Journal: Circulation

Article Title: CD73/Ecto-5′-Nucleotidase Protects Against Vascular Inflammation and Neointima Formation

doi: 10.1161/circulationaha.105.595249

Figure Lengend Snippet: Figure 2. CD73/ ECs display a proinflammatory phenotype. A, mRNA expression of vWF, CD73, and VCAM-1 (compared with aldo- lase) in wild-type and CD73/ aortal ECs was analyzed by RT-PCR. Agarose gels are representative of 3 independent experiments. B and C, Protein expression of vWF, CD73, and VACM-1 in wild-type and CD73/ aortic ECs was analyzed by flow cytometry and was compared with expression in isotype control ECs. Histograms are rep- resentative of 3 independent experiments (B). Specific VCAM-1 mean fluorescence intensity (sMFI) and median were analyzed (C, n3 to 5). D, Immunofluorescence staining of p65 in wild-type and CD73/ ECs is shown. Images are representative of 3 independent experiments; arrows indicate nuclear p65. Bars100 m. E, ChIP analysis of lysates of CD73/and CD73/ ECs was performed using Abs against NF-B subunits, acetylated histone H3, or cytochrome c. The region of the VCAM-1 promoter containing the NF-B binding site was amplified by PCR. For comparison, serial dilutions of input chro- matin DNA were analyzed. F, Quantitative p65-DNA binding assays were performed with nuclear extracts of wild-type and CD73/ ECs (n6). *P0.05 versus CD73/.

Article Snippet: Confluent ECs on glass slides were fixed in methanol, permeabilized with 0.5% Triton-X, and reacted with p65 mAb (C-20, Santa Cruz Biotechnology, Inc).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cytometry, Control, Staining, Binding Assay, Comparison

Journal: Scientific reports

Article Title: Polyphenols journey through blood-brain barrier towards neuronal protection.

doi: 10.1038/s41598-017-11512-6

Figure Lengend Snippet: Figure 4. Effects on neuroinflammation by Cat-sulf and Pyr-sulf. Pro-inflammatory markers were evaluated, namely (a) TNF-α release, (b) intracellular superoxide production, (c) nitric oxide, and (d) CD40 quantified in N9 microglial cells. Cells were pre-incubated for 6 h with each of the bioavailable (poly)phenol metabolite and then challenged with 300ng/mL of LPS. Statistical differences are denoted as ***p < 0.001, **p < 0.01 and *p < 0.05 relatively to lesion (LPS). (e) Microglial NF-κB p65 translocation into the nucleus after 60 minutes of LPS stimulation. Cells were pre-treated with Cat-sulf or Pyr-sulf for 6 h before LPS-stimulation. NF-κB (red); Nuclei (blue) stained with DAPI. Each capture is representative of at least 3 independent biological replicates. Scale bar: 10 µm. (f–i) Microglial NF-κB p65 phosphorylation ratio and IκBα fold change in protein levels. (f) IkBα protein levels along time after LPS stimulation and (g) after 60 min of LPS stimulation with representative western blots. (h) NF-κB activation profile along time after LPS stimulation looking at NF-κB p65 phosphorylation (ser536) ratio along time after LPS stimulation and (i)after 60 min of LPS stimulation with representative western blots. Cells were pre-treated either with Pyr-sulf or Cat-sulf before LPS stimulation. Control cells (white triangles, solid line), LPS-stimulated cells (black triangles, solid line), cells treated with Cat-sulf prior to LPS stimulation (black circles, dashed line), cells treated with Pyr-sulf prior to LPS stimulation (black squares, dotted line). Statistical differences are denoted as *p < 0.05 and **p < 0.01 relatively to lesion (LPS). Western blots were analyzed under the same experimental conditions. Data are presented as the means ± SD, n = 3.

Article Snippet: Briefly, HBMEC coverslips were incubated overnight at 4 °C with primary antibodies anti-P-gp (1:50, Calbiochem), anti-MRP1 (1:100, Millipore) and anti-BCRP (1:100, Millipore) and N9 cells coverslips were incubated overnight at 4 °C with rabbit polyclonal anti-NF-κB p65 (C-20) (1:200, Santa Cruz Biotechnology).

Techniques: Incubation, Translocation Assay, Staining, Phospho-proteomics, Western Blot, Activation Assay, Control

Journal: Basic & clinical pharmacology & toxicology

Article Title: Effects of insulin-like growth factor-1 on rotenone-induced apoptosis in human lymphocyte cells.

doi: 10.1111/j.1742-7843.2009.00472.x

Figure Lengend Snippet: Fig. 4. Rotenone (ROT) induced simultaneous NF-jB, p53 and c-Jun transcription factor activation in lymphocytes. Peripheral blood lymphocytes cells were left untreated (A–C), exposed to 250 lM rotenone (D–F), 250 nM IGF-1 (G–I), and to 250 nM IGF-1 + 250 lM rotenone (J–L) for 24 hr. After the incubation period, cells were stained with anti-NF-jB-p65 (A,D,G,J), anti-p53 (B,E,H,K) and anti-c-Jun (C,F,I,L) antibodies according to the procedure described in Materials and Methods. Notice that NF-jB, p53 and c-Jun positive-nuclei (dark brown) reflect their nuclear translocation ⁄ activation. Magnification 400· (A–L).

Article Snippet: The supplier’s protocol (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; goat ABC staining System: cat # sc-2023) was followed for the immunocytochemistry using primary goat polyclonal antibodies NF-jB p65 (C-20)-G (Santa Cruz 2009 The Authors Journal compilation 2009 Nordic Pharmacological Society.

Techniques: Activation Assay, Incubation, Staining, Translocation Assay

Journal: PLoS ONE

Article Title: How the Venom from the Ectoparasitoid Wasp Nasonia vitripennis Exhibits Anti-Inflammatory Properties on Mammalian Cell Lines

doi: 10.1371/journal.pone.0096825

Figure Lengend Snippet: A. Western blot analysis of nuclear extracts from Raw264.7. After pre-incubation with N. vitripennis venom (5 µg/ml) for 15 minutes, cells were induced for the indicated times with LPS (1 µg/ml). Subsequently, nuclear extracts were subjected to Western blot analysis to determine p65 levels. Separation of nuclear and cytoplasmic fractions was verified using PARP as control for the nuclear fractions. B. After 15 minutes pre-incubation with N. vitripennis venom, cells were induced for the indicated times with LPS (1 µg/ml). Immunofluorescence staining was performed to visualize the trafficking of the p65 subunit. Representative results from three separate experiments are shown.

Article Snippet: Antibodies to rabbit IκBα (C-21), mouse A20 (A-12), rabbit PARP-1/2 (H-250 and rabbit NF-κB p65 (C-20) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Incubation, Control, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: How the Venom from the Ectoparasitoid Wasp Nasonia vitripennis Exhibits Anti-Inflammatory Properties on Mammalian Cell Lines

doi: 10.1371/journal.pone.0096825

Figure Lengend Snippet: HEK293T cells were transiently transfected by the PEI method with 100(Gal)2-50.huIL6P-luc+ and with various expression plasmids, the total amount of DNA being fixed at 250 ng, i.e., pGal4 (10 ng) or pGal4-p65 (10 ng) whether or not with pGR (25 ng). 10 −6 M DEX or 5 µg/ml of venom was added 24 hours before analysis. Lysates were made and the relative luciferase activity was determined by using β-gal values as a basis for normalization. The data are expressed as the mean ±S.D. of three biological replicates.

Article Snippet: Antibodies to rabbit IκBα (C-21), mouse A20 (A-12), rabbit PARP-1/2 (H-250 and rabbit NF-κB p65 (C-20) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Expressing, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: How the Venom from the Ectoparasitoid Wasp Nasonia vitripennis Exhibits Anti-Inflammatory Properties on Mammalian Cell Lines

doi: 10.1371/journal.pone.0096825

Figure Lengend Snippet: TNF stimulation leads to activation of the canonical NF-κB signal transduction pathway marked by IκBα degradation and translocation of the p65-p50 dimer to the nucleus. Subsequent NF-κB DNA binding enables transcription of different genes, among which the cytokine IL-6. Venom treatment leads to the inhibition of TNF induced IL-6 gene expression. In addition, TNF activates the MAPKs,JNK, ERK1/2 and p38, and pretreatment with venom results in prolonged JNK activation. The negative NF-κB regulators A20 and IκBα are indicated and both were suppressed by the venom. Glucocorticoid binding to the cytosolic Glucocorticoid Receptor (GR) results in the dissociation of chaperoning proteins, followed by GR translocation to the nucleus, where it can interfere with the activity of NF-κB. Activated GR can also directly bind to the DNA, stimulating transcription of FKBP5, MKP1 and GILZ target genes. Only the two latter genes are transcriptionally induced by the venom. The red arrows mark the various levels at which venom of N. vitripennis interferes with the represented cellular signaling pathways.

Article Snippet: Antibodies to rabbit IκBα (C-21), mouse A20 (A-12), rabbit PARP-1/2 (H-250 and rabbit NF-κB p65 (C-20) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Transduction, Translocation Assay, Binding Assay, Inhibition, Gene Expression, Activity Assay, Protein-Protein interactions

Journal: The Journal of nutrition

Article Title: 3,3'-Diindolylmethane suppresses the inflammatory response to lipopolysaccharide in murine macrophages.

doi: 10.1093/jn/138.1.17

Figure Lengend Snippet: FIGURE 2 Effects of DIM on LPS-induced NF-kB signaling in RAW264.7 cells. Serum-deprived cells were treated with DIM for 18 h in DMEM containing 1% FBS. LPS was then added and incubated for another 20 min. (A) Cell lysates were subjected to western blotting with an anti-IkBa antibody. Nuclear extracts were prepared for western blotting with an anti-p65 antibody (B) and electrophoretic mobility shift assay (C). Photographs of chemiluminescent detectionof theblots (A,B) or an autoradiography of the dried gels (C), which were representative of 3 independent experiments, are shown. The relative abundance of each band was quantified and the control levels (0 mmol/L DIM) were set at 100%. The adjusted means 6 SEM, n ¼ 3 of each band is shown above each blot. (D) Cells werecotransfected with NF-kB-Luc reporter plasmid and pCMV-b-galactosidase vector and plated in 6-well plates at 2 3 105

Article Snippet: The following reagents were purchased from the indicated suppliers: DIM, LKT Laboratories; antibodies against iNOS and COX-2, BD Transduction Laboratories; anti-NF-kB p65 (C-20) antibody, Santa Cruz Biotechnology; antibodies against inhibitor of kB (IkB)a, stress-activated protein kinase/Jun-N-terminal kinase (SAPK/JNK), p-SAPK/JNK (Thr-183/Tyr-185), c-Jun, p-c-Jun (Ser-63), extracellular signal-regulated kinase (ERK)-1/2, p-ERK-1/2 (Thr-202/Tyr-204), p38 mitogen-activated protein kinase (MAPK), and p-p38 MAPK (Thr-180/ Tyr-182), Cell Signaling Technology; pNF-kB-Luc and pAP-1-Luc containing the firefly luciferase (luc) coding sequence from Photinus pyralis (21), and pCMV-b, Takara Bio. pNF-kB-Luc contains multiple copies of NF-kB consensus sequence and pAP-1-Luc contains multiple copies of the AP-1 enhancer.

Techniques: Incubation, Western Blot, Electrophoretic Mobility Shift Assay, Autoradiography, Control, Plasmid Preparation

Journal: The Journal of Experimental Medicine

Article Title: The Cdx2 homeobox gene suppresses intestinal tumorigenesis through non–cell-autonomous mechanisms

doi: 10.1084/jem.20170934

Figure Lengend Snippet: NF-κB activation and iNOS induction in the cecal lesions of AhCre ERT ::Cdx2 f/f mice. Coimmunodetection of Cdx2 and β-catenin, RelA (p65 NF-κB) and β-catenin, iNOS and β-catenin, RelA and Cdx2, and RelA and Cdx2 in the normal cecal mucosa of wild-type mice and the cecal lesions of AhCre ERT ::Cdx2 f/f mice. Open and closed arrows respectively show Cdx2 -devoid and Cdx2 -expressing surface epithelium. The asterisk points to a Cdx2 -depleted gland underneath the surface epithelium. Pictures correspond to serial sections. They were obtained in four mice of each genotype from two independent crossings. Bars, 100 µm.

Article Snippet: Primary antibodies were as follows: mouse anti–β-catenin (clone 14; dilution 1:500; BD Transduction Lab), mouse anti-CD4 (50134-M08H; dilution 1:500; Sino Biological), goat anti-CD8b (M-20, sc-1144; dilution 1:500, Santa Cruz Biotechnology), rabbit anti-Cdx1 ( ; dilution 1:1,000), mouse anti-Cdx2 (CDX2-88, F/MU392A-UC; dilution 1:500; Biogenex), rabbit anti-Cdx2 (EPR2764Y, ab76541; dilution 1:10,000; Thermo Fisher Scientific), rabbit anti-Cldn18 (38-8000; dilution 1:500; Invitrogen), rat anti-FoxP3 (FJK-16s, 14-5773-80; dilution 1:500; Affymetrix eBioscience), rabbit anti-Iba1 (orb10863; dilution 1:500; Biorbyt), rabbit anti-iNOS (M-19, sc-650; dilution 1:500; Santa Cruz Biotechnology), rabbit anti-Ki67 (RM9106-S; dilution 1:500; Thermo Fisher Scientific), rabbit anti-Muc2 (H-300, sc-15334; dilution 1:1,000; Santa Cruz Biotechnology), rabbit anti-Olfm4 (D6Y5A, mouse-specific; dilution 1:500; Cell Signaling Technology), rabbit anti-Olfm4 (ab85046, human-specific; dilution 1:500; Abcam), rabbit anti-p-Erk1/2 (D11A8, mAb5683; dilution 1:500; Cell Signaling Technology), rabbit anti–p-STAT3 (ab76315; dilution 1:500; Abcam), rabbit anti-RelA (NF-κB p65; C-20, sc-372; dilution 1:500; Santa Cruz Biotechnology), rabbit anti-Sox2 (AB5603; dilution 1:500; Millipore), rabbit anti-Sox9 ( ; dilution 1:500), rabbit anti-Tff1 ( ; dilution 1:500), rabbit anti-Tff2 ( ; dilution 1:500), and rat anti-Tomato (clone 16D7; dilution 1:250; KerFast).

Techniques: Activation Assay, Expressing