Journal: NPJ Aging and Mechanisms of Disease

Article Title: Clampdown of inflammation in aging and anticancer therapies by limiting upregulation and activation of GPCR, CXCR4

doi: 10.1038/s41514-018-0028-0

Figure Lengend Snippet: CXCR4 receptor regulates DNA-damage associated inflammation. For all experiments, media from treated cells (as indicated), was used for sandwich ELISA for determining levels of IL8 or IL6 pg/ml secreted per 10 3 cells, represented as fold change over control untreated cells. a Effect of activation of CXCR4-CXCL12 signaling during DNA damage on IL8 and IL6 cytokine secretion ( n > 6). b Effect of CXCR4 inhibition (AMD treatment) on IL6 production from senescent cells post-CXCL12 stimulation. Cells were treated with various compounds as indicated and IL6 levels in supernatant were analysed ( n = 3). c Effect of inhibition of Gαi by PTx treatment. Comparison of IL8 levels between HeLa cells treated with BrdU; BrdU + CXCL12 or BrdU + CXCL12 in presence of pertussis toxin (PTx) ( n = 3). d Effect of inhibition of Gαi by PTx treatment on MRC5 cells. Comparison of IL8 levels in MRC5, primary cells treated with BrdU; BrdU + CXCL12 and BrdU + CXCL12 in presence of PTx ( n = 3). For all experiments, results are represented as mean ± s.e.m. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.001 (Student’s t -test)

Article Snippet: For estimating extracellular levels of various cytokines using media collected from treated cells as indicated, BD OptiEIA™ Human IL8, IL6 and TGFβ ELISA kits (BD Biosciences, USA) were used as per manufacturer’s instructions.

Techniques: Sandwich ELISA, Activation Assay, Inhibition

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

Journal: Inflammation Research

Article Title: Staphylococcal protein A modulates inflammation by inducing interferon signaling in human nasal epithelial cells

doi: 10.1007/s00011-022-01656-1

Figure Lengend Snippet: S. aureus increased IFN signaling pathway in a CRS mouse model

Article Snippet: Interleukin-6 (IL-6) and Interleukin-8 (IL-8) protein levels were determined with IL-6 and IL-8 enzyme-linked immunosorbent assay (ELISA) kits (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer’s instructions after treatment of HNECs with 5% bacterial planktonic supernatants and purified SpA or control for 24 hours.

Techniques:

Journal: Inflammation Research

Article Title: Staphylococcal protein A modulates inflammation by inducing interferon signaling in human nasal epithelial cells

doi: 10.1007/s00011-022-01656-1

Figure Lengend Snippet: IL-6 and IL-8 secretion after challenge with SpA and bacterial supernatants. HNECs ( n = 3 donors) were treated with SpA and S. aureus supernatants (H1, H2, L1, L2) for 24 h. Interleukin-6 (IL-6) A and Interleukin-8 (IL-8) B protein levels were determined with enzyme-linked immunosorbent assays (ELISA). H1, H2 and L1, L2 = S. aureus clinical isolates having high (H1, H2) and low (L1, L2) SpA concentrations in planktonic supernatants. C = untreated control, KD-C = lipo3000 without siRNA control, KD = Ifgr 1 knockdown. The values are shown as means ± SEM. * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001; **** = P ≤ 0.0001, one-way ANOVA followed by Tukey’s multiple comparisons test

Article Snippet: Interleukin-6 (IL-6) and Interleukin-8 (IL-8) protein levels were determined with IL-6 and IL-8 enzyme-linked immunosorbent assay (ELISA) kits (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer’s instructions after treatment of HNECs with 5% bacterial planktonic supernatants and purified SpA or control for 24 hours.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Inhibition of adiponectin-mediated gene expression in vitro by monoclonal antibodies (mAbs). To test the ability of the mAb KH4–8 to block adiponectin function, ( a ) human osteoblasts and ( b ) human umbilical vein endothelial cells (HUVECs) were treated with adiponectin (ADIPO) or KH4–8 mAb (mAb) or both. The mAb (~120 μg/mL) and recombinant adiponectin (2.5 μg/mL) were mixed and incubated for 1 h before being used to treat cells. After 24-h treatment, the culture supernatants were collected and frozen, and interleukin-6 (IL-6) and IL-8 were measured by using enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA). The experiments were performed in quadruplicate. The data shown are representative of three independent experiments, and similar results were obtained with all three mAbs. Values are expressed as mean ± standard error of the mean. The expression levels of the factors were compared between groups by using the Mann–Whitney test. * P <0.05, ** P <0.01 versus the untreated group, # P <0.05, ## P <0.01 versus the group treated with adiponectin and mAb

Article Snippet: Culture supernatants from cells treated with mAb plus adiponectin were analyzed with IL-6 and IL-8 ELISA kits (BD Bioscience Korea, Seoul, Korea) in accordance with the protocol of the manufacturer.

Techniques: Inhibition, Expressing, In Vitro, Blocking Assay, Recombinant, Incubation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Anti-inflammatory effect of monoclonal antibodies (mAbs) KH4–8 and KH7–33 on the expression of serum pro-inflammatory cytokines of the collagen-induced arthritis mouse model. The serum levels of adiponectin, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and receptor activator of nuclear factor-kappa Β ligand (RANKL) were analyzed by using the Luminex system. Values are expressed as mean ± standard error of the mean. The expression levels of the factors were compared between groups ( n = 8) by using the Mann–Whitney test. ** P <0.01, * P <0.05 versus normal (NOR) group, and ## P <0.01, ## P <0.05 versus the control (CON) group. Abbreviations: ns not significant, pre prednisolone

Article Snippet: Culture supernatants from cells treated with mAb plus adiponectin were analyzed with IL-6 and IL-8 ELISA kits (BD Bioscience Korea, Seoul, Korea) in accordance with the protocol of the manufacturer.

Techniques: Expressing, Luminex, MANN-WHITNEY

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Louqin Zhisou Decoction Inhibits Mucus Hypersecretion for Acute Exacerbation of Chronic Obstructive Pulmonary Disease Rats by Suppressing EGFR-PI3K-AKT Signaling Pathway and Restoring Th17/Treg Balance

doi: 10.1155/2019/6471815

Figure Lengend Snippet: Primers sequence used for RT-qPCR.

Article Snippet: The primers of MUC5AC, ROR γ t, Foxp3, IL-17, IL-10, NE, MCP-1, and GAPDH served as internal control were designed and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and sequences were shown in .

Techniques: Sequencing