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 & Disease

Article Title: PARL stabilizes mitochondrial BCL-2 via Nur77-mediated scaffolding as a therapeutic strategy for Parkinson’s disease

doi: 10.1038/s41419-025-08035-8

Figure Lengend Snippet: A Western blot analysis of extracts Nur77, PARL, BCL-2, and BAX. Overexpression of Nur77 increased PARL and reduced apoptosis of SH-SY5Y cells. Data are representative of three independent experiments, which gave similar results. B SH-SY5Y cells were co-transfected with Nur77 plasmid for 24 h and treated with (or without) MPP + for an additional 24 h. Cell lysates were extracted for immunoblot analysis of Nur77, PARL, BCL-2, and BAX. Representative WB is shown. C Nur77 was overexpressed in SH-SY5Y cells with low PARL expression, and the expressions of BCL-2, BAX, Caspase3 and Cleaved-Caspase3 were detected by western blotting. D , E Fluorescence MitoTracker Green staining in Control and MPP + -treated SH-SY5Y cells; mitochondria (GREEN) and Nur77 (RED). Bar = 5 μm. F , G Fluorescence MitoTracker Green staining in Nur77-Flag and MPP + -treated Nur77-Flag cells; mitochondria (GREEN) and Nur77-Flag (RED). Bar = 5 μm. (ns not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 compared with the control group; n = 3, mean ± SEM).

Article Snippet: The PVDF membranes was incubated overnight at 4 °C with primary antibodies (1:2000) against PARL (Abcam Cat# ab118554, RRID:AB_10903849), Nur77 (Cell Signaling Technology Cat# 3960, RRID:AB_2153738), BCL-2 (Proteintech Cat# 26593-1-AP, RRID:AB_2818996), BAX (Abcam Cat# ab32503, RRID:AB_725631), Caspase3 (Proteintech Cat# 66470-2-Ig, RRID:AB_2876892), Cleaved-Caspase3 (Cell Signaling Technology Cat# 9664/9664 P, RRID:AB_2070042).

Techniques: Western Blot, Over Expression, Transfection, Plasmid Preparation, Expressing, Fluorescence, Staining, Control

Journal: Cell Death & Disease

Article Title: PARL stabilizes mitochondrial BCL-2 via Nur77-mediated scaffolding as a therapeutic strategy for Parkinson’s disease

doi: 10.1038/s41419-025-08035-8

Figure Lengend Snippet: A Representative immunofluorescence images of TH (green; tyrosine hydroxylase) and TUNEL (red; apoptotic nuclei) co-staining in the substantia nigra pars compacta (SNpc) of PD mice. Nuclei were counterstained with DAPI (blue). Scale bar: 50 µm. Top panel : Individual channels; bottom panels : Merged channels. Quantification of TH-positive neurons ( B ) and TUNEL-positive apoptotic nuclei ( C ) in the SNpc across experimental groups. D Western blot analysis of TH, PARL, Nur77, BCL-2, BAX, Caspase 3, and Cleaved-Caspase 3 protein levels in midbrain lysates. β-actin was used as a loading control. Low PARL expression abrogates the neuroprotective effects of Nur77 overexpression in PD mice. (ns not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 compared with the control group; n = 3, mean ± SEM).

Article Snippet: The PVDF membranes was incubated overnight at 4 °C with primary antibodies (1:2000) against PARL (Abcam Cat# ab118554, RRID:AB_10903849), Nur77 (Cell Signaling Technology Cat# 3960, RRID:AB_2153738), BCL-2 (Proteintech Cat# 26593-1-AP, RRID:AB_2818996), BAX (Abcam Cat# ab32503, RRID:AB_725631), Caspase3 (Proteintech Cat# 66470-2-Ig, RRID:AB_2876892), Cleaved-Caspase3 (Cell Signaling Technology Cat# 9664/9664 P, RRID:AB_2070042).

Techniques: Immunofluorescence, TUNEL Assay, Staining, Western Blot, Control, Expressing, Over Expression

Journal: Cell Death & Disease

Article Title: PARL stabilizes mitochondrial BCL-2 via Nur77-mediated scaffolding as a therapeutic strategy for Parkinson’s disease

doi: 10.1038/s41419-025-08035-8

Figure Lengend Snippet: A Predicted Nur77-PARL binding interface by AlphaFold2. Nur77 (purple) interacts with PARL (blue) via key residues: salt bridge (Nur77-GLU525/PARL-ARG41), hydrogen bonds (Nur77-GLN519/PARL-ARG42; Nur77-ARG318/PARL-ARG9). B Co-immunoprecipitation (co-IP) validation of Nur77-PARL interaction in MPP + (1 mM, 24 h)-treated SH-SY5Y cells. Lysates immunoprecipitated with anti-Nur77 antibody. IgG: isotype control. C Schematic of PARL truncations (GST-tagged fragments) used for binding assays. Numbers denote amino acid positions. D GST pull-down assay showing Nur77 binds preferentially to PARL-GST1 (1-167 aa). Input: 100% lysate; GST control: empty vector. E , F Western blot analysis of BCL-2 levels after PARL peptide inhibition. β-actin: loading control. G AlphaFold2-predicted PARL-BCL-2 complex. PARL (yellow) binds BCL-2 (blue) via hydrogen bonds (PARL-TRP158/BCL-2-GLU13; PARL-ARG76/BCL-2-SER50) and a π-π interaction (PARL-TRP11/BCL-2-HIS20). H Co-IP confirms PARL-BCL-2 interaction in MPP + -treated cells. Lysates immunoprecipitated with anti-PARL antibody. I , J GST pull-down assays demonstrate Nur77 knockdown disrupts PARL-BCL-2 binding. PARL-GST2 (1-167 aa) shows strongest BCL-2 affinity.

Article Snippet: The PVDF membranes was incubated overnight at 4 °C with primary antibodies (1:2000) against PARL (Abcam Cat# ab118554, RRID:AB_10903849), Nur77 (Cell Signaling Technology Cat# 3960, RRID:AB_2153738), BCL-2 (Proteintech Cat# 26593-1-AP, RRID:AB_2818996), BAX (Abcam Cat# ab32503, RRID:AB_725631), Caspase3 (Proteintech Cat# 66470-2-Ig, RRID:AB_2876892), Cleaved-Caspase3 (Cell Signaling Technology Cat# 9664/9664 P, RRID:AB_2070042).

Techniques: Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Biomarker Discovery, Control, Pull Down Assay, Plasmid Preparation, Western Blot, Inhibition, Knockdown

Journal: Scientific Reports

Article Title: Pro-angiogenic Ginsenosides F1 and Rh1 Inhibit Vascular Leakage by Modulating NR4A1

doi: 10.1038/s41598-019-41115-2

Figure Lengend Snippet: F1 and Rh1 regulate NR4A1 expression and transcriptional activity. ( A ) Effect of F1 and Rh1 on VEGF-induced NR4A1 mRNA expression in HUVECs. The mRNA expression of NR4A1 after treatment with F1 or Rh1 in VEGF-stimulated cells was analyzed by Real-time RT PCR. β-actin was used as a reference gene in the analysis. Data are presented as mean ± SD (n = 3). ( B ) Effect of F1 and Rh1 on VEGF-induced NR4A1 protein expression in HUVECs. The protein expressions of NR4A1 after treatment with F1 or Rh1 in VEGF-stimulated cells were assessed with by Western blot analysis. Data are presented as mean ± SD (n = 3). Full-length blots are presented in Supplementary Fig. ( C ) Effect of F1 and Rh1 on NR4A1 transcriptional activity. NurRE-Luc reporter plasmid-transfected HEK293T cells were treated with the indicated condition for 1 hr (0.1 μM of Csn-B and 10 μM of F1 or Rh1). NR4A1 transcriptional activities were analyzed using the dual luciferase reporter gene assay. Data are presented as mean ± SD (n = 3). ( D ) Effect of F1 and Rh1 on Csn-B-induced NR4A1 mRNA expression in HUVECs. NR4A1 mRNA expressions after co-treatment of F1 or Rh1 with Csn-B were analyzed by Real-time RT PCR. β-actin was used as a reference gene in the analysis. Data are presented as mean ± SD (n = 3). ( E ) Effect of F1 and Rh1 on the VEGF-induced junctional disruption in HUVECs. The cells were treated with F1 or Rh1 (10 μM, 1 hr) after VEGF stimulation (2.5 nM, 1 hr). VE-cadherin (green) was stained with immunofluorescence and the nucleus (blue) was stained by DAPI. Scale bars, 10 μm. ( F ) Effect of F1 and Rh1 on the Csn-B-induced junctional disruption in HUVECs. The cells were co-treated with 1 μM of Csn-B and 10 μM of F1 or Rh1 for an hour. VE cadherin (green) was stained by immunofluorescence and the nucleus (blue) was stained by DAPI. Scale bars, 10 μm. ( G ) Schematic representation of the inhibitory effect of F1 and Rh1 on VEGF-induced vascular leakage in ECs. Statistical significance was calculated based on three independent experiments (* P < 0.05; ** P < 0.01; *** P < 0.001, P-values between depicted groups).

Article Snippet: NR4A1 was detected with anti-NR4A1 antibody (1: 100 dilution; Cat#sc-365113, Santa Cruz) and the anti-β-actin antibody (1:1000 dilution; Cat#ab8227, Abcam) was used to normalize the loading cell lysates.

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Transfection, Luciferase, Reporter Gene Assay, Disruption, Staining, Immunofluorescence

Journal: Cell reports

Article Title: Lactic acid induces transcriptional repression of macrophage inflammatory response via histone acetylation

doi: 10.1016/j.celrep.2024.113746

Figure Lengend Snippet:

Article Snippet: NR4A1 (Nur77) siRNA , Santa Cruz Biotechnology , Sc36110.

Techniques: Recombinant, Luciferase, Reporter Assay, Staining, Control, Software

Journal: Scientific reports

Article Title: Orphan nuclear receptor Nur77 affects cardiomyocyte calcium homeostasis and adverse cardiac remodelling.

doi: 10.1038/srep15404

Figure Lengend Snippet: Figure 1. Isoproterenol increases Nur77 expression in cardiomyocytes and Nur77 knockdown enhances isoproterenol-induced cardiomyocyte hypertrophy. (a) Northern blot analysis of Nur77 expression in healthy human tissues. Heart and skeletal muscle exhibited highest endogenous Nur77 expression relative to loading control β -actin. Blots have been prepared under the same experimental conditions. (b) Significantly up-regulated NR4A gene expression in NRVMs after 1 h of isoproterenol (ISO) stimulation (n = 3), with Nur77 mRNA levels showing highest abundance. (c) Representative example of nuclear Nur77 protein expression in cardiomyocytes of healthy WT C57Bl/6 mice as assessed by immunohistochemistry. Photomicrograph shown at 200× magnification. Nur77 protein expression is significantly enhanced in nuclei of NRVMs after isoproterenol stimulation for 1 and 3 h (n = 4). (d) NRVMs are significantly larger after siRNA-mediated Nur77 knockdown (siNur77) compared to control-transfected (siCon) cells under both control conditions and 48 h of isoproterenol stimulation, as assessed by α -Actinin staining in > 250 cells per group (n = 3). Photomicrographs are shown at 200× magnification. Boxplots represent median, inter-quartile range and minimum/maximum values; bars represent mean + SEM; *p < 0.05, **p< 0.01, ***p < 0.001.

Article Snippet: Nur77 protein expression was assessed by immunohistochemistry using anti-Nur77 antibody (Santa Cruz) and anti-rabbit-IgG-HRP polymer (BrightVision, ImmunoLogic, Duiven, The Netherlands).

Techniques: Expressing, Knockdown, Northern Blot, Control, Gene Expression, Immunohistochemistry, Transfection, Staining

Journal: Scientific reports

Article Title: Orphan nuclear receptor Nur77 affects cardiomyocyte calcium homeostasis and adverse cardiac remodelling.

doi: 10.1038/srep15404

Figure Lengend Snippet: Figure 5. Nur77-KO mice exhibit enhanced adverse cardiac remodelling after chronic β-adrenergic stimulation. WT (n = 16) and 13 surviving Nur77-KO mice were analysed after 1 week of chronic isoproterenol (ISO) infusion. (a) Heart weight/tibia length (HW/TL) ratio was higher after isoproterenol stimulation and further increased in Nur77-KO mice compared to WT mice. Tibia length did not differ between WT and Nur77-KO mice. (b) Cardiomyocyte cross-sectional area was significantly larger in isoproterenol-treated Nur77-KO mice compared to treated WT mice, as assessed by fluorescent wheat germ agglutinin staining in > 75 cells per heart. Photomicrographs are shown at 630× magnification. (c) RT-PCR analysis showed significant up-regulation of foetal gene expression after isoproterenol treatment. Nur77-KO mice exhibited a significant increase in acta1 and myh7 after isoproterenol, when compared to WT. anp: atrial natriuretic peptide; bnp: brain natriuretic peptide; acta1: α 1 skeletal actin; myh7: β -myosin heavy chain. (d) Collagen deposition was assessed by Masson’s Trichrome staining assessed by quantitative morphometry, photomicrographs are shown at 50× magnification. The left ventricle and septum of Nur77-KO mice was affected stronger by interstitial fibrosis (blue) after isoproterenol than WT mice. (e) Calcineurin phosphatase activity is significantly higher in myocardium of control Nur77-KO mice. After chronic isoproterenol stimulation, calcineurin activity is significantly enhanced in WT hearts, while it does not further increase in Nur77-KO mice compared to Nur77-KO control mice (n = 3–4 in each group). Boxplots represent median, inter-quartile range and minimum/ maximum values; bars represent mean+ SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Nur77 protein expression was assessed by immunohistochemistry using anti-Nur77 antibody (Santa Cruz) and anti-rabbit-IgG-HRP polymer (BrightVision, ImmunoLogic, Duiven, The Netherlands).

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Activity Assay, Control

Journal: Scientific reports

Article Title: Orphan nuclear receptor Nur77 affects cardiomyocyte calcium homeostasis and adverse cardiac remodelling.

doi: 10.1038/srep15404

Figure Lengend Snippet: Figure 6. Attenuated pressure overload-induced adverse cardiac remodelling in Nur77-KO mice. WT (n = 12) and Nur77-KO (n = 11) mice were analysed after 28 days of TAC. (a) Left ventricle/tibia length (LV/TL) ratio was significantly lower in Nur77-KO mice after TAC than in WT mice. Tibia length did not differ between WT and Nur77-KO mice. (b) Cardiomyocytes from Nur77-KO mice were significantly smaller compared to cardiomyocytes from WT mice, as assessed by fluorescent wheat germ agglutinin staining in > 75 cells per heart. Photomicrographs are shown at 630× magnification. (c) Foetal gene expression after TAC was significantly down-regulated in Nur77-KO mice, as assessed by RT-PCR. anp: atrial natriuretic peptide; bnp: brain natriuretic peptide; acta1: α 1 skeletal actin; myh7: β -myosin heavy chain. (d) Collagen deposition as assessed by Masson’s Trichrome staining, photomicrograph shown at 100× magnification. Significantly less perivascular fibrosis was present in Nur77-KO mice compared to WT, while areas of interstitial fibrosis were not different. *p < 0.05, **p < 0.01. (e) Proposed mechanism for the role of Nur77 in cardiac remodelling induced by different insults. ISO: isoproterenol; TAC: transverse aortic constriction; AngII: angiotensin II; LAD: left anterior descending coronary artery; ROS: reactive oxygen species.

Article Snippet: Nur77 protein expression was assessed by immunohistochemistry using anti-Nur77 antibody (Santa Cruz) and anti-rabbit-IgG-HRP polymer (BrightVision, ImmunoLogic, Duiven, The Netherlands).

Techniques: Staining, Gene Expression, Reverse Transcription Polymerase Chain Reaction

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