comparison test.

Article Snippet: CD8α −/− and NR4A1 −/− mice were originally purchased from Cyagen Biosciences (Suzhou), Nur77–EGFP mice were originally purchased from the Jackson Laboratory and bred in house.

Techniques: Infection, Control, Expressing, Two Tailed Test, Comparison

Journal: Nature communications

Article Title: Dendrogenin A drives LXR to trigger lethal autophagy in cancers.

doi: 10.1038/s41467-017-01948-9

Figure Lengend Snippet: Fig. 1 Nur77 and NOR1 are required for the induction of cell death by DDA in melanoma. a Chemical structure of DDA and C17. b B16F10 and SKMEL-28 cells were treated or not for 24, 48, or 72 h with DDA or C17. DDA- or C17-induced cell death was determined by a trypan blue assay and expressed as the percentage cell death relative to control (vehicle). c Cells were treated with 5 µM DDA for 48 h or 72 h with or without 500 µM vitamin E (Vit E), 50 µM z- VAD-fmk, 50 µM z-DEVD-fmk, 1 µg/ml actinomycin D (Act D) or 2.5 µg/ml cycloheximide (CHX), and cell death was measured as in b. d DDA induced the accumulation of free sterols in cells. Cells were treated with solvent vehicle or 2.5 µM DDA for 48 h, then fixed and stained with filipin and analyzed by fluorescence microscopy. e Representative EM images of B16F10 (panels 1–3) and SKMEL-28 (panels 2–4) cells treated for 24 h with 2.5 µM DDA. Ly: multilamellar body-derived lysosomes, AP: autophagosomes, AL: autolysosomes, N: nucleus, C: cytoplasm. Bars: 250 nm for panel 1 and 100 nm for inserts; 500 nm for panel 2 and 100 nm for inserts; 500 nm for panel 3; and 250 nm for panel 4. f Heat map depicting transcription of genes encoding nuclear receptors (NR) and their co-regulators, using PCR arrays in B16F10 and SKMEL-28 cells treated or not with 2.5 µM DDA for 5 h, using PCR arrays. g Immunoblot for Nur77 and NOR1 protein expression in melanoma cells treated or not with DDA for 24 h. h Analysis of DDA cytotoxicity in B16F10 and SKMEL-28 cells transfected with control scramble siRNA (siSC), siNur77 and/or siNOR1. 72 h after transfection, cells were treated or not for 24 h with 2.5 µM DDA. Cell death is expressed as a percentage relative to the level of cell death induced by DDA in cells transfected with a scramble control siRNA (siSC). Data from b–d and h are the means ± S.E.M. of three independent experiments performed in triplicate, **P < 0.01, ***P < 0.001, t-test. All images and densitometry values are representative of three independent experiments

Article Snippet: For the KD of Nur77 and NOR1 in KG1, 29 mer shRNA constructs in pRS retroviral vectors targeting Nur77 and NOR1 were purchased from Origene.

Techniques: Control, Solvent, Staining, Microscopy, Derivative Assay, Western Blot, Expressing, Transfection

Journal: Nature communications

Article Title: Dendrogenin A drives LXR to trigger lethal autophagy in cancers.

doi: 10.1038/s41467-017-01948-9

Figure Lengend Snippet: Fig. 2 DDA induces Nur77- and NOR1-dependent lethal autophagy in melanoma cells. a DDA triggers the accumulation of autophagic vesicles. Cells were treated for 24 h with or without 2.5 µM DDA then stained with monodansylcadaverine (MDC) and observed by fluorescence microscopy. MDC-specific activity was measured by fluorescence photometry. b Cells were treated for 24 h with solvent vehicle or increasing concentrations of DDA then analyzed for autophagic protein expression by immunoblotting. Blots are representative of three independent experiments. c Long-life protein degradation was determined in cells treated with solvent vehicle (control) or 1 µM DDA for 18 h in the presence or absence of the autolysosomal inhibitors bafilomycin A1 (Baf A1) and hydroxychloroquine (HCQ). Autophagic activity was measured as the level of degradation of long-lived proteins. Starvation for 18 h in Hank’s balanced salt solution (HBSS) was used as a positive control. d Immunoblots of LC3 proteins from cells treated for 24 h with or without 2.5 µM DDA and with or without E64 + pepstatin A (10 µg/ml). Images are representative of three independent experiments. e DDA induced the formation of punctate LC3 cells. Cells were transfected with a plasmid-expressing GFP-LC3 and then treated for 24 h with the solvent vehicle or 2.5 µM DDA, with or without E64 + pepstatin A (10 µg/ml) and observed by fluorescent microscopy. The percentage of GFP-LC3-positive cells with GFP-LC3 puncta was calculated. f Analysis of DDA cytotoxicity in cells transfected with scramble siRNA (siSC), siATG7, siVPS34, or siBECN1. Seventy-two hours after transfection, cells were treated or not for 24 h with 2.5 µM DDA. g Analysis of DDA cytotoxicity in SKMEL-28 cells permanently transfected with control shRNA (shCTRL) or shRNA against VPS34 (shVPS34). Cells were treated for 72 h with solvent vehicle (CTRL) or 2.5 µM DDA. h Cells were treated with 2.5 µM DDA for 24, 48 and 72 h in the presence or absence of the autolysosome inhibitors Baf A1 or HCQ. Cell death is expressed as in Fig. 1a. Data from a, c, e, f, g are the means ± S. E.M. of three experiments performed in triplicate, *P < 0.05, **P < 0.01, ***P < 0.001, t-test

Article Snippet: For the KD of Nur77 and NOR1 in KG1, 29 mer shRNA constructs in pRS retroviral vectors targeting Nur77 and NOR1 were purchased from Origene.

Techniques: Staining, Microscopy, Activity Assay, Solvent, Expressing, Western Blot, Control, Positive Control, Transfection, Plasmid Preparation, shRNA

Journal: Nature communications

Article Title: Dendrogenin A drives LXR to trigger lethal autophagy in cancers.

doi: 10.1038/s41467-017-01948-9

Figure Lengend Snippet: Fig. 4 DDA induces autophagy in melanoma in vivo and in vitro in an LXRβ-dependent manner. a, b Mice engrafted with human SKMEL-28 or mouse B16F10 cells (10 per group) were treated with DDA (i.p. 20 mg/kg/day) or vehicle. Mean tumor volumes ± S.E.M. are shown, **P < 0.01, analysis of variance (ANOVA). Data are representative of three independent experiments. At the end of treatments, tumors were analyzed for Nur77, NOR1 and LC3 protein expression by a, b immunoblotting or c immunohistochemistry (brown staining). c Representative immunohistochemical analysis. Magnification ×40. Insert is 4× digital amplification. d, e TEM images of cells transfected with shCTRL, sh3LXRβ, or sh4LXRβ and treated with control vehicle or 2.5 µM DDA for 24 h. e TEM images of cells transfected with shCTRL, sh3LXRβ, or sh4LXRβ and treated with 2.5 µM DDA for 24 h. d, e N, nucleus; C, cytoplasm; AM, amphisome

Article Snippet: For the KD of Nur77 and NOR1 in KG1, 29 mer shRNA constructs in pRS retroviral vectors targeting Nur77 and NOR1 were purchased from Origene.

Techniques: In Vivo, In Vitro, Expressing, Western Blot, Immunohistochemistry, Staining, Immunohistochemical staining, Transfection, Control

Journal: Nature communications

Article Title: Dendrogenin A drives LXR to trigger lethal autophagy in cancers.

doi: 10.1038/s41467-017-01948-9

Figure Lengend Snippet: Fig. 6 DDA induces lethal autophagy in AML cells via LXRβ. a DDA-induced cell death in KG1 and HL60 cells was determined over time as in Fig. 1b. b Quantification of the Δ8-sterols that had accumulated in KG1 and HL60 cells treated without or with 2.5 µM DDA. c May–Grünwald–Giemsa staining of KG1 and HL60 cells treated or not with DDA. d Immunoblots of LC3 proteins in cells treated or not with 2.5 µM DDA, and E64 + pepstatin A (Pep). e Effect of the pharmacological inhibitor of autophagy Baf A1 on DDA cytotoxicity at 48 and 72 h. f DDA cytotoxicity in KG1 or HL60 cells permanently transfected with control shRNA (shC) or shRNA against VPS34 (shVPS34) after 72 h treatment. g Immunoblots of ATG3 and LC3 proteins in KG1 cells transfected with control scramble siRNA (siSC) or siRNA against ATG3 (siATG3). Seventy-two hours after transfection, cells were treated for 24 h with 5 µM DDA. h Analysis of DDA cytotoxicity in KG1 cells transfected with control scramble siRNA (siSC), siATG3, siATG7, or siBECN1. Seventy-two hours after transfection, cells were treated for 24 h with 5 µM DDA. i Analysis of DDA cytotoxicity in KG1 cells transfected with shCTRL, sh3LXRβ, or sh4LXRβ. Cells were treated for 24 h with 5 µM DDA or vehicle. j Immunoblots of LC3 protein expression in cells transfected with shCTRL, sh3LXRβ, or sh4LXRβ and treated with 5 µM DDA or vehicle for 24 h. Analysis of the cytotoxicity k and acridine orange-positive vesicles l in KG1 cells treated or not with 5 µM DDA, 2 µM TO, 2 µM GW, or 10 µM 22(R)HC. The presence of Nur77 and NOR1 is required in DDA cytotoxicity (m) and autophagy (n). Data from a, b, e, f, h, i, k, l, m, n are the means ± S.E.M. of three experiments performed in triplicate, *P < 0.05, **P < 0.01, ***P < 0.001 t-test. All images and densitometry values are representative of three independent experiments

Article Snippet: For the KD of Nur77 and NOR1 in KG1, 29 mer shRNA constructs in pRS retroviral vectors targeting Nur77 and NOR1 were purchased from Origene.

Techniques: Staining, Western Blot, Transfection, Control, shRNA, Expressing

Journal: Nature communications

Article Title: Dendrogenin A drives LXR to trigger lethal autophagy in cancers.

doi: 10.1038/s41467-017-01948-9

Figure Lengend Snippet: Fig. 7 DDA induces an LXRβ-, Nur77-, and NOR1-dependent lethal autophagy in AML. a Tumor volume curves of xenografts of cells transfected with shCTRL, sh3LXRβ, and sh4LXRβ and implanted into NOD/SCID mice (20 per group) who were then treated daily with DDA (20 mg/kg/day, i.p.) or solvent vehicle. b HL60 cells were injected i.v. into irradiated NSG mice (n = 10 per group) who were then treated daily with DDA (20 mg/kg/day, i.p. or 40 mg/ kg/day, p.o.) for 16 days. Analyses of HL60 cell contents and viability in bone marrow (BM) and brain (BR) of NSG mice. Cells were quantified by flow cytometry using human anti-CD45 and human anti-CD33 antibodies (left panel) and viability was determined by Annexin-V staining (right panel). c Overall survival was determined for NSG mice (n = 10 per group) engrafted with HL60 cells and treated, after disease establishment, with control (vehicle) or DDA (40 mg/kg/day, p.o.), *P < 0.05, log-rank test. Samples from AML patients (n = 61, Supplementary Data 1) were exposed to increasing concentrations of DDA (d) or cytarabine (e) for 48 h. Cell death was assessed both in the AML bulk and in the progenitor/LSC cells (CD34+CD38−CD123 +) using Annexin-V/7AAD staining. Data are represented as the percentage of survival. Scatter plots comparing DDA efficacy in primary AML patients according to f their prognostic risk category (LR: low risk, IR: intermediate risk, and HR: high risk), g CFU-L formation, h total white blood count, and i their Flt3-ITD and NMP1 status. Samples from AML patients were exposed to 10 nM daunorubicin (DNR) j or 100 µM cytarabine k with or without 5 µM DDA for 72 h. “daunorubicin insensitive” or “cytarabine insensitive” when cell death was lower than 20%, and “sensitive” when cell death was over 70%. Bars represent S.E.M. l Images of primary AML cells stained with MGG after treatment with vehicle or DDA for 24 h. Images are representative of three independent experiments. Data from a-e are means ± S.E.M., and is representative of 3–5 independent experiments, **P < 0.01, t-test. d–k Bars represent S. E.M., *P < 0.05, t-test

Article Snippet: For the KD of Nur77 and NOR1 in KG1, 29 mer shRNA constructs in pRS retroviral vectors targeting Nur77 and NOR1 were purchased from Origene.

Techniques: Transfection, Solvent, Injection, Irradiation, Cytometry, Staining, Control

Journal: Nature communications

Article Title: Dendrogenin A drives LXR to trigger lethal autophagy in cancers.

doi: 10.1038/s41467-017-01948-9

Figure Lengend Snippet: Fig. 8 DDA exerts anti-leukemic activity in vivo in patient AML samples. Primary cells from AML patients were injected i.p. into irradiated NSG mice (three AML patients were tested separately). After validation of tumor engraftment, mice were treated with DDA (20 mg/kg/d, i.p.) or control (vehicle) for 19 days. a Representative flow cytometry analysis showing the selection of the human AML population (hCD45+hCD33+) and analysis of cell viability by Annexin-V/7AAD staining. b Human leukemic cell content in the hind limb bone marrow (BM) and spleen (SP) was measured by flow cytometry using human anti-CD45 and anti-CD33 antibodies. The viability of human CD45+CD33+ cells was determined by Annexin-V staining and flow cytometry analysis, *P < 0.05, **P < 0.01, ***P < 0.001, t-test. c Histological analysis of femur and sternum sections from mice injected with primary AML cells (AML#1), stained with Goldner (femur) and HE (sternum). d Histological analysis of bone marrow staining for Nur77, NOR1, and P62. Normal human CD34+ cells from a healthy donor were implanted intravenously into NSG mice and treated with vehicle or DDA for 3 weeks (20 mg/kg/day, i.p.). e Engraftment was quantified by assessing the percentage of hCD45+ cells in the BM. f The percentage of human myeloid (CD45+/CD33+) and lymphoid (CD45+/CD19+) cells was determined by flow cytometry. e, f Bars represent S.E.M

Article Snippet: For the KD of Nur77 and NOR1 in KG1, 29 mer shRNA constructs in pRS retroviral vectors targeting Nur77 and NOR1 were purchased from Origene.

Techniques: Activity Assay, In Vivo, Injection, Irradiation, Biomarker Discovery, Control, Cytometry, Selection, Staining

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Consumption of a high fat diet promotes protein O -GlcNAcylation in mouse retina via NR4A1-dependent GFAT2 expression

doi: 10.1016/j.bbadis.2018.09.006

Figure Lengend Snippet: Primers used for RT-PCR. Primer specificity to human (H), mouse (M), and rat (R) is indicated.

Article Snippet: Plasmids for expression of NR4A1 (Cat. # MR209316) and XBP1 (Myc-DDK-tagged ORF, Cat. # RR208206) were obtained from OriGene Technologies Inc (Rockville, MD, USA).

Techniques:

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Consumption of a high fat diet promotes protein O -GlcNAcylation in mouse retina via NR4A1-dependent GFAT2 expression

doi: 10.1016/j.bbadis.2018.09.006

Figure Lengend Snippet: A. NR4A1 mRNA expression was assessed in the retina of mice fed either a control chow (Chow) or HFD for 4 weeks (n=6). B. TR-MUL retinal cells were exposed to culture medium containing either BSA or palmitate bound to BSA (PAL) for 8 h (n=3). C. TR-MUL cells were exposed to culture medium containing the cell permeable ceramide Cer6 for up to 16 h (n=3). NR4A1 mRNA expression was assessed by RT-PCR. Results are expressed as means ± SEM. Statistical significance is denoted by the presence of different letters above bars on the graphs. Bars with different letters are statistically different; p<0.05. Results in B & C are representative of two experiments; within each experiment, three independent samples were analyzed.

Article Snippet: Plasmids for expression of NR4A1 (Cat. # MR209316) and XBP1 (Myc-DDK-tagged ORF, Cat. # RR208206) were obtained from OriGene Technologies Inc (Rockville, MD, USA).

Techniques: Expressing, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Consumption of a high fat diet promotes protein O -GlcNAcylation in mouse retina via NR4A1-dependent GFAT2 expression

doi: 10.1016/j.bbadis.2018.09.006

Figure Lengend Snippet: A-E. TR-MUL cells were transfected with either an empty vector (EV) or NR4A1 expression plasmid for 48 h. A. FLAG-tagged NR4A1, GFAT1, GFAT2, and GAPDH protein expression were assessed by Western blotting. Protein molecular mass (kDa) is indicated at the right of blots. Protein Staining (Protein S.) is shown as a loading control. NR4A1 (B.), GFAT1 (C.), and GFAT2 (D.) mRNA expression were assessed using RT-PCR. E. TR-MUL cells were transfected with either EV or NR4A1 plasmid for 48 h prior to treatment with the GFAT inhibitor DON for 16 h. Protein O-GlcNAcylation and GAPDH expression were measured by Western blotting. F-H. TR-MUL cells were exposed to culture medium containing either DMSO (Veh) or Cer6 for 16 h in the presence or absence of the NR4A1 inhibitor DIM-C-pPhOH (DIM). NR4A1 (F.), GFAT1 (G.), and GFAT2 (H.) mRNA expression were assessed by RT-PCR. Results are expressed as means ± SEM (n=3). Statistical significance is denoted by the presence of different letters above bars on the graphs. Bars with different letters are statistically different; p<0.05. Results are representative of two experiments; within each experiment, three independent samples were analyzed. I. Working model for mechanism whereby high fat diet (HFD) promotes retinal O-GlcNAcylation via enhanced NR4A1 and GFAT2 expression.

Article Snippet: Plasmids for expression of NR4A1 (Cat. # MR209316) and XBP1 (Myc-DDK-tagged ORF, Cat. # RR208206) were obtained from OriGene Technologies Inc (Rockville, MD, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Staining, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Endocrinology

Article Title: Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy

doi: 10.1210/endocr/bqaf144

Figure Lengend Snippet: Roles of NR4A1 and NR4A2 in endometriotic cell growth and apoptosis. IHEEC and IHESC cells were transfected with siNR4A1 and siNR4A2, and effects on protein levels (A) and cell viability (B) were determined as outlined in the Methods. Apoptosis was analyzed 72 hours after transfection of IHEEC and IHESC cells with siRNAs targeting NR4A1 (siNR4A1) or NR4A2 (siNR4A2). Representative flow cytometry dot plots showing apoptosis profiles in IHEEC and IHESC are presented (C, D). In each plot: the upper left quadrant (Q1) represents necrotic cells (PI-positive only), the upper right quadrant (Q2) shows late apoptotic cells (Annexin V-FITC and PI double-positive), the lower right quadrant (Q3) indicates early apoptotic cells (Annexin V-FITC–positive only), and the lower left quadrant (Q4) represents viable cells (double-negative). Quantification of early apoptotic (E) and late apoptotic (F) cell populations is shown. Statistical significance was determined as follows: * P < .05, ** P < .01, *** P < .001. Abbreviations: FITC, fluorescein isothiocyanate; IHEEC, immortalized human endometriotic epithelial cells; IHESC, immortalized human endometriotic stromal cells; NR4A1, nuclear receptor 4A1; NR4A2, nuclear receptor 4A2; PI, propidium iodide; siRNA, small interfering RNA.

Article Snippet: For immunohistochemistry staining, antibodies against NR4A1 (#NB100-56745, RRID:AB_2153757) and NR4A2 (#AF2156, RRID:AB_2153894) from Novus Biologicals (Centennial, CO, USA) and Ki-67 (#ab16667, RRID:AB_302459) from Abcam were used.

Techniques: Transfection, Flow Cytometry, Small Interfering RNA

Journal: Endocrinology

Article Title: Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy

doi: 10.1210/endocr/bqaf144

Figure Lengend Snippet: Effects of DIM-3,5 analogs on proliferation and migration of IHEEC and IHESC cells. The structure of the DIM-3,5 analogs (A) and corresponding dissociation constants (Kd) values of selected DIM-3,5 analogs for NR4A1 and NR4A2 are shown (B). IC 50 values for IHEEC and IHESC cells at 48 hours, determined from XTT assays (C). IHEEC and stromal IHESC cells were treated with selected DIM-3,5 analogs, and cell proliferation was assessed using XTT assays, as described in the Methods section (D, E). IHEEC (F) and IHESC (G) cells were transfected with oligonucleotides targeting NR4A1 or NR4A2 or treated with DIM-3,5 analogs (6.5 µM) for 24 hours, and effects on cell migration were determined in a scratch assay as outlined in the Materials and Methods. Data are presented as means ± SD for 3 replicate determinations, and statistical differences ( P < .05) of treated vs controls (DMSO) are indicated (*). Abbreviations: DIM-3,5, 1,1-bis(3′-indolyl)-(3,5-disubstitutedphenyl)methane; DMSO, dimethyl sulfoxide; IHEEC, immortalized human endometriotic epithelial cells; IHESC, immortalized human endometriotic stromal cells; NR4A1, nuclear receptor 4A1; NR4A2, nuclear receptor 4A2.

Article Snippet: For immunohistochemistry staining, antibodies against NR4A1 (#NB100-56745, RRID:AB_2153757) and NR4A2 (#AF2156, RRID:AB_2153894) from Novus Biologicals (Centennial, CO, USA) and Ki-67 (#ab16667, RRID:AB_302459) from Abcam were used.

Techniques: Migration, Transfection, Wound Healing Assay

Journal: Endocrinology

Article Title: Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy

doi: 10.1210/endocr/bqaf144

Figure Lengend Snippet: Effects of DIM-3,5 analogs and NR4A knockdown on EGFR and C-PARP expression in IHEEC and IHESC cells. IHEEC cells were treated for 24 hours with DMSO (vehicle control) or selected DIM-3,5 analogs (DIM-3,5-Br 2 , DIM-3,5-Cl 2 , and DIM-3-Cl-5-CF 3 ) (A) and oligonucleotides that target NR4A1 and NR4A2 (B). Whole-cell lysates were collected and analyzed by Western blotting, as described in the Methods section. IHESC cells were also treated with DIM-3,5 ligands (C) and transfected with siNR4A1 or siNR4A2 (D), and whole-cell lysates were obtained and analyzed by Western blots as outlined in the Methods. Protein expression levels were quantified by measuring relative band intensities compared to DMSO controls (set at 1.0) and normalized to β-actin (A-D). The same lysates from IHEEC cells were used for the blots shown in and , and the same lysates from IHESC cells were used for the blots shown in and ; therefore, the β-actin loading controls are identical. Data are presented as mean ± SD. * P < .05. Abbreviations: C-PARP, cleaved poly(ADP-ribose) polymerase; DIM-3,5, 1,1-bis(3′-indolyl)-(3,5-disubstitutedphenyl)methane; DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; IHEEC, immortalized human endometriotic epithelial cells; IHESC, immortalized human endometriotic stromal cells; NR4A1, nuclear receptor 4A1; NR4A2, nuclear receptor 4A2.

Article Snippet: For immunohistochemistry staining, antibodies against NR4A1 (#NB100-56745, RRID:AB_2153757) and NR4A2 (#AF2156, RRID:AB_2153894) from Novus Biologicals (Centennial, CO, USA) and Ki-67 (#ab16667, RRID:AB_302459) from Abcam were used.

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

Journal: Endocrinology

Article Title: Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy

doi: 10.1210/endocr/bqaf144

Figure Lengend Snippet: Effects of DIM-3,5 analogs and NR4A1/NR4A2 knockdown on EMT-related markers expression in IHEEC and IHESC cells. IHEEC cells were treated for 24 hours with DMSO (vehicle control) or selected DIM-3,5 analogs (DIM-3,5-Br 2 , DIM-3,5-Cl 2 , and DIM-3-Cl-5-CF 3 ) (A) for 24 hours and transfected with siNR4A1 or siNR4A2 (B) for 72 hours. Whole-cell lysates were collected and analyzed by Western blotting, as described in the Methods section. IHESC cells were treated with DIM-3,5 analogs (C) for 24 hours, and transfected with siNR4A1 or siNR4A2 (D) for 72 hours, and whole-cell lysates were analyzed by Western blots. Protein expression levels of EMT markers were quantified by measuring relative band intensities compared to DMSO controls (set at 1.0) and normalized to β-actin (A-D). Data are presented as mean ± SD. * P < .05. Abbreviations: DIM-3,5, 1,1-bis(3′-indolyl)-(3,5-disubstitutedphenyl)methane; DMSO, dimethyl sulfoxide; EMT, epithelial-to-mesenchymal transition; IHEEC, immortalized human endometriotic epithelial cells; IHESC, immortalized human endometriotic stromal cells.

Article Snippet: For immunohistochemistry staining, antibodies against NR4A1 (#NB100-56745, RRID:AB_2153757) and NR4A2 (#AF2156, RRID:AB_2153894) from Novus Biologicals (Centennial, CO, USA) and Ki-67 (#ab16667, RRID:AB_302459) from Abcam were used.

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

Journal: Endocrinology

Article Title: Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy

doi: 10.1210/endocr/bqaf144

Figure Lengend Snippet: Effects of DIM-3,5 analogs NR4A1 and NR4A2 knockdown on β1-integrin, estrogen receptor β, and mTOR signaling in IHEEC and IHESC cells. IHEEC cells were treated with DIM-3,5 analogs for 24 hours (A) and transfected with siRNAs targeting NR4A1 (siNR4A1) or NR4A2 (siNR4A2) (B) for 72 hours. Whole-cell lysates were then collected and analyzed by Western blotting, as described in the Methods section. IHESC cells were treated with DIM-3,5 analogs for 24 hours (C) and transfected with siNR4A1 or siNR4A2 for 72 hours (D). Whole-cell lysates were analyzed by Western blots. Protein expression levels were quantified by measuring relative band intensities compared to control siRNA-treated cells (set at 1.0) and normalized to β-actin (A–D). Data are presented as mean ± SD. * P < .05. Abbreviations: DIM-3,5, 1,1-bis(3′-indolyl)-(3,5-disubstitutedphenyl)methane; IHEEC, immortalized human endometriotic epithelial cells; IHESC, immortalized human endometriotic stromal cells; mTOR, mechanistic target of rapamycin; NR4A1, nuclear receptor 4A1; NR4A2, nuclear receptor 4A2; siRNA, small interfering RNA.

Article Snippet: For immunohistochemistry staining, antibodies against NR4A1 (#NB100-56745, RRID:AB_2153757) and NR4A2 (#AF2156, RRID:AB_2153894) from Novus Biologicals (Centennial, CO, USA) and Ki-67 (#ab16667, RRID:AB_302459) from Abcam were used.

Techniques: Knockdown, Transfection, Western Blot, Expressing, Control, Small Interfering RNA

Journal: Endocrinology

Article Title: Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy

doi: 10.1210/endocr/bqaf144

Figure Lengend Snippet: Effects of DIM-3,5 analogs and NR4A1/NR4A2 knockdown on fibrosis markers in IHEEC and IHESC cells. IHEEC cells were treated for 24 hours with DMSO (vehicle control) or selected DIM-3,5 analogs (DIM-3,5-Br 2 , DIM-3,5-Cl 2 , and DIM-3-Cl-5-CF 3 ) (A) and transfected with siNR4A1 or siNR4A2 (B). Whole-cell lysates were collected and analyzed by Western blotting, as described in the Methods section. IHESC cells were treated with DIM-3,5 compound for 24 hours (C) and transfected with siNR4A1 or siNR4A2 for 72 hours (D), and whole-cell lysates were analyzed by Western blots. Protein expression levels were quantified by measuring relative band intensities compared to DMSO controls (set at 1.0) and normalized to β-actin (A-D). The same lysates from IHESC cells were used for the blots shown in and , as well as and ; therefore, the β-actin loading controls are identical. Data are presented as mean ± SD. * P < .05. Abbreviations: DIM-3,5, 1,1-bis(3′-indolyl)-(3,5-disubstitutedphenyl)methane; DMSO, dimethyl sulfoxide; IHEEC, immortalized human endometriotic epithelial cells; IHESC, immortalized human endometriotic stromal cells; NR4A1, nuclear receptor 4A1; NR4A2, nuclear receptor 4A2.

Article Snippet: For immunohistochemistry staining, antibodies against NR4A1 (#NB100-56745, RRID:AB_2153757) and NR4A2 (#AF2156, RRID:AB_2153894) from Novus Biologicals (Centennial, CO, USA) and Ki-67 (#ab16667, RRID:AB_302459) from Abcam were used.

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

Journal: Endocrinology

Article Title: Dual Targeting of Orphan Nuclear Receptors NR4A1 and NR4A2 for Nonhormonal Endometriosis Therapy

doi: 10.1210/endocr/bqaf144

Figure Lengend Snippet: Suppression of ectopic lesion growth in a mouse model of endometriosis by DIM-3,5-Cl 2 treatment. Mice with surgically induced endometriosis were treated once daily with DIM-3,5-Cl 2 (2.5 mg/kg) or vehicle control for 34 days (A). Following treatment, ectopic lesions were harvested, and their volumes were calculated using the formula: 0.5 × Length × Width 2 (B). Luciferase activity of ectopic lesions was monitored throughout the treatment period using an in vivo imaging system. Luciferase signal intensity for each lesion was quantified and plotted in the corresponding graph (C). Proliferation in ectopic lesions was assessed by Ki-67 IHC (D). The levels of NR4a1 and Nr4a2 in epithelial and stromal compartments were determined by IHC (F, G), and H-scores were calculated using QuPath software . Apoptosis was evaluated by TUNEL assay (E), and the percentage of TUNEL-positive cells in ectopic lesions was quantified using QuPath. H-scores for Nr4a1 and Nr4a2 expression in each cell compartment were also determined using QuPath. Abbreviations: DIM-3,5, 1,1-bis(3′-indolyl)-(3,5-disubstitutedphenyl)methane; IHC, immunohistochemistry; N.S., not significant; TUNEL, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling.

Article Snippet: For immunohistochemistry staining, antibodies against NR4A1 (#NB100-56745, RRID:AB_2153757) and NR4A2 (#AF2156, RRID:AB_2153894) from Novus Biologicals (Centennial, CO, USA) and Ki-67 (#ab16667, RRID:AB_302459) from Abcam were used.

Techniques: Control, Luciferase, Activity Assay, In Vivo Imaging, Software, TUNEL Assay, Expressing, Immunohistochemistry, End Labeling

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