Journal: Frontiers in Immunology

Article Title: NR4A Receptors Differentially Regulate NF-κB Signaling in Myeloid Cells

doi: 10.3389/fimmu.2017.00007

Figure Lengend Snippet: NR4As positively regulate TLR4 driven MIP-3α in human and murine monocyte/macrophage cells . (A) Undifferentiated THP-1 cells transduced with shRNA directed against scrambled non-target control (Sc), NR4A2 (A2), or NR4A3 (A3) were treated with 1 µg/ml lipopolysaccharide (LPS) for 8 h, followed by media collection. (B) Undifferentiated THP-1 cells transduced with shRNA directed against scrambled non-target control, NR4A2, or NR4A3 were treated with 1 µg/ml LPS for 0, 2, and 8 h. (C) Human primary PBMCs were exposed to 1 µg/ml LPS for 2 h. (D) Undifferentiated THP-1 cells were pretreated with 10 µM NF-κB inhibitor (NF-κBi) for 1 h followed by the addition of 1 µg/ml LPS for 2 h. (E,F) Mouse embryonic fibroblast (MEF) cells lacking p65 −/− and wild-type (WT) controls were exposed to 1 µg/ml LPS for 4 h (E) and 2 h (F) . (G) Murine raw mac 264.7 cells transduced with shRNA directed against scrambled non-target control (pretreated with 10 µM NF-κB inhibitor (NF-κBi) for 1 h) and NR4A2 were subsequently treated with of 1 µg/ml LPS for 1 h. (H) MEF cells lacking p65 −/− and WT controls were exposed to 1 µg/ml LPS for 2 h. (I,J) Undifferentiated THP-1 cells were pretreated with 100 nM Cytosporone-B (Csn-b) for 30 min followed by the addition of 100 ng/ml LPS for a further 1.5 h. Analysis: ELISA was performed at time indicated for MIP-3α protein (A) . RNA was isolated and RT-PCR was performed at indicated times to assess levels of MIP-3α, NR4A2, NR4A3, TNFα, and control gene GAPDH (B–D,F,H,I,J) . Whole-cell lysates were prepared at indicated time followed by Western blot analysis performed for both p65 and loading control β-tubulin (E) . Densitometric analysis included for Western blot data was determined using LI-COR ® Image Studio Lite version 3.1 and band density of target protein (p65) normalized to loading control (β-Tubulin) are displayed above relevant treatments. Un, untreated control. Data are expressed as fold over untreated control (FOC) or pg/ml ± SEM for n = minimum of three individual experiments. ** p < 0.01, *** p < 0.001 treatments compared to untreated control (Un). # p < 0.05, ## p < 0.01, ### p < 0.001 treatments compared displayed here using a bar attachment.

Article Snippet: MCP-1 and MIP-3α (RayBiotech) protein levels were measured using an enzyme-linked immunosorbent assay.

Techniques: Transduction, shRNA, Enzyme-linked Immunosorbent Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Immunology

Article Title: NR4A Receptors Differentially Regulate NF-κB Signaling in Myeloid Cells

doi: 10.3389/fimmu.2017.00007

Figure Lengend Snippet: NR4A2 does not require DNA binding in order to regulate TLR4-driven MIP-3α . (A) Graphical representation [showing ≈500 bp upstream and ≈200 downstream of the transcription start site (TSS) (TSS is indicated on graphic as a black arrowhead)] of the NR4A (shown as yellow box) and NF-κB (shown as purple box) DNA-binding motifs (NBRE and NRE, respectively) on the MIP-3α human and murine promoter analyzed using the Genomatix software suite program MatInspector for 1,000 bp upstream of the TSS. Expanded out regions marked by a blue lined box is ≈200 bp upstream of TSS showing the exact sequence of the NR4A and NF-κB-binding motifs. Human and murine promoter are shown aligned using the ApE plasmid editor software, red boxes indicate gaps, and red boxes with a # indicate a mismatch. (B) Mouse embryonic fibroblast (MEF) cells were transfected with 500 ng of wild-type (WT) p-CMX-NR4A2, p-CMX-NR4A2 mutant (C283G), and backbone control plasmid (BB). Following 48 h incubation, cells were treated with 1 µg/ml lipopolysaccharide (LPS) for 2 h. (C) MEF cells lacking p65 −/− and WT controls were exposed to 1 µg/ml LPS for 2 h. Analysis: RNA was isolated and subsequent qRT-PCR performed to assess levels of MIP-3, IL-6, and control gene β-actin (B,C) . Data are expressed as fold over untreated control (FOC) ± SEM for n = minimum of 2 (B) and 6 (C) individual experiments. * p < 0.05, ** p < 0.01 treatments compared to untreated control (Un). # p < 0.05 treatments compared displayed here using a bar attachment.

Article Snippet: MCP-1 and MIP-3α (RayBiotech) protein levels were measured using an enzyme-linked immunosorbent assay.

Techniques: Binding Assay, Software, Sequencing, Plasmid Preparation, Transfection, Mutagenesis, Incubation, Isolation, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: NR4A Receptors Differentially Regulate NF-κB Signaling in Myeloid Cells

doi: 10.3389/fimmu.2017.00007

Figure Lengend Snippet: NR4As are regulators of Relb expression during TLR4 stimulation . (A) Mouse embryonic fibroblast (MEF) cells lacking p65 −/− and wild-type (WT) control were exposed to 1 µg/ml lipopolysaccharide (LPS) for 2 h. (B) MEF cells lacking p65 −/− , Relb −/− , and WT control were exposed to 1 µg/ml LPS for 4 h. (C) MEF cells lacking Relb −/− and WT control were exposed to 1 µg/ml LPS for 2 h. (D) Undifferentiated THP-1 cells were pretreated with 10 µM NF-κB inhibitor (NF-κBi) for 1 h followed by the addition of 1 µg/ml LPS for 2 h. (E) Human primary PBMCs were exposed to 1 µg/ml LPS for 2 h. (F) Undifferentiated THP-1 cells transduced with shRNA directed against scrambled non-target control, NR4A2, or NR4A3 were treated with 1 µg/ml LPS for 2 h. Analysis: RNA was isolated and RT-PCR was performed at indicated times to assess levels of MIP-3α, Relb, and control gene GAPDH (A,C–F) . Whole-cell lysates were prepared at indicated time followed by Western blot analysis performed for both Relb and loading control β-tubulin (B) . Densitometric analysis included for Western blot data was determined using LI-COR ® Image Studio Lite version 3.1, and band density of target protein (Relb) normalized to loading control (β-Tubulin) are displayed above relevant treatments. Un, untreated control. Data are expressed as fold over untreated control (FOC) ± SEM for n = minimum of three individual experiments. ** p < 0.01, *** p < 0.001 treatments compared to untreated control (Un). # p < 0.05, ### p < 0.001 treatments compared displayed here using a bar attachment.

Article Snippet: MCP-1 and MIP-3α (RayBiotech) protein levels were measured using an enzyme-linked immunosorbent assay.

Techniques: Expressing, Transduction, shRNA, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Immunology

Article Title: NR4A Receptors Differentially Regulate NF-κB Signaling in Myeloid Cells

doi: 10.3389/fimmu.2017.00007

Figure Lengend Snippet: Summary diagram . TLR4 or TNFα receptor stimulation increases the activity of NF-κB and the expression of its target genes NR4A1–3 (NR4A), MCP-1, MIP-3α, and Relb. Loss of NR4A potentiates this induction of MCP-1, while reducing the induction of Relb and MIP-3α. Furthermore using mouse embryonic cells, we identify MIP-3α is a novel Relb target gene. Therefore, NR4A receptors act as repressors of inflammatory-NF-κB-driven MCP-1 (indicated by a red T bar) and enhancers of NF-κB-driven Relb and subsequently MIP-3α. Using a NR4A2 DNA-binding mutant reveals that NR4A2 does not require the ability to bind DNA in order to enhance/repress target genes simultaneously.

Article Snippet: MCP-1 and MIP-3α (RayBiotech) protein levels were measured using an enzyme-linked immunosorbent assay.

Techniques: Activity Assay, Expressing, Binding Assay, Mutagenesis