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: 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: 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: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: α-Lipoic Acid Prevents Neointimal Hyperplasia Via Induction of p38 Mitogen-Activated Protein Kinase/Nur77-Mediated Apoptosis of Vascular Smooth Muscle Cells and Accelerates Postinjury Reendothelialization

doi: 10.1161/atvbaha.110.212308

Figure Lengend Snippet: Figure 2. ALA enhances the expression and cytoplasmic localization of Nur77. A, ALA increased Nur77 expression at a tran- scriptional level. At 24 hours after seeding of VSMCs, medium was replaced with fresh complete medium containing ALA. Cells were then incubated for indicated times and harvested for semiquantitative RT-PCR (left). Levels of Nur77 mRNA were normal- ized against those of -actin, and data in the bar graph are the meanSE of 3 inde- pendent measurements. *P0.05 and **P0.01 vs control. Transient transfection assay results are also given (right). VSMCs were transfected with Nur77 pro-Luc and treated with ALA for 24 hours. Luciferase activity was normalized to -galactosidase activity. *P0.05 vs reporter alone. B, Immunocytochemical staining for Nur77 in VSMCs treated with ALA, 2 mmol/L. C, Subcellular localization of Nur77 was detected by immunofluorescence. VSMCs were treated with ALA for 2 hours and im- munostained with anti-Nur77 and anti–heat shock protein (HSP) 60 antibodies. Cells were stained with 4,6-diamidino-2-phenylindole (DAPI) to identify nuclei. Nur77, HSP60 (mito- chondria), and DAPI (nuclei) were visualized by confocal microscopy; and the images were overlaid. D, ALA stimulates the interac- tion between Nur77 and Bcl-2. VSMCs were treated with ALA, cell lysates were immuno- precipitated using anti–Nur77 antibody, and the immune complex was subjected to West- ern blotting with anti–Bcl-2 antibody. Con indicates control; Nur77 pro-Luc, Nur77 promoter-driven luciferase reporter.

Article Snippet: Sections were then incubated with goat polyclonal anti-Nur77 (C-19) antibody (Santa Cruz Biotechnology) or mouse monoclonal anti- CD31/PECAM-1 antibody (Millipore, MAB1393, 1:50 dilution) followed by Polink-2 Plus HRP Anti-Goat DAB Detection kit (Golden Bridge International, Inc., Mukilteo, WA) or UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen (Thermo Fisher Scientific Inc., Fremont, CA), respectively, according to the manufacturers’ instructions.

Techniques: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Control, Transient Transfection Assay, Transfection, Luciferase, Activity Assay, Staining, Confocal Microscopy

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: α-Lipoic Acid Prevents Neointimal Hyperplasia Via Induction of p38 Mitogen-Activated Protein Kinase/Nur77-Mediated Apoptosis of Vascular Smooth Muscle Cells and Accelerates Postinjury Reendothelialization

doi: 10.1161/atvbaha.110.212308

Figure Lengend Snippet: Figure 5. ALA prevents neointimal hyperplasia via induction of Nur77- mediated VSMC apoptosis. A, Repre- sentative photomicrographs of cross- sections of uninjured (con) and BI carotid arteries of rats given saline or ALA, 50 and 100 mg/kg per day, from 3 days before to 14 days after BI (hematoxylin-eosin [H&E] stain, original magnification 100). B, TUNEL staining (left) and immunohistochemical staining (right) for Nur77 were performed on the cross-sections (hematoxylin stain, origi- nal magnification 200). C, Neointimal VSMCs are more sensitive to ALA- induced apoptosis than medial VSMCs. Neointimal and medial VSMCs were incubated with ALA, 1 and 2 mmol/L, for 72 hours and analyzed by flow cytometry. D, Adenoviruses encoding shRNA against Nur77 (Ad-shNur77) markedly diminished the inhibitory effect of ALA on neointimal hyperplasia. Ad-shNur77 or adenoviruses encoding control shRNA (Ad-shRNA) were infused into BI carotid arteries of rats immediately after injury, and rats were given ALA from 3 days before to 14 days after BI (H&E stain, original magni- fication 100). Immunohistochemical staining for Nur77 (brown) was per- formed (hematoxylin stain, original magnification 200).

Article Snippet: Sections were then incubated with goat polyclonal anti-Nur77 (C-19) antibody (Santa Cruz Biotechnology) or mouse monoclonal anti- CD31/PECAM-1 antibody (Millipore, MAB1393, 1:50 dilution) followed by Polink-2 Plus HRP Anti-Goat DAB Detection kit (Golden Bridge International, Inc., Mukilteo, WA) or UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen (Thermo Fisher Scientific Inc., Fremont, CA), respectively, according to the manufacturers’ instructions.

Techniques: Saline, Staining, TUNEL Assay, Immunohistochemical staining, Incubation, Cytometry, shRNA, Control

Journal: Psychiatry Investigation

Article Title: Translational Approach to Social Isolation During a Global Pandemic: Hippocampal Somatic Mutation and Stress

doi: 10.30773/pi.2024.0178

Figure Lengend Snippet: Changes in the behavior and protein expression in maternal separation and socially isolated mice. A: Description of the mouse behavior experiment method. Mouse behavioral experiments were conducted at week 7. B-E: There was no difference in the OFT between the CTL and the MS. In EPM to measure anxiety-like behavior, the time spent in the open arm increased in the MS compared to CTL. Considering fear-related behavior, the MS showed increased freezing in the contextual fear test compared to the CTL (N=11 mice [CTL], 8 [MS], Student’s t-test, *p<0.05). F: Compared to the CTL, the MS+SS showed significant increase in the CRHR1 and CRHR2, which are related to stress and hypothalamic-pituitary-adrenal axis. Moreover, neurogenesis-related SOX2 and DCX demonstrated an increase in the social isolation group (CTL+SS). TrKB did not show a difference between the groups. In the t-test, CRHR1 increased in the MS and CTL+SS, and DCX increased in the CTL+SS group. It was standardized based on the protein expression ratio of the hippocampi of the control mice group (N=9 mice [CTL], 9 [CTL+SS], 9 [MS], 9 [MS+SS]; one-way analysis of variance,*p<0.05; **p<0.01, post-hoc Tukey; Student’s t-test, † p<0.05). G: Representative images of immunoblots in the mouse hippocampus. OFT, locomotion; CTL, control group; MS, maternal separation group; SS, social isolation after maternal separation group; EPM, elevated-plus maze; CRHR1, corticotropin-releasing hormone receptor 1; CRHR2, corticotropin- releasing hormone receptor 2; TrKB, tropomyosin receptor kinase B (mouse); SOX2, SRY (sex-determining region Y)-box 2; DCX, doublecortin.

Article Snippet: The cell lysates (20 μg) were electrophoresis using SDS gels and transferred to nitrocellulose membranes and the incubated with tropomyosin receptor kinase B (TrkB) antibody (1:1,000, goat, AF1494; R&D systems, Minneapolis, MN, USA), corticotropin-releasing hormone receptor 1 (CRHR1) antibody (1:1,000, goat, GRX88961; GeneTex, Irvine, CA, USA), CRHR2 antibody (1:1,000, rabbit, AP17244PU-N; OriGene Technologies Inc., Rockville, MD, USA), Grin2B antibody (1:1,000, rabbit, 21920-1-AP; Proteintech, Rosemont, IL, USA), HTR1B antibody (1:1,000, rabbit, OAAF02797; Aviva Systems Biology, San Diego, CA, USA), doublecortin (DCX) antibody (1:1,000, rabbit, #4604; Cell Signaling Technology, Danvers, MA, USA), MAP2 antibody (1:1,000, rabbit, #4542; Cell Signaling Technology), SOX2 antibody (1:1,000, rabbit, #23064; Cell Signaling Technology), NeuN antibody (1:1,000, rabbit, #24307; Cell Signaling Technology), Erk antibody (1:1,000, rabbit, #4695; Cell Signaling Technology), p-Erk (thy202/204) antibody (1:1,000, rabbit, #9101; Cell Signaling Technology), mTOR antibody (1:1,000, rabbit, #2983; Cell Signaling Technology), p-mTOR antibody (1:1,000, rabbit, #5536; Cell Signaling Technology) and β-actin antibody (1:10,000, mouse, Sc-47778 HRP; Santa Cruz Biotechnology, Dallas, TX, USA) for 16 hours at 4°C.

Techniques: Expressing, Isolation, Control, Western Blot