Journal: ACS Omega

Article Title: Conformationally Restricted Dipeptide-Based Nanoparticles for Delivery of siRNA in Experimental Liver Cirrhosis

doi: 10.1021/acsomega.2c05292

Figure Lengend Snippet: (a) Gel retardation assay showing complete complexation of siRN4A1 with RΔFRGD NPs at 1:40 (siNR4A1/RΔFRGD, w/w). (b) ζ-Potential of the RΔFRGD-siNR4A1 nanocomplex, 1.95 ± 0.18 mV ( n = 3). (c) Transmission electron microscopy image of the RΔFRGD-siNR4A1 nanocomplex ( n = 3).

Article Snippet: siNR4A1 was provided by Eurogentec (Seraing, Belgium).

Techniques: Electrophoretic Mobility Shift Assay, Transmission Assay, Electron Microscopy

Journal: ACS Omega

Article Title: Conformationally Restricted Dipeptide-Based Nanoparticles for Delivery of siRNA in Experimental Liver Cirrhosis

doi: 10.1021/acsomega.2c05292

Figure Lengend Snippet: Cytotoxicity of RΔFRGD NPs and the RΔFRGD-siNR4A1 nanocomplex treated with four cell lines and monitored by the MTT assay. Percentage cell viability against WRL 68, Hep G2, Huh7, and HEK 293T cells treated with different concentrations of (a) RΔFRGD NPs and (c) RΔFRGD-siNR4A1 nanocomplex. (b, d) Percentage hemolysis for RΔFRGD NPs and the RΔFRGD-siNR4A1 nanocomplex. (e) IFN-γ response for RΔFRGD at different concentrations and two well-known peptide-based antigens as the positive control, NYESO-1 and MAGE-3, in vitro coculture of T cells with DCs. The data has been presented as mean ± standard deviation.

Article Snippet: siNR4A1 was provided by Eurogentec (Seraing, Belgium).

Techniques: MTT Assay, Positive Control, In Vitro, Standard Deviation

Journal: ACS Omega

Article Title: Conformationally Restricted Dipeptide-Based Nanoparticles for Delivery of siRNA in Experimental Liver Cirrhosis

doi: 10.1021/acsomega.2c05292

Figure Lengend Snippet: (a) Confocal microscopy images of healthy and cirrhotic rats intravenously administered with labeled RΔFRGD NPs, showing significant accumulation of the NPs in the liver of cirrhotic rats ( n = 3). (b) NR4A1 mRNA expression in healthy and experimental liver cirrhotic rats ( n = 6). (c) Quantitative real-time PCR ( n = 6) and (d) relative protein expression of NR4A1 in cirrhotic liver treated with the RΔFRGD-siNR4A1 nanocomplex, determined by western blotting. Histopathological images for cirrhotic liver sections stained with (e) hematoxylin and eosin stain (H&E) indicating the presence of enhanced inflammatory cell infiltration in the group treated with the RΔFRGD-siNR4A1 nanocomplex (periceptal nuclei in blue) as compared to the vehicle group. (f) Masson’s trichrome (MT) showing significant aggravation in hepatic fibrosis in the RΔFRGD-siNR4A1-treated group as the red stain has been pulled out by collagen in this group. *BV, blood vessels.

Article Snippet: siNR4A1 was provided by Eurogentec (Seraing, Belgium).

Techniques: Confocal Microscopy, Labeling, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, H&E Stain

Journal: ACS Omega

Article Title: Conformationally Restricted Dipeptide-Based Nanoparticles for Delivery of siRNA in Experimental Liver Cirrhosis

doi: 10.1021/acsomega.2c05292

Figure Lengend Snippet: Relative mRNA expression analysis of inflammatory markers for (a) IL-6, (b) TNF-α, and (c) α-SMA for RΔFRGD-Veh and RΔFRGD-siNR4A1 nanocomplexes.

Article Snippet: siNR4A1 was provided by Eurogentec (Seraing, Belgium).

Techniques: Expressing

Journal: ACS Omega

Article Title: Conformationally Restricted Dipeptide-Based Nanoparticles for Delivery of siRNA in Experimental Liver Cirrhosis

doi: 10.1021/acsomega.2c05292

Figure Lengend Snippet: Effects of RΔFRGD-veh/siNR4A1 on Hepatic and Systemic Hemodynamic in CCl 4 Experimental Cirrhotic Rats a

Article Snippet: siNR4A1 was provided by Eurogentec (Seraing, Belgium).

Techniques:

Journal: ACS Omega

Article Title: Conformationally Restricted Dipeptide-Based Nanoparticles for Delivery of siRNA in Experimental Liver Cirrhosis

doi: 10.1021/acsomega.2c05292

Figure Lengend Snippet: Effects of RΔFRGD-veh/siNR4A1 on Biochemical Parameters in CCl 4 Experimental Cirrhotic Rats a

Article Snippet: siNR4A1 was provided by Eurogentec (Seraing, Belgium).

Techniques:

Journal: American Journal of Cancer Research

Article Title: Bis-indole derived nuclear receptor 4A1 (NR4A1) antagonists inhibit TGFβ-induced invasion of embryonal rhabdomyosarcoma cells

doi:

Figure Lengend Snippet: NR4A1-Bcl-2 interactions and mitochondrial damage. RD cells were transfected with siCtrl/siBcl-2 oligonucleotides (A), treated with DMSO, CDIM8 alone or in combination with GSH (5 mM) (B) or transfected with siCtrl/siBcl-2 and treated with DMSO or CDIM8 (C) and at the end of the treatment period whole cell lysates were analyzed by western blots. (D) RD cells were treated with DMSO, CDIM8 (15 µM), siCtrl or siBcl-2/CDIM8 and the levels of ROS were determined as outlined in the Materials and Methods. (E) Electron microscopic analysis of mitochondria in RD cells treated with DMSO or 15 µM CDIM8 was determined as outlined in the Methods.

Article Snippet: Formaldehyde, MG-132, β-actin antibody, M2 Flag antibody, β-catenin primers for PCR, and oligonucleotides for RNAi experiments (siβ-catenin, siNR4A1, siIL-24, siBcl-2) were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Transfection, Western Blot

Journal: Endocrinology

Article Title: Bis-Indole–Derived NR4A1 Ligands and Metformin Exhibit NR4A1-Dependent Glucose Metabolism and Uptake in C2C12 Cells

doi: 10.1210/en.2017-03049

Figure Lengend Snippet: Confocal microscopic analysis of Glut4 induction and effects of NR4A1 knockdown. C2C12 cells were transfected with si-Ctl (nonspecific oligonucleotide) or siNR4A1. After 72 hours, cells were treated with DMSO (control), 10 μM DIM-C-pPhOH-3-Cl-5-OCH3, or 1 mM metformin. Cells were then fixed, immunostained with β-actin, Glut4 antibodies, and DAPI, merged, and analyzed by confocal microscopy.

Article Snippet: Both siNR4A1 (Santa Cruz Biotechnology, Dallas, TX) and nontargeted control small interfering RNAs were used.

Techniques: Knockdown, Transfection, Control, Confocal Microscopy

Journal: Endocrinology

Article Title: Bis-Indole–Derived NR4A1 Ligands and Metformin Exhibit NR4A1-Dependent Glucose Metabolism and Uptake in C2C12 Cells

doi: 10.1210/en.2017-03049

Figure Lengend Snippet: NR4A1 knockdown attenuates the effects of DIM-C-pPhOH-3-Cl-5-OCH3 and metformin. C2C12 cells were transfected with si-Ctl (nonspecific oligonucleotide; white bar) or siNR4A1. After 72 hours, cells were treated with DIM-C-pPhOH-3-Cl-5-OCH3 or metformin, and effects on (A) NR4A1/Glut4 and (B) glycolytic gene expression were determined. (C) C2C12 cells were treated as described in (A), and whole cell lysates were analyzed by western blots. (D) Cells were treated as described in (A–C), and effects of siNR4A1 on compound-induced glucose uptake were determined (glucose uptake in control cells was set at 1.0). (A, B, D) Results are expressed as means ± SE for three replicate determinations. Significantly (P < 0.05) decreased activity due to NR4A1 knockdown is indicated (*). Conc, concentration.

Article Snippet: Both siNR4A1 (Santa Cruz Biotechnology, Dallas, TX) and nontargeted control small interfering RNAs were used.

Techniques: Knockdown, Transfection, Gene Expression, Western Blot, Control, Activity Assay, Concentration Assay

Journal: PLoS ONE

Article Title: Interaction of ApoA-IV with NR4A1 and NR1D1 Represses G6Pase and PEPCK Transcription: Nuclear Receptor-Mediated Downregulation of Hepatic Gluconeogenesis in Mice and a Human Hepatocyte Cell Line

doi: 10.1371/journal.pone.0142098

Figure Lengend Snippet: (A) HepG2 cells were treated with r-h-apoA-IV-GFP or GFP (control) for 6 h. Nuclear proteins were extracted from the cells, and immunoprecipitation was performed using an anti-GFP antibody. The precipitates were analyzed for the presence of ApoA-IV-GFP and NR4A1 by western blotting. (B) The colocalization of exogenous ApoA-IV and endogenous NR4A1 at the human G6Pase promoter was detected using ChIP. HepG2 cells were treated as described above for coimmunoprecipitation. Immunoprecipitation was performed using anti-NR4A1 and anti-apoA-IV antibodies. Primers were used to amplify the RORE sequence in the human G6Pase promoter or the GAPDH promoter (control) by PCR. The mean ± SE of three samples is shown (* P < 0.05 vs. vehicle control). (C) The effect of NR4A1 on G6Pase transcription in HEK-293 cells was examined using a luciferase reporter assay. HEK-293 cells were transfected with the G6Pase -luciferase reporter plasmid, human NR4A1 plasmid, renilla luciferase control reporter plasmid, and siNR4A1, control siRNA, or an equivalent volume of solvent for 24 h. Cells were also transfected with the G6Pase-luciferase and renilla luciferase reporter plasmids and the pcDNA3.1 plasmid as a control. The transfected cells were treated with recombinant human ApoA-IV protein or vehicle control for 24 h, and relative luciferase activity was measured. Relative luciferase activities (right) are presented as the mean ± SE of at least three samples from three independent experiments (*** P < 0.001 vs. pcDNA or siC controls). (D) Western blotting of cells transfected with the pcDNA plasmid and no siRNA (pcDNA), cells transfected with the NR4A1 expression plasmid and no siRNA (NR4A1), cells cotransfected with the control siRNA (siC), and cells cotransfected with the siNR4A1 (siNR4A1).

Article Snippet: The siNR1D1 (Cat. no. SASI_Mm01_00116940), which targeted the mouse NR1D1 mRNA, and the siNR4A1 (Cat. no. SASI_Mm01_00077215), which targeted the mouse NR4A1 mRNA, were purchased from Sigma-Aldrich.

Techniques: Immunoprecipitation, Western Blot, Sequencing, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Recombinant, Activity Assay, Expressing

Journal: PLoS ONE

Article Title: Interaction of ApoA-IV with NR4A1 and NR1D1 Represses G6Pase and PEPCK Transcription: Nuclear Receptor-Mediated Downregulation of Hepatic Gluconeogenesis in Mice and a Human Hepatocyte Cell Line

doi: 10.1371/journal.pone.0142098

Figure Lengend Snippet: Primary mouse hepatocytes were transfected with siNR4A1 or control siRNA (siC) for 48 h, and treated with 20 μg/mL r-m-apoA-IV or an equivalent volume of PBS (vehicle control) for 6 h or overnight. The levels of the (A) PEPCK and (B) G6Pase mRNAs were measured by qRT-PCR at 6 h posttreatment. (C) The levels of the NR4A1 and GAPDH (control) proteins were assessed by western blotting to demonstrate the inhibition of NR4A1 protein expression at 6 h posttreatment. (D) The level of glucose in the culture medium was also measured at 24 h posttreatment. Data are presented as the mean ± SE of at least three samples from three independent experiments (* P < 0.05, ** P < 0.01, and *** P < 0.001 vs. vehicle or siC control).

Article Snippet: The siNR1D1 (Cat. no. SASI_Mm01_00116940), which targeted the mouse NR1D1 mRNA, and the siNR4A1 (Cat. no. SASI_Mm01_00077215), which targeted the mouse NR4A1 mRNA, were purchased from Sigma-Aldrich.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Inhibition, Expressing

Journal: PLoS ONE

Article Title: Interaction of ApoA-IV with NR4A1 and NR1D1 Represses G6Pase and PEPCK Transcription: Nuclear Receptor-Mediated Downregulation of Hepatic Gluconeogenesis in Mice and a Human Hepatocyte Cell Line

doi: 10.1371/journal.pone.0142098

Figure Lengend Snippet: (A) Primary mouse hepatocytes were transiently transfected with siNR4A1, siNR1D1, or control siRNA (siC) for 48 h. (Lower panel) The levels of the NR1D1 and NR4A1 mRNAs were measured, relative to the cyclophilin control, using qRT-PCR. (Upper panel) The levels of the NR1D1 and NR4A1 proteins were measured, relative to the GAPDH control, by western blotting. Data are presented as the mean ± SE of at least three samples from three independent experiments (* P < 0.05, and ** P < 0.01 vs. vehicle or siC control). (B) Diagram depicting the roles of ApoA-IV, NR4A1, and NR1D1 in the downregulation of hepatic glucose production. The ApoA-IV-induced expression of NR4A1 and NR1D1 represses the transcription of G6Pase and PEPCK in hepatocytes, which in turn reduces glucose output. The expression of NR4A1 and NR1D1 is also regulated by bidirectional feedback, in which each NR represses the expression of the other.

Article Snippet: The siNR1D1 (Cat. no. SASI_Mm01_00116940), which targeted the mouse NR1D1 mRNA, and the siNR4A1 (Cat. no. SASI_Mm01_00077215), which targeted the mouse NR4A1 mRNA, were purchased from Sigma-Aldrich.

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