recombinant human tnf α  (R&D Systems)

 
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  • 99
    Name:
    Recombinant Human TNF alpha Protein
    Description:
    The Recombinant Human TNF alpha Protein from R D Systems is derived from E coli The Recombinant Human TNF alpha Protein has been validated for the following applications Bioactivity
    Catalog Number:
    210-ta-005
    Price:
    99
    Applications:
    Bioactivity
    Purity:
    >97%, by SDS-PAGE under reducing conditions and visualized by silver stain.
    Conjugate:
    Unconjugated
    Size:
    5 ug
    Category:
    Proteins and Enzymes
    Source:
    E. coli-derived Recombinant Human TNF-alpha Protein
    Buy from Supplier


    Structured Review

    R&D Systems recombinant human tnf α
    Recombinant Human TNF alpha Protein
    The Recombinant Human TNF alpha Protein from R D Systems is derived from E coli The Recombinant Human TNF alpha Protein has been validated for the following applications Bioactivity
    https://www.bioz.com/result/recombinant human tnf α/product/R&D Systems
    Average 99 stars, based on 361 article reviews
    Price from $9.99 to $1999.99
    recombinant human tnf α - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Leukadherin-1 ameliorates endothelial barrier damage mediated by neutrophils from critically ill patients"

    Article Title: Leukadherin-1 ameliorates endothelial barrier damage mediated by neutrophils from critically ill patients

    Journal: Journal of Intensive Care

    doi: 10.1186/s40560-018-0289-5

    LA-1 inhibits migration of neutrophils on endothelial monolayers. HUVEC monolayers were grown on delta T dishes coated with 40 μg/mL type I collagen and TNF-α activated. Neutrophils were either TNF-α-activated and given no treatment, 15 μM LA-1, or DMSO vehicle control. Cell interactions were allowed to adhere at 37 °C and neutrophil migration dynamics were imaged. × 20 bright-field images were captured every 30 s for 20 min. Neutrophil migration paths were tracked in ImageJ and analyzed using ibidi’s Chemotaxis and Migration Tool ( a ). LA-1 treatment reduced neutrophil path length ( b ), displacement ( c ), and speed ( d ) regardless of donor. Two-way ANOVA with post hoc Tukey analysis, * p
    Figure Legend Snippet: LA-1 inhibits migration of neutrophils on endothelial monolayers. HUVEC monolayers were grown on delta T dishes coated with 40 μg/mL type I collagen and TNF-α activated. Neutrophils were either TNF-α-activated and given no treatment, 15 μM LA-1, or DMSO vehicle control. Cell interactions were allowed to adhere at 37 °C and neutrophil migration dynamics were imaged. × 20 bright-field images were captured every 30 s for 20 min. Neutrophil migration paths were tracked in ImageJ and analyzed using ibidi’s Chemotaxis and Migration Tool ( a ). LA-1 treatment reduced neutrophil path length ( b ), displacement ( c ), and speed ( d ) regardless of donor. Two-way ANOVA with post hoc Tukey analysis, * p

    Techniques Used: Migration, Chemotaxis Assay

    2) Product Images from "Transcriptional Repression of the Transforming Growth Factor β (TGF-β) Pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) by Nuclear Factor κB (NF-κB) p50 Enhances TGF-β Signaling in Hepatic Stellate Cells *"

    Article Title: Transcriptional Repression of the Transforming Growth Factor β (TGF-β) Pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) by Nuclear Factor κB (NF-κB) p50 Enhances TGF-β Signaling in Hepatic Stellate Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.543769

    Mechanism of BAMBI repression by HDAC1. A , the knockdown efficiencies of siRNA HDAC1 were estimated by Western blot analysis. LX2 cells were transected with siRNA HDAC1 or a control for 72 h, followed by treatment with LPS (100 ng/ml) or TNF-α
    Figure Legend Snippet: Mechanism of BAMBI repression by HDAC1. A , the knockdown efficiencies of siRNA HDAC1 were estimated by Western blot analysis. LX2 cells were transected with siRNA HDAC1 or a control for 72 h, followed by treatment with LPS (100 ng/ml) or TNF-α

    Techniques Used: Western Blot

    Identification of the element in the promoter region responsible for repression of BAMBI by LPS and TNF-α . Nucleotide sequence positions are indicated relative to the transcriptional start site. LX2 cells were transfected with the indicated h
    Figure Legend Snippet: Identification of the element in the promoter region responsible for repression of BAMBI by LPS and TNF-α . Nucleotide sequence positions are indicated relative to the transcriptional start site. LX2 cells were transfected with the indicated h

    Techniques Used: Sequencing, Transfection

    LPS enhances TGF-β signaling in hHSCs through down-regulation of BAMBI. A , LX2 cells were pretreated with LPS (100 ng/ml) or TNF-α (10 ng/ml) for 12 h and subsequently treated with TGF-β1 (5 ng/ml) for 30 min. A Western blot analysis
    Figure Legend Snippet: LPS enhances TGF-β signaling in hHSCs through down-regulation of BAMBI. A , LX2 cells were pretreated with LPS (100 ng/ml) or TNF-α (10 ng/ml) for 12 h and subsequently treated with TGF-β1 (5 ng/ml) for 30 min. A Western blot analysis

    Techniques Used: Western Blot

    NF-κBp50 mediates BAMBI repression by LPS and TNF-α . A and B , BAMBI mRNA levels in LX2 cells after 4 h of stimulation with LPS (100 ng/ml) ( A ) or TNF-α (10 ng/ml) ( B ) were determined by qPCR. Before treatment, LX2 cells were transfected
    Figure Legend Snippet: NF-κBp50 mediates BAMBI repression by LPS and TNF-α . A and B , BAMBI mRNA levels in LX2 cells after 4 h of stimulation with LPS (100 ng/ml) ( A ) or TNF-α (10 ng/ml) ( B ) were determined by qPCR. Before treatment, LX2 cells were transfected

    Techniques Used: Real-time Polymerase Chain Reaction, Transfection

    3) Product Images from "Largazole, a class I histone deacetylase inhibitor, enhances TNF-α to induce ICAM-1 and VCAM-1 expression in Rheumatoid Arthritis Synovial Fibroblasts"

    Article Title: Largazole, a class I histone deacetylase inhibitor, enhances TNF-α to induce ICAM-1 and VCAM-1 expression in Rheumatoid Arthritis Synovial Fibroblasts

    Journal: Toxicology and applied pharmacology

    doi: 10.1016/j.taap.2013.04.014

    Tub-A suppresses TNF-α+LAR-induced ICAM-1 and VCAM-1 expression and coordinates to inhibit MMP-2 activity in RA synovial fibroblasts. (A) RA synovial fibroblasts were preincubated with LAR (2–5 μM), SAHA (5 μM), or Tub-A (10 μM) in serum-free RPMI for 2 h followed by TNF-α (20 ng/ml) stimulation for 24 h to determine the effect on ICAM-1 and VCAM-1 expression by Western blotting method. Densitometric analysis of the expression was quantified by determining the intensity of the bands using Image-J software (NIH). (B) Conditioned media was utilized for the assessment of MMP-2 activity using gelatin zymography. (C) RA synovial fibroblasts were treated as described in section (A), except that the reaction was terminated within 30 min of TNF-α stimulation to analyze signaling pathways. The results represent values obtained from 3-4 independent donors’ cells under similar conditions. **p
    Figure Legend Snippet: Tub-A suppresses TNF-α+LAR-induced ICAM-1 and VCAM-1 expression and coordinates to inhibit MMP-2 activity in RA synovial fibroblasts. (A) RA synovial fibroblasts were preincubated with LAR (2–5 μM), SAHA (5 μM), or Tub-A (10 μM) in serum-free RPMI for 2 h followed by TNF-α (20 ng/ml) stimulation for 24 h to determine the effect on ICAM-1 and VCAM-1 expression by Western blotting method. Densitometric analysis of the expression was quantified by determining the intensity of the bands using Image-J software (NIH). (B) Conditioned media was utilized for the assessment of MMP-2 activity using gelatin zymography. (C) RA synovial fibroblasts were treated as described in section (A), except that the reaction was terminated within 30 min of TNF-α stimulation to analyze signaling pathways. The results represent values obtained from 3-4 independent donors’ cells under similar conditions. **p

    Techniques Used: Expressing, Activity Assay, Western Blot, Software, Zymography

    Synergistic induction of TNF-α-induced ICAM-1 and VCAM-1 expression by LAR or SAHA in RA synovial fibroblasts. (A B) RA synovial fibroblasts were pretreated with LAR (2–5 μM) or SAHA (5 μM) for 2 h followed by TNF-α (20 ng/ml) stimulation for 24 h in serum-free RPMI. Cells were lysed in extraction buffer containing protease inhibitors and utilized for the determination of ICAM-1 and VCAM-1 expression using Western blotting. The intensity of the bands was quantified using Image-J software (NIH). (C D) For ICAM-1 and VCAM-1 mRNA analysis, RA synovial fibroblasts (2× 10 5 /well) were treated as described above and analyzed for mRNA expression by a qRT-PCR method. Results represent the mean ± SEM of experiments performed using 3-4 different RA synovial fibroblast donors. *p
    Figure Legend Snippet: Synergistic induction of TNF-α-induced ICAM-1 and VCAM-1 expression by LAR or SAHA in RA synovial fibroblasts. (A B) RA synovial fibroblasts were pretreated with LAR (2–5 μM) or SAHA (5 μM) for 2 h followed by TNF-α (20 ng/ml) stimulation for 24 h in serum-free RPMI. Cells were lysed in extraction buffer containing protease inhibitors and utilized for the determination of ICAM-1 and VCAM-1 expression using Western blotting. The intensity of the bands was quantified using Image-J software (NIH). (C D) For ICAM-1 and VCAM-1 mRNA analysis, RA synovial fibroblasts (2× 10 5 /well) were treated as described above and analyzed for mRNA expression by a qRT-PCR method. Results represent the mean ± SEM of experiments performed using 3-4 different RA synovial fibroblast donors. *p

    Techniques Used: Expressing, Western Blot, Software, Quantitative RT-PCR

    Inhibition of TNF-α-induced MMP-2 activity by LAR in RA synovial fibroblasts. RA synovial fibroblasts were pre-incubated with the LAR (2-5 μM) or SAHA (5 μM) for 2 h, followed by stimulation with TNF-α (20 ng/ml) for 24 h. MMP-2 activity in the cell-free supernatants from different treatment combinations was estimated by gelatin zymography. Values are the mean ± SEM of experiments performed using RA synovial fibroblast obtained from 5 different donors. **p
    Figure Legend Snippet: Inhibition of TNF-α-induced MMP-2 activity by LAR in RA synovial fibroblasts. RA synovial fibroblasts were pre-incubated with the LAR (2-5 μM) or SAHA (5 μM) for 2 h, followed by stimulation with TNF-α (20 ng/ml) for 24 h. MMP-2 activity in the cell-free supernatants from different treatment combinations was estimated by gelatin zymography. Values are the mean ± SEM of experiments performed using RA synovial fibroblast obtained from 5 different donors. **p

    Techniques Used: Inhibition, Activity Assay, Incubation, Zymography

    4) Product Images from "Shikonin Promotes Skin Cell Proliferation and Inhibits Nuclear Factor-κB Translocation via Proteasome Inhibition In Vitro"

    Article Title: Shikonin Promotes Skin Cell Proliferation and Inhibits Nuclear Factor-κB Translocation via Proteasome Inhibition In Vitro

    Journal: Chinese Medical Journal

    doi: 10.4103/0366-6999.162512

    Effect of shikonin on tumor necrosis factor-α (TNF-α)-induced phosphorylated-inhibitor κB-α (p-IκB-α) protein expression in HDFs. (a) TNF-α-induced increase in p-IκB-α expression in HDFs; (b) Representative pictures of p-IκB-α protein expression in indicated groups detected by immunoblot analysis. SKN: Shikonin. Results are shown as the mean ± standard deviation from three independent experiments. * P
    Figure Legend Snippet: Effect of shikonin on tumor necrosis factor-α (TNF-α)-induced phosphorylated-inhibitor κB-α (p-IκB-α) protein expression in HDFs. (a) TNF-α-induced increase in p-IκB-α expression in HDFs; (b) Representative pictures of p-IκB-α protein expression in indicated groups detected by immunoblot analysis. SKN: Shikonin. Results are shown as the mean ± standard deviation from three independent experiments. * P

    Techniques Used: Expressing, Standard Deviation

    Immunofluorescence study of expression of nuclear factor-κB (NF-κB) in human dermal fibroblasts (HDFs). (a) Purified normal rabbit IgG (negative control); (b-d) Representative immunofluorescence images for NF-κB p65 of HDFs. Vehicle (0.1% dimethyl sulfoxide) (b). Stimulation with tumor necrosis factor-α (TNF-α) alone for 15 min (c) and 30 min (d); (e and f) Effect of pretreatment with shikonin. Stimulation with TNF-α for 15 min (e) and 30 min; (f) SKN: Shikonin, scale bars: 200 μm.
    Figure Legend Snippet: Immunofluorescence study of expression of nuclear factor-κB (NF-κB) in human dermal fibroblasts (HDFs). (a) Purified normal rabbit IgG (negative control); (b-d) Representative immunofluorescence images for NF-κB p65 of HDFs. Vehicle (0.1% dimethyl sulfoxide) (b). Stimulation with tumor necrosis factor-α (TNF-α) alone for 15 min (c) and 30 min (d); (e and f) Effect of pretreatment with shikonin. Stimulation with TNF-α for 15 min (e) and 30 min; (f) SKN: Shikonin, scale bars: 200 μm.

    Techniques Used: Immunofluorescence, Expressing, Purification, Negative Control

    5) Product Images from "Molecular and Biochemical Characterization of Recombinant Guinea Pig Tumor Necrosis Factor-Alpha"

    Article Title: Molecular and Biochemical Characterization of Recombinant Guinea Pig Tumor Necrosis Factor-Alpha

    Journal: Mediators of Inflammation

    doi: 10.1155/2015/619480

    Amino acid sequence comparison of TNF- α from different species showing the presence of conserved putative phosphorylation sites. Amino acid sequences of human (AAA63207), chimpanzee (ABM91951), Squirrel monkey (AAK92045), Cow (AAA19011), Pig (AAA74410), Woodchuck (AY253723), Mouse (AAA39275), Rat (AAA41425), and Guinea pig (JN020146) were aligned by EBI Clustal W. Shading emphasizes identical amino acids and numbers on the right represent the position of amino acid.
    Figure Legend Snippet: Amino acid sequence comparison of TNF- α from different species showing the presence of conserved putative phosphorylation sites. Amino acid sequences of human (AAA63207), chimpanzee (ABM91951), Squirrel monkey (AAK92045), Cow (AAA19011), Pig (AAA74410), Woodchuck (AY253723), Mouse (AAA39275), Rat (AAA41425), and Guinea pig (JN020146) were aligned by EBI Clustal W. Shading emphasizes identical amino acids and numbers on the right represent the position of amino acid.

    Techniques Used: Sequencing

    (a) Coomassie blue-stained SDS-PAGE analysis of eukaryotic protein elutions from cells transfected with 18 μ g of pCEP-Pu plasmid DNA without any gene (vector control) or pCEP-Pu plasmid DNA (18 μ g) containing TNF- α gene. The arrow indicates a ~19 kDa band which was recognized as rgpTNF- α . Absence of band in the vector control is also shown. MM: molecular marker; V: vector control elutions, and T: TNF-alpha elutions. (b) Coomassie blue-stained SDS-PAGE analysis of prokaryotic expressed rgp-TNF- α . rgpTNF- α was expressed using prokaryotic expression system (pQE-15) and analyzed on SDS-PAGE gel. MM: molecular marker. (c) Identification of eukaryotic expressed rgpTNF- α by polyclonal antiserum (1 : 2000 dilution) from immunized rabbits. Approximately 200 nanograms of rgpTNF- α was run on 10–20% tricine gel and analyzed by western blot analysis for checking its specificity. The ladder was stained with Coomassie Brilliant Blue.
    Figure Legend Snippet: (a) Coomassie blue-stained SDS-PAGE analysis of eukaryotic protein elutions from cells transfected with 18 μ g of pCEP-Pu plasmid DNA without any gene (vector control) or pCEP-Pu plasmid DNA (18 μ g) containing TNF- α gene. The arrow indicates a ~19 kDa band which was recognized as rgpTNF- α . Absence of band in the vector control is also shown. MM: molecular marker; V: vector control elutions, and T: TNF-alpha elutions. (b) Coomassie blue-stained SDS-PAGE analysis of prokaryotic expressed rgp-TNF- α . rgpTNF- α was expressed using prokaryotic expression system (pQE-15) and analyzed on SDS-PAGE gel. MM: molecular marker. (c) Identification of eukaryotic expressed rgpTNF- α by polyclonal antiserum (1 : 2000 dilution) from immunized rabbits. Approximately 200 nanograms of rgpTNF- α was run on 10–20% tricine gel and analyzed by western blot analysis for checking its specificity. The ladder was stained with Coomassie Brilliant Blue.

    Techniques Used: Staining, SDS Page, Transfection, Plasmid Preparation, Marker, Expressing, Western Blot

    Bioactivity of prokaryotic and eukaryotic expressed guinea pig TNF- α . Prokaryotic and eukaryotic expressed rgpTNF- α proteins in the concentration ranging from 0 to 10 μ g/mL were analyzed for their cytotoxicity on L-929 fibroblasts and the percentage of cytotoxicity was calculated.
    Figure Legend Snippet: Bioactivity of prokaryotic and eukaryotic expressed guinea pig TNF- α . Prokaryotic and eukaryotic expressed rgpTNF- α proteins in the concentration ranging from 0 to 10 μ g/mL were analyzed for their cytotoxicity on L-929 fibroblasts and the percentage of cytotoxicity was calculated.

    Techniques Used: Concentration Assay

    6) Product Images from "Functional Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Production by Avian Influenza Virus–Infected Macrophages"

    Article Title: Functional Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Production by Avian Influenza Virus–Infected Macrophages

    Journal: The Journal of Infectious Diseases

    doi: 10.1086/500954

    Sensitivity of Jurkat T cells to tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL)– and Fas ligand (FasL)–induced apoptosis but not to TNF-α. A Jurkat T cells cultured in the presence of recombinant TNF-α, TRAIL, and FasL at concentrations of 0–100 ng/mL for 24 h. Cell death was determined by active caspase–3 staining. B Jurkat T cell death, induced by 100 ng/mL recombinant human (rh) TRAIL and 100 ng/mL FasL and inhibited by specific antibodies at a final concentration of 1 μg/mL. Data are representative of 3 independent experiments. TRAIL-R2, TRAIL receptor 2
    Figure Legend Snippet: Sensitivity of Jurkat T cells to tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL)– and Fas ligand (FasL)–induced apoptosis but not to TNF-α. A Jurkat T cells cultured in the presence of recombinant TNF-α, TRAIL, and FasL at concentrations of 0–100 ng/mL for 24 h. Cell death was determined by active caspase–3 staining. B Jurkat T cell death, induced by 100 ng/mL recombinant human (rh) TRAIL and 100 ng/mL FasL and inhibited by specific antibodies at a final concentration of 1 μg/mL. Data are representative of 3 independent experiments. TRAIL-R2, TRAIL receptor 2

    Techniques Used: Cell Culture, Recombinant, Staining, Concentration Assay

    H9N2/G1 and H5N1/97 infection sensitizing Jurkat T cells to induction of apoptosis by tumor necrosis factor (TNF)–α, TNF-related apoptosis-inducing ligand (TRAIL), and Fas ligand (FasL). After 1 h of virus adsorption, H9N2/G1- and H5N1/97-infected Jurkat T cells were cultured in presence of exogenous TNF-α (500 ng/mL), TRAIL (10 ng/mL), or FasL (10 ng/mL), respectively, for another 24-h culture. A significantly higher percentage of apoptotic cells was observed (P
    Figure Legend Snippet: H9N2/G1 and H5N1/97 infection sensitizing Jurkat T cells to induction of apoptosis by tumor necrosis factor (TNF)–α, TNF-related apoptosis-inducing ligand (TRAIL), and Fas ligand (FasL). After 1 h of virus adsorption, H9N2/G1- and H5N1/97-infected Jurkat T cells were cultured in presence of exogenous TNF-α (500 ng/mL), TRAIL (10 ng/mL), or FasL (10 ng/mL), respectively, for another 24-h culture. A significantly higher percentage of apoptotic cells was observed (P

    Techniques Used: Infection, Adsorption, Cell Culture

    mRNA expression of tumor necrosis factor (TNF)–α, TNF-related apoptosis-inducing ligand (TRAIL), and Fas ligand (FasL) in influenza virus–infected monocyte-derived macrophages (MDMs). A, B and C The kinetics of TNF-α, TRAIL, and FasL mRNA expression in H1N1/98- and H9N2/G1-infected MDMs. Total RNA was harvested at 4, 8, and 12 h after influenza virus infection. The target genes were quantified by quantitative reverse-transcription polymerase chain reaction and normalized to 1×10 4 copies of β-actin mRNA. Data are the mean ± SE from 6 independent experiments. D, E and F TNF-α, TRAIL, and FasL mRNA expression in MDMs treated by H5N1 and UV-irradiated H5N1 (UVH1N1) at 7 h after infection. Data are the mean ± SE from 4 independent experiments. *P
    Figure Legend Snippet: mRNA expression of tumor necrosis factor (TNF)–α, TNF-related apoptosis-inducing ligand (TRAIL), and Fas ligand (FasL) in influenza virus–infected monocyte-derived macrophages (MDMs). A, B and C The kinetics of TNF-α, TRAIL, and FasL mRNA expression in H1N1/98- and H9N2/G1-infected MDMs. Total RNA was harvested at 4, 8, and 12 h after influenza virus infection. The target genes were quantified by quantitative reverse-transcription polymerase chain reaction and normalized to 1×10 4 copies of β-actin mRNA. Data are the mean ± SE from 6 independent experiments. D, E and F TNF-α, TRAIL, and FasL mRNA expression in MDMs treated by H5N1 and UV-irradiated H5N1 (UVH1N1) at 7 h after infection. Data are the mean ± SE from 4 independent experiments. *P

    Techniques Used: Expressing, Infection, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Irradiation

    7) Product Images from "Pro-Inflammatory Cytokines Induce Expression of Matrix-Metabolizing Enzymes in Human Cervical Smooth Muscle Cells"

    Article Title: Pro-Inflammatory Cytokines Induce Expression of Matrix-Metabolizing Enzymes in Human Cervical Smooth Muscle Cells

    Journal: The American Journal of Pathology

    doi:

    Effect of TNF-α on the time course of expression of MMP, TIMP and cathepsin S mRNAs. A Northern blot analysis of total RNA (40 μg/lane) extracted from CSMCs cultured in the absence (Control) or presence of TNF-α (10 ng/ml) for the indicated time periods is shown. The blot was sequentially probed with the indicated cDNAs.
    Figure Legend Snippet: Effect of TNF-α on the time course of expression of MMP, TIMP and cathepsin S mRNAs. A Northern blot analysis of total RNA (40 μg/lane) extracted from CSMCs cultured in the absence (Control) or presence of TNF-α (10 ng/ml) for the indicated time periods is shown. The blot was sequentially probed with the indicated cDNAs.

    Techniques Used: Expressing, Northern Blot, Cell Culture

    MMP and TIMP production by CSMC cultured in the absence or presence of TNF-α. Cells were incubated without or with TNF-α (10 ng/ml) for 48 hours in serum-free medium and aliquots of conditioned media were analyzed by Western blotting using specific monoclonal antibodies as described in Materials and Methods.
    Figure Legend Snippet: MMP and TIMP production by CSMC cultured in the absence or presence of TNF-α. Cells were incubated without or with TNF-α (10 ng/ml) for 48 hours in serum-free medium and aliquots of conditioned media were analyzed by Western blotting using specific monoclonal antibodies as described in Materials and Methods.

    Techniques Used: Cell Culture, Incubation, Western Blot

    8) Product Images from "Epigallocatechin-3-gallate Suppresses Galactose-?1,4-galactose-?1,4-glucose Ceramide Expression in TNF-? Stimulated Human Intestinal Epithelial Cells Through Inhibition of MAPKs and NF-?B"

    Article Title: Epigallocatechin-3-gallate Suppresses Galactose-?1,4-galactose-?1,4-glucose Ceramide Expression in TNF-? Stimulated Human Intestinal Epithelial Cells Through Inhibition of MAPKs and NF-?B

    Journal: Journal of Korean Medical Science

    doi: 10.3346/jkms.2005.20.4.548

    TNF-α enhanced Gb3 content through MAPK phosphorylation and NF-κB activation, and that then the transcription factor of NF-κB induced the activity of all of the three enzymes involved in Gb3 glycosphingolipid precursor synthesis. EGCG suppresses Gb3 expression in a TNF-α stimulated HT-29 cells via inhibition of NF-κB. TRADD, TNF receptor-associated death domain; RIP, Receptor-interacting protein
    Figure Legend Snippet: TNF-α enhanced Gb3 content through MAPK phosphorylation and NF-κB activation, and that then the transcription factor of NF-κB induced the activity of all of the three enzymes involved in Gb3 glycosphingolipid precursor synthesis. EGCG suppresses Gb3 expression in a TNF-α stimulated HT-29 cells via inhibition of NF-κB. TRADD, TNF receptor-associated death domain; RIP, Receptor-interacting protein

    Techniques Used: Activation Assay, Activity Assay, Expressing, Inhibition

    Flow cytometric analysis of HT29 cell death. The HT29 cells pretreated with EGCG (25 µM) were cultured for 48 hr with TNF-α (50 ng/mL) and were analyzed by flow cytometric analysis using annexin V-FITC and propidium iodide. LL quadrant: viable cells (annexin V and propidium iodide negative cells); LR quadrant: apoptotic cells (annexin V positive and propidium iodide negative cells); UR quadrant: dead cells (annexin V and propidium iodide positive cells). Numbering refers to the cell percentage of each population. Number of counted cells: 10,000.
    Figure Legend Snippet: Flow cytometric analysis of HT29 cell death. The HT29 cells pretreated with EGCG (25 µM) were cultured for 48 hr with TNF-α (50 ng/mL) and were analyzed by flow cytometric analysis using annexin V-FITC and propidium iodide. LL quadrant: viable cells (annexin V and propidium iodide negative cells); LR quadrant: apoptotic cells (annexin V positive and propidium iodide negative cells); UR quadrant: dead cells (annexin V and propidium iodide positive cells). Numbering refers to the cell percentage of each population. Number of counted cells: 10,000.

    Techniques Used: Flow Cytometry, Cell Culture

    Effect of TNF-α on Gb3 induction in HT29 cells. ( A ) Dose-dependent effect of TNF-α on Gb3 production. TNF-α treated dose-dependently (10, 25, 50, and 100 ng/mL) for 2 days in HT29 cells. And time-dependent effect of TNF-α on Gb3 production. The cells were cultured with TNF-α (50 ng/mL) for 0, 12, 24, 48, and 72 hr. ( B ) The intensity was measured by densitometry. Isotype control represents the thick lines. The values are the mean±S.E. of duplicate determinations from three separate experiments. The sigmificance was determined by Student's t-test ( * , p
    Figure Legend Snippet: Effect of TNF-α on Gb3 induction in HT29 cells. ( A ) Dose-dependent effect of TNF-α on Gb3 production. TNF-α treated dose-dependently (10, 25, 50, and 100 ng/mL) for 2 days in HT29 cells. And time-dependent effect of TNF-α on Gb3 production. The cells were cultured with TNF-α (50 ng/mL) for 0, 12, 24, 48, and 72 hr. ( B ) The intensity was measured by densitometry. Isotype control represents the thick lines. The values are the mean±S.E. of duplicate determinations from three separate experiments. The sigmificance was determined by Student's t-test ( * , p

    Techniques Used: Cell Culture

    Effect of EGCG on Gb3 induction in TNF-α stimulated HT29 cells. ( A ) The HT29 cells pretreated with EGCG (10, 25, and 50 µM) were cultured for 2 days with TNF-α (50 ng/mL), and were analyzed by FACS. ( B ) The inhibitory effect of EGCG on Gb3 induction by TNF-α was shown in a dose-dependent manner. Isotype control represents the thin lines. Analysis of Gb3 content was carried out using FACS. MFI, mean fluorescence intensity.
    Figure Legend Snippet: Effect of EGCG on Gb3 induction in TNF-α stimulated HT29 cells. ( A ) The HT29 cells pretreated with EGCG (10, 25, and 50 µM) were cultured for 2 days with TNF-α (50 ng/mL), and were analyzed by FACS. ( B ) The inhibitory effect of EGCG on Gb3 induction by TNF-α was shown in a dose-dependent manner. Isotype control represents the thin lines. Analysis of Gb3 content was carried out using FACS. MFI, mean fluorescence intensity.

    Techniques Used: Cell Culture, FACS, Fluorescence

    Effect of EGCG on ERK1/2, JNK1/2, and p38 phosphorylation in TNF-α-stimulated HT29 cells. ( A ) The HT29 cells pretreated with EGCG (25 µM) were cultured for 15 min with TNF-α (50 ng/mL), and were analyzed by Western blot using phospho-p38, phospho-JNK1/2 and phospho-ERK1/2 antibodies. ( B ) The intensity was measured by densitometry. The values are the mean±S.E. from three separate experiments ( * p
    Figure Legend Snippet: Effect of EGCG on ERK1/2, JNK1/2, and p38 phosphorylation in TNF-α-stimulated HT29 cells. ( A ) The HT29 cells pretreated with EGCG (25 µM) were cultured for 15 min with TNF-α (50 ng/mL), and were analyzed by Western blot using phospho-p38, phospho-JNK1/2 and phospho-ERK1/2 antibodies. ( B ) The intensity was measured by densitometry. The values are the mean±S.E. from three separate experiments ( * p

    Techniques Used: Cell Culture, Western Blot

    Effect of EGCG on CGT, GalT2, and GalT6 mRNA expression in TNF-α-stimulated HT29 cells. ( A ) The HT29 cells pretreated with EGCG (25 µM) were cultured for 24 hr with TNF-α (50 ng/mL), and were analyzed by RT-PCR for CGT, GalT2, GalT6, and α-galactosidase mRNA. ( B ) A PCR using housekeeping gene β-actin mRNA was carried out in parallel to confirm the equivalency of cDNA preparation. Relative intensity represents mRNA levels of CGT, GalT2, GalT6, and α-galactosidase/β-actin. The values are the mean±S.E. from three separate experiments ( * p
    Figure Legend Snippet: Effect of EGCG on CGT, GalT2, and GalT6 mRNA expression in TNF-α-stimulated HT29 cells. ( A ) The HT29 cells pretreated with EGCG (25 µM) were cultured for 24 hr with TNF-α (50 ng/mL), and were analyzed by RT-PCR for CGT, GalT2, GalT6, and α-galactosidase mRNA. ( B ) A PCR using housekeeping gene β-actin mRNA was carried out in parallel to confirm the equivalency of cDNA preparation. Relative intensity represents mRNA levels of CGT, GalT2, GalT6, and α-galactosidase/β-actin. The values are the mean±S.E. from three separate experiments ( * p

    Techniques Used: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Effect of EGCG on NF-κB p65 nuclear translocation in TNF-α-stimulated HT29 cells. ( A ) The EGCG (25 µM)-pretreated cells were stimulated for 2 hr with TNF-α (50 ng/mL), and then were analyzed by Western blot using the anti-NF-κB p65 antibody. ( B ) The optical density unit was measured by densitometry. The values are the mean±S.E. from three separate experiments ( * p
    Figure Legend Snippet: Effect of EGCG on NF-κB p65 nuclear translocation in TNF-α-stimulated HT29 cells. ( A ) The EGCG (25 µM)-pretreated cells were stimulated for 2 hr with TNF-α (50 ng/mL), and then were analyzed by Western blot using the anti-NF-κB p65 antibody. ( B ) The optical density unit was measured by densitometry. The values are the mean±S.E. from three separate experiments ( * p

    Techniques Used: Translocation Assay, Western Blot

    9) Product Images from "CFH Y402H polymorphism is associated with elevated vitreal GM-CSF and choroidal macrophages in the postmortem human eye"

    Article Title: CFH Y402H polymorphism is associated with elevated vitreal GM-CSF and choroidal macrophages in the postmortem human eye

    Journal: Molecular Vision

    doi:

    The RPE secreted higher levels of GM-CSF after stimulation with HNE and TNF-α. The GM-CSF secreted into the culture supernatant was increased when primary RPE cells were exposed to 4-hydroxynonenal (HNE), an agent that promotes oxidative stress, at 10 µM for 6 h (mean ± SEM, 145.88±5.06 pg/ml versus 123.27±4.05 pg/ml, n=3, Student t test, *p
    Figure Legend Snippet: The RPE secreted higher levels of GM-CSF after stimulation with HNE and TNF-α. The GM-CSF secreted into the culture supernatant was increased when primary RPE cells were exposed to 4-hydroxynonenal (HNE), an agent that promotes oxidative stress, at 10 µM for 6 h (mean ± SEM, 145.88±5.06 pg/ml versus 123.27±4.05 pg/ml, n=3, Student t test, *p

    Techniques Used:

    10) Product Images from "Tumour necrosis factor-?- and interleukin-1?-stimulated cell proliferation through activation of mitogen-activated protein kinase in canine tracheal smooth muscle cells"

    Article Title: Tumour necrosis factor-?- and interleukin-1?-stimulated cell proliferation through activation of mitogen-activated protein kinase in canine tracheal smooth muscle cells

    Journal: British Journal of Pharmacology

    doi: 10.1038/sj.bjp.0703359

    Concentration-dependence of TNF-α- and IL-1β-stimulated p42/p44 MAPK phosphorylation in TSMCs. The cells were grown to confluence, made quiescent by serum-deprivation for 24 h and incubated with various concentrations of TNF-α (A) and IL-1β (B) for 15 min. The cell lysates were subjected to 10% SDS–PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed using an antiserum reactive with an anti-phospho-p42/p44 MAPK polyclonal antibody. Bands were visualized by an ECL method and quantified by a densitometer. Similar results were obtained in three independent experiments. Data are expressed as the mean±s.e.mean of three independent experiments. (Bar graph). * P
    Figure Legend Snippet: Concentration-dependence of TNF-α- and IL-1β-stimulated p42/p44 MAPK phosphorylation in TSMCs. The cells were grown to confluence, made quiescent by serum-deprivation for 24 h and incubated with various concentrations of TNF-α (A) and IL-1β (B) for 15 min. The cell lysates were subjected to 10% SDS–PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed using an antiserum reactive with an anti-phospho-p42/p44 MAPK polyclonal antibody. Bands were visualized by an ECL method and quantified by a densitometer. Similar results were obtained in three independent experiments. Data are expressed as the mean±s.e.mean of three independent experiments. (Bar graph). * P

    Techniques Used: Concentration Assay, Incubation, SDS Page, Western Blot

    Schematic pathway for TNF-α and IL-1β signaling of cellular proliferation. Each solid line and arrow represents a step in an activating pathway. Each T-shaped and dashed line represents inactivation or inhibition. TNF-α and IL-1β bind to their receptors (R) and activate phosphatidylcholine-phospholipase C (PC-PLC) and phosphatidylinositide-phospholipase C (PI-PLC) through the phosphorylation of tyrosine kinase (TK) to induce protein kinase C (PKC) activation. Activation of PKC leads to sequential phosphorylation of MEK1/2 and p42/p44 MAPK that relays extracellular signalling into nuclei. TNF-α and IL-1β induce activation of the components of downstream p42/p44 MAPK and enhance DNA synthesis and cell proliferation in TSMCs.
    Figure Legend Snippet: Schematic pathway for TNF-α and IL-1β signaling of cellular proliferation. Each solid line and arrow represents a step in an activating pathway. Each T-shaped and dashed line represents inactivation or inhibition. TNF-α and IL-1β bind to their receptors (R) and activate phosphatidylcholine-phospholipase C (PC-PLC) and phosphatidylinositide-phospholipase C (PI-PLC) through the phosphorylation of tyrosine kinase (TK) to induce protein kinase C (PKC) activation. Activation of PKC leads to sequential phosphorylation of MEK1/2 and p42/p44 MAPK that relays extracellular signalling into nuclei. TNF-α and IL-1β induce activation of the components of downstream p42/p44 MAPK and enhance DNA synthesis and cell proliferation in TSMCs.

    Techniques Used: Inhibition, Planar Chromatography, Activation Assay, DNA Synthesis

    [ 3 H]-Thymidine incorporation induced by cytokines in TSMCs. For time course, after 24 h in serum-free medium, the cells were stimulated with vehicle (basal), 30 ng ml −1 TNF-α or 50 ng ml −1 IL-1β. The cells were labeled with 1 μCi ml −1 [ 3 H]-thymidine for the times indicated in the continuous presence of cytokines (A). For concentration dependence, the cells were stimulated with various concentrations of TNF-α (B) and IL-1β (C). After stimulation for 6 h, cells were labeled with 1 μCi ml −1 [ 3 H]-thymidine for another 18 h in the presence of cytokines. The incorporation of [ 3 H]-thymidine was determined as described in Methods. Data are expressed as the mean±s.e.mean of three separate experiments determined in triplicate. * P
    Figure Legend Snippet: [ 3 H]-Thymidine incorporation induced by cytokines in TSMCs. For time course, after 24 h in serum-free medium, the cells were stimulated with vehicle (basal), 30 ng ml −1 TNF-α or 50 ng ml −1 IL-1β. The cells were labeled with 1 μCi ml −1 [ 3 H]-thymidine for the times indicated in the continuous presence of cytokines (A). For concentration dependence, the cells were stimulated with various concentrations of TNF-α (B) and IL-1β (C). After stimulation for 6 h, cells were labeled with 1 μCi ml −1 [ 3 H]-thymidine for another 18 h in the presence of cytokines. The incorporation of [ 3 H]-thymidine was determined as described in Methods. Data are expressed as the mean±s.e.mean of three separate experiments determined in triplicate. * P

    Techniques Used: Labeling, Concentration Assay

    11) Product Images from "TAK1-Mediated Serine/Threonine Phosphorylation of Epidermal Growth Factor Receptor via p38/Extracellular Signal-Regulated Kinase: NF-κB-Independent Survival Pathways in Tumor Necrosis Factor Alpha Signaling "

    Article Title: TAK1-Mediated Serine/Threonine Phosphorylation of Epidermal Growth Factor Receptor via p38/Extracellular Signal-Regulated Kinase: NF-κB-Independent Survival Pathways in Tumor Necrosis Factor Alpha Signaling

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00375-09

    EGFR is essential for protection from TNF-α-induced proapoptotic signals. (A) HeLa-shLuc or HeLa-shTAK1 cells were treated with TNF-α for 4 h. Whole-cell lysates were immunoblotted with phospho-JNK, JNK, PARP, TAK1, and tubulin antibodies. Arrows show cleaved forms of PARP. (B) HeLa cells were transfected with Luc siRNA or two different siRNAs against EGFR (#1 and #2). At 72 h posttransfection, cells were stimulated with TNF-α for 4 h. Whole-cell lysates were immunoblotted with caspase-3, PARP, EGFR, and actin antibodies. Arrows show cleaved forms of caspase-3 and PARP. (C and D) HeLa cells were pretreated with PD153035 (PD) (1 μM), 5Z-7-oxozeaenol (5Z) (0.3 μM), SB203580 (SB) (10 μM), or U0126 (U) (5 μM) for 30 min and then stimulated with TNF-α for another 4 h. Whole-cell lysates were immunoblotted with the indicated antibodies. DMSO, dimethyl sulfoxide. (E) HeLa cells were transfected with siRNAs against CHC, EGFR (#1), and Luc. At 72 h posttransfection, cells were stimulated with TNF-α for 4 h. Whole-cell lysates were immunoblotted with the indicated antibodies.
    Figure Legend Snippet: EGFR is essential for protection from TNF-α-induced proapoptotic signals. (A) HeLa-shLuc or HeLa-shTAK1 cells were treated with TNF-α for 4 h. Whole-cell lysates were immunoblotted with phospho-JNK, JNK, PARP, TAK1, and tubulin antibodies. Arrows show cleaved forms of PARP. (B) HeLa cells were transfected with Luc siRNA or two different siRNAs against EGFR (#1 and #2). At 72 h posttransfection, cells were stimulated with TNF-α for 4 h. Whole-cell lysates were immunoblotted with caspase-3, PARP, EGFR, and actin antibodies. Arrows show cleaved forms of caspase-3 and PARP. (C and D) HeLa cells were pretreated with PD153035 (PD) (1 μM), 5Z-7-oxozeaenol (5Z) (0.3 μM), SB203580 (SB) (10 μM), or U0126 (U) (5 μM) for 30 min and then stimulated with TNF-α for another 4 h. Whole-cell lysates were immunoblotted with the indicated antibodies. DMSO, dimethyl sulfoxide. (E) HeLa cells were transfected with siRNAs against CHC, EGFR (#1), and Luc. At 72 h posttransfection, cells were stimulated with TNF-α for 4 h. Whole-cell lysates were immunoblotted with the indicated antibodies.

    Techniques Used: Transfection

    Phosphorylation-dependent endocytosis and subsequent dephosphorylation of EGFR. (A and B) HeLa-EGFR-GFP cells were pretreated with SB203580 (SB) (10 μM) and U0126 (U) (5 μM) for 30 min and then stimulated with TNF-α (A) or EGF (B) for another 15 min. Subcellular localization of EGFR-GFP was examined by confocal fluorescent microscopy. (C) Cells were pretreated with SB203580 (10 μM) for 30 min and then stimulated with 20 ng/ml TNF-α or 10 ng/ml EGF for 15 min. Cell surface expression of EGFR was investigated by FACS analysis. Cont, control. (D) HeLa cells were transfected with siRNAs against CHC and Luc. At 72 h posttransfection, cells were stimulated with TNF-α for 10 min. Whole-cell lysates were immunoblotted with the indicated antibodies. (E and F) HeLa cells were pretreated with U0126 (5 μM) (E) or SB203580 (10 μM) (F) for 30 min and then stimulated with TNF-α for another 15 and 60 min. Whole-cell lysates were immunoblotted with the indicated antibodies. DMSO, dimethyl sulfoxide.
    Figure Legend Snippet: Phosphorylation-dependent endocytosis and subsequent dephosphorylation of EGFR. (A and B) HeLa-EGFR-GFP cells were pretreated with SB203580 (SB) (10 μM) and U0126 (U) (5 μM) for 30 min and then stimulated with TNF-α (A) or EGF (B) for another 15 min. Subcellular localization of EGFR-GFP was examined by confocal fluorescent microscopy. (C) Cells were pretreated with SB203580 (10 μM) for 30 min and then stimulated with 20 ng/ml TNF-α or 10 ng/ml EGF for 15 min. Cell surface expression of EGFR was investigated by FACS analysis. Cont, control. (D) HeLa cells were transfected with siRNAs against CHC and Luc. At 72 h posttransfection, cells were stimulated with TNF-α for 10 min. Whole-cell lysates were immunoblotted with the indicated antibodies. (E and F) HeLa cells were pretreated with U0126 (5 μM) (E) or SB203580 (10 μM) (F) for 30 min and then stimulated with TNF-α for another 15 and 60 min. Whole-cell lysates were immunoblotted with the indicated antibodies. DMSO, dimethyl sulfoxide.

    Techniques Used: De-Phosphorylation Assay, Microscopy, Expressing, FACS, Transfection

    Schematic diagram of TNF-α-induced antiapoptotic signals. TNF-α binding to TNF-R1 rapidly induces the activation of TAK1. TAK1 induces two independent signaling pathways to EGFR through ERK and p38. TAK1 regulates two independent antiapoptotic pathways, NF-κB and p38-EGFR. These signals coordinately prevent death-inducing signaling complex (DISC)-mediated proapoptotic cleavage of caspase and PARP.
    Figure Legend Snippet: Schematic diagram of TNF-α-induced antiapoptotic signals. TNF-α binding to TNF-R1 rapidly induces the activation of TAK1. TAK1 induces two independent signaling pathways to EGFR through ERK and p38. TAK1 regulates two independent antiapoptotic pathways, NF-κB and p38-EGFR. These signals coordinately prevent death-inducing signaling complex (DISC)-mediated proapoptotic cleavage of caspase and PARP.

    Techniques Used: Binding Assay, Activation Assay

    Role of TAK1 and EGFR TK activity in TNF-α- and EGF-induced phosphorylation and endocytosis of EGFR. (A) HeLa cells were treated with 20 ng/ml TNF-α or 10 ng/ml EGF for 5 or 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Tyr-845, Tyr-974, and Tyr-1068) and EGFR antibodies. (B) HeLa-shLuc and HeLa-shTAK1 cells were stimulated with TNF-α for 10 min. Whole-cell lysates were immunoblotted with EGFR, TAK1, and PCNA antibodies. (C and D) HeLa cells were pretreated with 5Z-7-oxozeaenol (5Z) (0.3 μM) and PD153035 (PD) (1 μM) for 30 min and then stimulated with 20 ng/ml TNF-α (C) or 10 ng/ml EGF (D) for another 10 min. Whole-cell lysates were immunoblotted with EGFR antibody. (E and F) HeLa cells stably expressing EGFR-GFP were pretreated with 5Z-7-oxozeaenol (0.3 μM) and PD153035 (1 μM) for 30 min and then stimulated with TNF-α (C) or EGF (D) for another 15 min. Subcellular localization of EGFR-GFP was examined by confocal fluorescent microscopy.
    Figure Legend Snippet: Role of TAK1 and EGFR TK activity in TNF-α- and EGF-induced phosphorylation and endocytosis of EGFR. (A) HeLa cells were treated with 20 ng/ml TNF-α or 10 ng/ml EGF for 5 or 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Tyr-845, Tyr-974, and Tyr-1068) and EGFR antibodies. (B) HeLa-shLuc and HeLa-shTAK1 cells were stimulated with TNF-α for 10 min. Whole-cell lysates were immunoblotted with EGFR, TAK1, and PCNA antibodies. (C and D) HeLa cells were pretreated with 5Z-7-oxozeaenol (5Z) (0.3 μM) and PD153035 (PD) (1 μM) for 30 min and then stimulated with 20 ng/ml TNF-α (C) or 10 ng/ml EGF (D) for another 10 min. Whole-cell lysates were immunoblotted with EGFR antibody. (E and F) HeLa cells stably expressing EGFR-GFP were pretreated with 5Z-7-oxozeaenol (0.3 μM) and PD153035 (1 μM) for 30 min and then stimulated with TNF-α (C) or EGF (D) for another 15 min. Subcellular localization of EGFR-GFP was examined by confocal fluorescent microscopy.

    Techniques Used: Activity Assay, Stable Transfection, Expressing, Microscopy

    Phosphorylation of endogenous EGFR at Thr-669 and Ser-1046/1047. (A) HeLa cells were treated with TNF-α or EGF for 10, 30, or 60 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669, Ser-1046/1047, Tyr-845, Tyr-974, Tyr-1045, Tyr-1068, and Tyr-1173), EGFR, phospho-MAPKs (p38, JNK, and ERK), and tubulin antibodies. (B) Cells were exposed to a high concentration of salt (Osmo) (300 mM NaCl), TNF-α, or EGF for the indicated time periods. Whole-cell lysates were immunoblotted with the indicated antibodies. (C) A549 cells were treated with TNF-α, IL-1β, or EGF for 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669 and Ser-1046/1047), EGFR, and actin antibodies. (D) HEK293 cells were transfected with expression vectors for the wild type (WT) and a kinase dead mutant (ΚΚ/ΑΑ) of EGFR. Twenty-four hours after transfection, cells were treated with TNF-α for 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669 and Ser-1046/1047) and EGFR antibodies. Cont, control.
    Figure Legend Snippet: Phosphorylation of endogenous EGFR at Thr-669 and Ser-1046/1047. (A) HeLa cells were treated with TNF-α or EGF for 10, 30, or 60 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669, Ser-1046/1047, Tyr-845, Tyr-974, Tyr-1045, Tyr-1068, and Tyr-1173), EGFR, phospho-MAPKs (p38, JNK, and ERK), and tubulin antibodies. (B) Cells were exposed to a high concentration of salt (Osmo) (300 mM NaCl), TNF-α, or EGF for the indicated time periods. Whole-cell lysates were immunoblotted with the indicated antibodies. (C) A549 cells were treated with TNF-α, IL-1β, or EGF for 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669 and Ser-1046/1047), EGFR, and actin antibodies. (D) HEK293 cells were transfected with expression vectors for the wild type (WT) and a kinase dead mutant (ΚΚ/ΑΑ) of EGFR. Twenty-four hours after transfection, cells were treated with TNF-α for 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669 and Ser-1046/1047) and EGFR antibodies. Cont, control.

    Techniques Used: Concentration Assay, Transfection, Expressing, Mutagenesis

    Signaling pathways leading to Ser/Thr phosphorylation of EGFR. (A) HeLa cells stably expressing shRNA against TAK1 (T) and luciferase (L) were stimulated with TNF-α or EGF for 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669, Ser-1046/1047, Tyr-845, 974, 1045, 1068 and 1173), EGFR, phospho-MAPKs (p38, JNK and ERK), and tubulin antibodies. Cont, control. (B and C) HeLa cells were pretreated with PD153035 (PD) (1 μM), 5Z-7-oxozeaenol (5Z) (0.3 μM), SB203580 (SB) (10 μM), U0126 (U) (5 μM), and SP600125 (SP) (10 μM) for 30 min and then stimulated with TNF-α (B) or EGF (C) for another 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669, Ser-1046/1047, and Tyr845), EGFR, and tubulin antibodies. (D) HeLa cells were transfected with siRNAs against ERK2, p38α, and Luc. At 72 h posttransfection, cells were stimulated with TNF-α for 10 min. Whole-cell lysates were immunoblotted with the indicated antibodies. (E) Cells were stimulated with TNF-α for the indicated time periods (min), and phosphorylation of EGFR, p38, and ERK was detected by immunoblotting.
    Figure Legend Snippet: Signaling pathways leading to Ser/Thr phosphorylation of EGFR. (A) HeLa cells stably expressing shRNA against TAK1 (T) and luciferase (L) were stimulated with TNF-α or EGF for 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669, Ser-1046/1047, Tyr-845, 974, 1045, 1068 and 1173), EGFR, phospho-MAPKs (p38, JNK and ERK), and tubulin antibodies. Cont, control. (B and C) HeLa cells were pretreated with PD153035 (PD) (1 μM), 5Z-7-oxozeaenol (5Z) (0.3 μM), SB203580 (SB) (10 μM), U0126 (U) (5 μM), and SP600125 (SP) (10 μM) for 30 min and then stimulated with TNF-α (B) or EGF (C) for another 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669, Ser-1046/1047, and Tyr845), EGFR, and tubulin antibodies. (D) HeLa cells were transfected with siRNAs against ERK2, p38α, and Luc. At 72 h posttransfection, cells were stimulated with TNF-α for 10 min. Whole-cell lysates were immunoblotted with the indicated antibodies. (E) Cells were stimulated with TNF-α for the indicated time periods (min), and phosphorylation of EGFR, p38, and ERK was detected by immunoblotting.

    Techniques Used: Stable Transfection, Expressing, shRNA, Luciferase, Transfection

    NF-κB is not involved in EGFR phosphorylation. (A) HeLa-shLuc or HeLa-shTAK1 cells were treated with TNF-α for 5 min. Whole-cell lysates were immunoblotted with phospho-p65, p65, PARP, TAK1, and tubulin antibodies. (B to D) HeLa cells were transfected with siRNAs against p65, EGFR (#1), and Luc. At 72 h posttransfection, cells were stimulated with TNF-α for the indicated time periods (B and C) or 4 h (D). Whole-cell lysates were immunoblotted with the indicated antibodies.
    Figure Legend Snippet: NF-κB is not involved in EGFR phosphorylation. (A) HeLa-shLuc or HeLa-shTAK1 cells were treated with TNF-α for 5 min. Whole-cell lysates were immunoblotted with phospho-p65, p65, PARP, TAK1, and tubulin antibodies. (B to D) HeLa cells were transfected with siRNAs against p65, EGFR (#1), and Luc. At 72 h posttransfection, cells were stimulated with TNF-α for the indicated time periods (B and C) or 4 h (D). Whole-cell lysates were immunoblotted with the indicated antibodies.

    Techniques Used: Transfection

    12) Product Images from "Chemerin activates fibroblast-like synoviocytes in patients with rheumatoid arthritis"

    Article Title: Chemerin activates fibroblast-like synoviocytes in patients with rheumatoid arthritis

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar3475

    Expression of chemerin and ChemR23 by rheumatoid arthritis fibroblast-like synoviocytes . (A) and (B) Chemerin expression stimulated by TNF-α (A) or IFN-γ (B) and evaluated by ELISA using supernatants of cultured fibroblast-like synoviocytes (FLSs) isolated from synovial tissue samples taken from rheumatoid arthritis (RA) patients ( n = 4). FLSs (2 × 10 4 cells/well) were stimulated at 37°C for 48 hours with TNF-α (0.1, 1 or 10 ng/ml) (A) or IFN-γ (1, 10 or 100 ng/ml) (A). Data in (A) and (B) are presented as means (± SEM) of one of four independent experiments analyzed in triplicate. * P
    Figure Legend Snippet: Expression of chemerin and ChemR23 by rheumatoid arthritis fibroblast-like synoviocytes . (A) and (B) Chemerin expression stimulated by TNF-α (A) or IFN-γ (B) and evaluated by ELISA using supernatants of cultured fibroblast-like synoviocytes (FLSs) isolated from synovial tissue samples taken from rheumatoid arthritis (RA) patients ( n = 4). FLSs (2 × 10 4 cells/well) were stimulated at 37°C for 48 hours with TNF-α (0.1, 1 or 10 ng/ml) (A) or IFN-γ (1, 10 or 100 ng/ml) (A). Data in (A) and (B) are presented as means (± SEM) of one of four independent experiments analyzed in triplicate. * P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Isolation

    13) Product Images from "Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System"

    Article Title: Lipocalin-2 Is a Chemokine Inducer in the Central Nervous System

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.299248

    Induction of chemokine gene expression by LCN2 in astrocytes, microglia, endothelial cells, and neuron cells. Astrocytes ( A and E ), microglia ( B and E ), bEnd.3 endothelial cells ( C ), and neuron cells ( D ) were treated with the recombinant LCN2 protein (10 μg/ml) for 8 h, and the total RNA was isolated for traditional RT-PCR or real time PCR. The cells were also treated for 8 h with LPS (100 ng/ml), TNF-α (10 ng/ml), or LPS (100 ng/ml) plus IFN-γ (50 units/ml) for comparison purposes. The mRNA levels of chemokines (CCL4, CCL20, CXCL2, and CXCL10) and other inflammatory genes (IL-6, COX-2, iNOS, and PIAS3) were determined by traditional RT-PCR ( A–D ) or real time PCR ( E ). β-Actin or GAPDH was used as an internal control. The results are one representative of more than three independent experiments ( A–D ) or means ± S.D. ( n = 3) ( E ).
    Figure Legend Snippet: Induction of chemokine gene expression by LCN2 in astrocytes, microglia, endothelial cells, and neuron cells. Astrocytes ( A and E ), microglia ( B and E ), bEnd.3 endothelial cells ( C ), and neuron cells ( D ) were treated with the recombinant LCN2 protein (10 μg/ml) for 8 h, and the total RNA was isolated for traditional RT-PCR or real time PCR. The cells were also treated for 8 h with LPS (100 ng/ml), TNF-α (10 ng/ml), or LPS (100 ng/ml) plus IFN-γ (50 units/ml) for comparison purposes. The mRNA levels of chemokines (CCL4, CCL20, CXCL2, and CXCL10) and other inflammatory genes (IL-6, COX-2, iNOS, and PIAS3) were determined by traditional RT-PCR ( A–D ) or real time PCR ( E ). β-Actin or GAPDH was used as an internal control. The results are one representative of more than three independent experiments ( A–D ) or means ± S.D. ( n = 3) ( E ).

    Techniques Used: Expressing, Recombinant, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    14) Product Images from "Characterization of human fetal brain endothelial cells reveals barrier properties suitable for in vitro modeling of the BBB with syngenic co-cultures"

    Article Title: Characterization of human fetal brain endothelial cells reveals barrier properties suitable for in vitro modeling of the BBB with syngenic co-cultures

    Journal: Journal of Cerebral Blood Flow & Metabolism

    doi: 10.1177/0271678X17708690

    Functional assays of immune–endothelial interactions using fBMVEC s. (a) Adhesion assay: fBMVEC s were treated with TNF-α (20 ng/mL) for 18 h. Treatments were removed prior to addition of monocytes. Data are represented as fold difference (mean ± SEM) of adherent cells. Results show a 2.5-fold increased in adherent monocytes on activated as compared to unactivated ECs. (b) Trans-endothelial migration assay: Monocytes were added to the upper chamber of Transwell® membranes and allowed to migrate through fBMVEC s towards MCP-1 in the lower chamber. Data are represented as fold difference (mean ± SEM) of migrated cells. Results show a ∼ 2-fold increase in migrated monocytes with the addition of MCP-1. (c) Representative images of the underside of the Transwell® showing migrated Calcein-AM labeled monocytes with or without MCP-1 in the lower chamber. Scare bars 100 µm.
    Figure Legend Snippet: Functional assays of immune–endothelial interactions using fBMVEC s. (a) Adhesion assay: fBMVEC s were treated with TNF-α (20 ng/mL) for 18 h. Treatments were removed prior to addition of monocytes. Data are represented as fold difference (mean ± SEM) of adherent cells. Results show a 2.5-fold increased in adherent monocytes on activated as compared to unactivated ECs. (b) Trans-endothelial migration assay: Monocytes were added to the upper chamber of Transwell® membranes and allowed to migrate through fBMVEC s towards MCP-1 in the lower chamber. Data are represented as fold difference (mean ± SEM) of migrated cells. Results show a ∼ 2-fold increase in migrated monocytes with the addition of MCP-1. (c) Representative images of the underside of the Transwell® showing migrated Calcein-AM labeled monocytes with or without MCP-1 in the lower chamber. Scare bars 100 µm.

    Techniques Used: Functional Assay, Cell Adhesion Assay, Migration, Labeling

    TNF-α induces upregulation of adhesion molecule expression in fBMVEC s. Fetal and adult BMVECs were stimulated with TNF-α (100 ng/mL), harvested at 0, 6, 18, 24, 48 h and analyzed by flow cytometry. Bar graph and histograms show the upregulation of adhesion molecules ICAM-1 (a,c) and VCAM-1 (b,d) in response to TNF-α which was not statistically significant between fBMVEC s and aBMVEC s.
    Figure Legend Snippet: TNF-α induces upregulation of adhesion molecule expression in fBMVEC s. Fetal and adult BMVECs were stimulated with TNF-α (100 ng/mL), harvested at 0, 6, 18, 24, 48 h and analyzed by flow cytometry. Bar graph and histograms show the upregulation of adhesion molecules ICAM-1 (a,c) and VCAM-1 (b,d) in response to TNF-α which was not statistically significant between fBMVEC s and aBMVEC s.

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    15) Product Images from "Tumour necrosis factor-? stimulates HIV-1 replication in single-cycle infection of human term placental villi fragments in a time, viral dose and envelope dependent manner"

    Article Title: Tumour necrosis factor-? stimulates HIV-1 replication in single-cycle infection of human term placental villi fragments in a time, viral dose and envelope dependent manner

    Journal: Retrovirology

    doi: 10.1186/1742-4690-3-36

    Infection with VSV-G or delta-Env pseudotyped HIV-1 ofplacental chorionic villi in the absence or presence of TNF-α . Viral pseudotypes were left in contact with placental fragments overnight. After viral contact, fragments were cultured in medium supplemented with (grey histograms) or without (horizontal black line) TNF-α and cultures stopped at 48, 96, 120 hours post-infection. Fragments were homogenized and luciferase activity read from tissue lysate. Results are presented as ratio (mean ± SE) of luciferase activity with TNF-α stimulation to activity without stimulation in three to seven (n) placentas from different donors. A: VSV-G pseudotyped HIV-1 at 0.2 ng of p24/placental fragment; B: VSV-G pseudotyped HIV-1 at 2 ng of p24/placental fragment; C: VSV-G pseudotyped HIV-1 at 20 ng of p24/placental fragment; D: delta-Env pseudotyped HIV-1 at 20 ng of p24/placental fragment.
    Figure Legend Snippet: Infection with VSV-G or delta-Env pseudotyped HIV-1 ofplacental chorionic villi in the absence or presence of TNF-α . Viral pseudotypes were left in contact with placental fragments overnight. After viral contact, fragments were cultured in medium supplemented with (grey histograms) or without (horizontal black line) TNF-α and cultures stopped at 48, 96, 120 hours post-infection. Fragments were homogenized and luciferase activity read from tissue lysate. Results are presented as ratio (mean ± SE) of luciferase activity with TNF-α stimulation to activity without stimulation in three to seven (n) placentas from different donors. A: VSV-G pseudotyped HIV-1 at 0.2 ng of p24/placental fragment; B: VSV-G pseudotyped HIV-1 at 2 ng of p24/placental fragment; C: VSV-G pseudotyped HIV-1 at 20 ng of p24/placental fragment; D: delta-Env pseudotyped HIV-1 at 20 ng of p24/placental fragment.

    Techniques Used: Infection, Cell Culture, Luciferase, Activity Assay

    Infection with R5 or X4 envelope pseudotyped HIV-1 of placental chorionic villi in the absence or presence of TNF-α . Viral pseudotypes were left in contact with placental fragments overnight. After viral contact, fragments were cultured in medium supplemented with (grey histograms) or without (horizontal black line) TNF-α and cultures stopped at 120, 168 or 216 hours post-infection. Fragments were homogenised and luciferase activity read from tissue lysate. Results are presented as ratio (mean ± SE) of luciferase activity with TNF-α stimulation to activity without stimulation in three to seven (n) placentas from different donors. A: R5 (Ba-L) pseudotyped HIV-1 at 20 ng of p24/placental fragment; B: R5 (Ba-L) pseudotyped HIV-1 at 50 ng of p24/placental fragment; C: X4 (HXB2) pseudotyped HIV-1 at 50 ng of p24/placental fragment; D: X4 (HXB2) pseudotyped HIV-1 at 100 ng of p24/placental fragment.
    Figure Legend Snippet: Infection with R5 or X4 envelope pseudotyped HIV-1 of placental chorionic villi in the absence or presence of TNF-α . Viral pseudotypes were left in contact with placental fragments overnight. After viral contact, fragments were cultured in medium supplemented with (grey histograms) or without (horizontal black line) TNF-α and cultures stopped at 120, 168 or 216 hours post-infection. Fragments were homogenised and luciferase activity read from tissue lysate. Results are presented as ratio (mean ± SE) of luciferase activity with TNF-α stimulation to activity without stimulation in three to seven (n) placentas from different donors. A: R5 (Ba-L) pseudotyped HIV-1 at 20 ng of p24/placental fragment; B: R5 (Ba-L) pseudotyped HIV-1 at 50 ng of p24/placental fragment; C: X4 (HXB2) pseudotyped HIV-1 at 50 ng of p24/placental fragment; D: X4 (HXB2) pseudotyped HIV-1 at 100 ng of p24/placental fragment.

    Techniques Used: Infection, Cell Culture, Luciferase, Activity Assay

    Ex vivo bioluminescence imaging of HIV-1 infected placental chorionic villi stimulated or not with TNF-α . Placental chorionic villi were placed in contact with pseudotyped HIV-1 overnight. After the addition of luciferine for 4 h at 37°C, the tissues were analysed with the Xenogen system. Infection of chorionic villi with 2 ng of p24/fragment of VSV-G pseudotyped HIV-1 in the absence (well 1) or presence of 5 ng/ml of TNF-α (well 3). Infection of chorionic villi with 20 ng of p24/fragment of delta-Env pseudotyped HIV-1 in the absence (well 2) or presence of 5 ng/ml of TNF-α (well 4).
    Figure Legend Snippet: Ex vivo bioluminescence imaging of HIV-1 infected placental chorionic villi stimulated or not with TNF-α . Placental chorionic villi were placed in contact with pseudotyped HIV-1 overnight. After the addition of luciferine for 4 h at 37°C, the tissues were analysed with the Xenogen system. Infection of chorionic villi with 2 ng of p24/fragment of VSV-G pseudotyped HIV-1 in the absence (well 1) or presence of 5 ng/ml of TNF-α (well 3). Infection of chorionic villi with 20 ng of p24/fragment of delta-Env pseudotyped HIV-1 in the absence (well 2) or presence of 5 ng/ml of TNF-α (well 4).

    Techniques Used: Ex Vivo, Imaging, Infection

    16) Product Images from "Nanoliter high throughput quantitative PCR"

    Article Title: Nanoliter high throughput quantitative PCR

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl639

    Transcript analysis of TNF-α treated HUVEC. PCR arrays were used to make transcript measurements of cDNA generated from pooled HUVEC cells treated with either TNF-α or vehicle. Samples were normalized (ΔC T ) by subtracting the geometric mean of 13 housekeeping genes from the C T of each assay. ( A ) Correlates ΔC T between treated and untreated assays (circles), triangles showing significant difference were, from left-to-right genes CCL2 (NM_ 002982), SELE (NM_000450) , TNFAIP2 (NM_006291) , TNFAIP3 (NM_006290) , VCAM1 ( P
    Figure Legend Snippet: Transcript analysis of TNF-α treated HUVEC. PCR arrays were used to make transcript measurements of cDNA generated from pooled HUVEC cells treated with either TNF-α or vehicle. Samples were normalized (ΔC T ) by subtracting the geometric mean of 13 housekeeping genes from the C T of each assay. ( A ) Correlates ΔC T between treated and untreated assays (circles), triangles showing significant difference were, from left-to-right genes CCL2 (NM_ 002982), SELE (NM_000450) , TNFAIP2 (NM_006291) , TNFAIP3 (NM_006290) , VCAM1 ( P

    Techniques Used: Polymerase Chain Reaction, Generated

    17) Product Images from "Expression and regulation of the chemokine CXCL16 in Crohn's disease and models of intestinal inflammation"

    Article Title: Expression and regulation of the chemokine CXCL16 in Crohn's disease and models of intestinal inflammation

    Journal: Inflammatory bowel diseases

    doi: 10.1002/ibd.21306

    CXCL16 gene expression is upregulated in intestinal inflammation (A) The proinflammatory cytokines TNF-α (50 μg/mL), IL-1β (10 μg/mL) and IFN-γ (1000 U/mL) significantly increase expression of CXCL16 mRNA in HT-29 cells as determined by quantitative PCR (*p
    Figure Legend Snippet: CXCL16 gene expression is upregulated in intestinal inflammation (A) The proinflammatory cytokines TNF-α (50 μg/mL), IL-1β (10 μg/mL) and IFN-γ (1000 U/mL) significantly increase expression of CXCL16 mRNA in HT-29 cells as determined by quantitative PCR (*p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    18) Product Images from "Expression of MicroRNA-146 in Rheumatoid Arthritis Synovial Tissue"

    Article Title: Expression of MicroRNA-146 in Rheumatoid Arthritis Synovial Tissue

    Journal: Arthritis and rheumatism

    doi: 10.1002/art.23429

    Quantitative reverse transcription–polymerase chain reaction analysis of the expression of primary microRNA-146a/b (pri-miR-146a/b), tumor necrosis factor α (TNF α ), and mature miR-146a in synovial tissue from 5 patients with rheumatoid arthritis (RA), 5 patients with osteoarthritis (OA), and a normal control subject. GAPDH was used as an internal control for primary miR-146a/b and TNF α , and let-7a was used as an internal control for mature miR-146a. A and B, Primary miR-146a/b mRNA was strongly expressed in RA synovial tissue, except for that from patient RA4. In OA synovium, primary miR-146a/b expression was low. C, TNF α mRNA was expressed in the same pattern as that of primary miR-146a/b. Normal synovial tissue showed little primary miR-146a/b or TNF α mRNA expression. D, Mature miR-146a mRNA was more strongly expressed in synovial tissue from patients RA1, RA2, RA3, and RA5 than in tissue from patient RA4 and all of the OA patients.
    Figure Legend Snippet: Quantitative reverse transcription–polymerase chain reaction analysis of the expression of primary microRNA-146a/b (pri-miR-146a/b), tumor necrosis factor α (TNF α ), and mature miR-146a in synovial tissue from 5 patients with rheumatoid arthritis (RA), 5 patients with osteoarthritis (OA), and a normal control subject. GAPDH was used as an internal control for primary miR-146a/b and TNF α , and let-7a was used as an internal control for mature miR-146a. A and B, Primary miR-146a/b mRNA was strongly expressed in RA synovial tissue, except for that from patient RA4. In OA synovium, primary miR-146a/b expression was low. C, TNF α mRNA was expressed in the same pattern as that of primary miR-146a/b. Normal synovial tissue showed little primary miR-146a/b or TNF α mRNA expression. D, Mature miR-146a mRNA was more strongly expressed in synovial tissue from patients RA1, RA2, RA3, and RA5 than in tissue from patient RA4 and all of the OA patients.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    Induction of primary microRNA-146a/b (pri-miRNA-146a/b) and mature miR-146a microRNA expression in rheumatoid arthritis synovial fibroblasts (RASFs) stimulated with tumor necrosis factor α (TNF α ) and interleukin-1 β (IL-1 β ). A, Expression of mature miR-146a, as determined by reverse transcription–polymerase chain reaction (RT-PCR) analysis. Mature miR-146 expression in RASFs was significantly increased after TNF α and IL-1 β stimulation. B, Expression of mRNA for primary miR-146a (pri-miR-146a), primary miR-146b, and TNF α by RT-PCR analysis, normalized to GAPDH expression. Primary miR-146a/b and TNF α mRNA expression in RASFs increased following TNF α and IL-1 β stimulation. C and D, Expression of primary miR-146a ( C ) and primary miR-146b ( D ), as determined by quantitative RT-PCR analysis. Primary miR-146a/b expression was significantly up-regulated by TNF α and IL-1 β stimulation. Bars show the mean and SD of triplicate experiments. P values were determined by Mann-Whitney U test.
    Figure Legend Snippet: Induction of primary microRNA-146a/b (pri-miRNA-146a/b) and mature miR-146a microRNA expression in rheumatoid arthritis synovial fibroblasts (RASFs) stimulated with tumor necrosis factor α (TNF α ) and interleukin-1 β (IL-1 β ). A, Expression of mature miR-146a, as determined by reverse transcription–polymerase chain reaction (RT-PCR) analysis. Mature miR-146 expression in RASFs was significantly increased after TNF α and IL-1 β stimulation. B, Expression of mRNA for primary miR-146a (pri-miR-146a), primary miR-146b, and TNF α by RT-PCR analysis, normalized to GAPDH expression. Primary miR-146a/b and TNF α mRNA expression in RASFs increased following TNF α and IL-1 β stimulation. C and D, Expression of primary miR-146a ( C ) and primary miR-146b ( D ), as determined by quantitative RT-PCR analysis. Primary miR-146a/b expression was significantly up-regulated by TNF α and IL-1 β stimulation. Bars show the mean and SD of triplicate experiments. P values were determined by Mann-Whitney U test.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, MANN-WHITNEY

    19) Product Images from "An IMiD-induced SALL4 degron system for selective degradation of target proteins"

    Article Title: An IMiD-induced SALL4 degron system for selective degradation of target proteins

    Journal: Communications Biology

    doi: 10.1038/s42003-020-01240-5

    Analysis of RelA-dependent signaling in RelA-sfGFP-S4D-KI cells. a Quantitative RT-PCR for the expression of TNF-α-induced genes. Parental or RelA-sfGFP-S4D-KI cells were pretreated with DMSO or pomalidomide (Po) for 24 h. Then, the cells were stimulated with 20 ng/ml TNF-α for 1 h, and the expression of IκBα or IL-6 was measured by quantitative RT-PCR. The mRNA expression in untreated parental HeLa cells was set to 1.0. b , c Pomalidomide causes TNF-α-induced cell death. Parental and RelA-sfGFP-S4D-KI cells pretreated with DMSO or Po for 12 h were stimulated with 20 ng/ml TNF-α for 12 h, and the viability was measured by MTS assay ( b ) or trypan blue staining ( c ). d Immunoblot analysis of pomalidomide-dependent TNF-α-induced cell death. Parental and RelA-sfGFP-S4D-KI cells pretreated with pomalidomide for 12 h were stimulated with 50 ng/ml TNF-α for the indicated times, and effectors of cell death were analyzed by immunoblot. e , f zVAD-FMK treatment rescued TNF-α-induced cell death in pomalidomide-treated KI cells. Parental and RelA-sfGFP-S4D-KI cells pretreated with 10 µM Po for 12 h were then treated with DMSO or 10 µM zVAD-FMK. After 2 h of zVAD-FMK treatment, the cells were stimulated with 50 ng/ml TNF-α for 12 h and the viability was measured by trypan blue staining ( e ), or effectors of apoptosis were analyzed by immunoblot ( f ). Error bars in a – c and e represent the mean ± SD ( n = 3), and P values were calculated by one-way ANOVA with Tukey’s post-hoc tests (NS not significant; * P
    Figure Legend Snippet: Analysis of RelA-dependent signaling in RelA-sfGFP-S4D-KI cells. a Quantitative RT-PCR for the expression of TNF-α-induced genes. Parental or RelA-sfGFP-S4D-KI cells were pretreated with DMSO or pomalidomide (Po) for 24 h. Then, the cells were stimulated with 20 ng/ml TNF-α for 1 h, and the expression of IκBα or IL-6 was measured by quantitative RT-PCR. The mRNA expression in untreated parental HeLa cells was set to 1.0. b , c Pomalidomide causes TNF-α-induced cell death. Parental and RelA-sfGFP-S4D-KI cells pretreated with DMSO or Po for 12 h were stimulated with 20 ng/ml TNF-α for 12 h, and the viability was measured by MTS assay ( b ) or trypan blue staining ( c ). d Immunoblot analysis of pomalidomide-dependent TNF-α-induced cell death. Parental and RelA-sfGFP-S4D-KI cells pretreated with pomalidomide for 12 h were stimulated with 50 ng/ml TNF-α for the indicated times, and effectors of cell death were analyzed by immunoblot. e , f zVAD-FMK treatment rescued TNF-α-induced cell death in pomalidomide-treated KI cells. Parental and RelA-sfGFP-S4D-KI cells pretreated with 10 µM Po for 12 h were then treated with DMSO or 10 µM zVAD-FMK. After 2 h of zVAD-FMK treatment, the cells were stimulated with 50 ng/ml TNF-α for 12 h and the viability was measured by trypan blue staining ( e ), or effectors of apoptosis were analyzed by immunoblot ( f ). Error bars in a – c and e represent the mean ± SD ( n = 3), and P values were calculated by one-way ANOVA with Tukey’s post-hoc tests (NS not significant; * P

    Techniques Used: Quantitative RT-PCR, Expressing, MTS Assay, Staining

    Analysis of IκBα-dependent signaling in IκBα-sfGFP-S4D-KI cells. a Nuclear and cytoplasmic fractions were obtained from parental and IκBα-sfGFP-S4D-KI (IκBα-KI) cells after treatment with 10 µM pomalidomide for the indicated times and were analyzed by immunoblot. b Parental and IκBα-sfGFP-S4D-KI (IκBα-KI) cells were pretreated with 10 µM pomalidomide for 24 h, and then stimulated with TNF-α for the indicated times. Nuclear and cytoplasmic fractions were analyzed by immunoblot.
    Figure Legend Snippet: Analysis of IκBα-dependent signaling in IκBα-sfGFP-S4D-KI cells. a Nuclear and cytoplasmic fractions were obtained from parental and IκBα-sfGFP-S4D-KI (IκBα-KI) cells after treatment with 10 µM pomalidomide for the indicated times and were analyzed by immunoblot. b Parental and IκBα-sfGFP-S4D-KI (IκBα-KI) cells were pretreated with 10 µM pomalidomide for 24 h, and then stimulated with TNF-α for the indicated times. Nuclear and cytoplasmic fractions were analyzed by immunoblot.

    Techniques Used:

    20) Product Images from "TNF-α Suppresses α-Smooth Muscle Actin Expression in Human Dermal Fibroblasts: An Implication for Abnormal Wound Healing"

    Article Title: TNF-α Suppresses α-Smooth Muscle Actin Expression in Human Dermal Fibroblasts: An Implication for Abnormal Wound Healing

    Journal: The Journal of investigative dermatology

    doi: 10.1038/sj.jid.5700890

    TNF- α affects α -SMA mRNA stability
    Figure Legend Snippet: TNF- α affects α -SMA mRNA stability

    Techniques Used:

    TNF- α inhibits Smad3 phosphorylation in normal human dermal fibroblasts
    Figure Legend Snippet: TNF- α inhibits Smad3 phosphorylation in normal human dermal fibroblasts

    Techniques Used:

    TNF- α decreases TGF- β 1 induction of α -SMA expression in a JNK-dependent pathway
    Figure Legend Snippet: TNF- α decreases TGF- β 1 induction of α -SMA expression in a JNK-dependent pathway

    Techniques Used: Expressing

    TNF- α suppresses TGF- β 1 promotion of myofibroblast structural elements
    Figure Legend Snippet: TNF- α suppresses TGF- β 1 promotion of myofibroblast structural elements

    Techniques Used:

    TNF- α affects the mechanical response of the fibroblast collagen constructs
    Figure Legend Snippet: TNF- α affects the mechanical response of the fibroblast collagen constructs

    Techniques Used: Construct

    TNF- α suppresses TGF- β 1-induced gene expression at the mRNA level
    Figure Legend Snippet: TNF- α suppresses TGF- β 1-induced gene expression at the mRNA level

    Techniques Used: Expressing

    TNF- α abolishes TGF- β 1-induced myofibroblast contractility
    Figure Legend Snippet: TNF- α abolishes TGF- β 1-induced myofibroblast contractility

    Techniques Used:

    TNF- α suppresses TGF- β 1 induction in human dermal fibroblasts
    Figure Legend Snippet: TNF- α suppresses TGF- β 1 induction in human dermal fibroblasts

    Techniques Used:

    A proposed mechanism of TNF- α suppression of TGF- β 1-induced gene expression
    Figure Legend Snippet: A proposed mechanism of TNF- α suppression of TGF- β 1-induced gene expression

    Techniques Used: Expressing

    21) Product Images from "The Role of IFN-? and TNF-?-Responsive Regulatory Elements in the Synergistic Induction of Indoleamine Dioxygenase"

    Article Title: The Role of IFN-? and TNF-?-Responsive Regulatory Elements in the Synergistic Induction of Indoleamine Dioxygenase

    Journal:

    doi: 10.1089/jir.2005.25.20

    TNF-α synergistically enhances IFN-γ-induced IDO transcription. HeLa cells were treated in triplicate with increasing concentrations of TNF-α alone (0.5–50 ng/ml) or combined with IFN-γ (10 ng/ml) for 48 h and analyzed
    Figure Legend Snippet: TNF-α synergistically enhances IFN-γ-induced IDO transcription. HeLa cells were treated in triplicate with increasing concentrations of TNF-α alone (0.5–50 ng/ml) or combined with IFN-γ (10 ng/ml) for 48 h and analyzed

    Techniques Used:

    Effect of TNF-α on C/EBP-β nuclear translocation. HeLa cells were cultivated with medium alone (medium), IFN-γ at 10 ng/ml (IFN), TNF-α at 5 ng/ml (TNF), or combined IFN-γ and TNF-α (IFN/TNF) in culture
    Figure Legend Snippet: Effect of TNF-α on C/EBP-β nuclear translocation. HeLa cells were cultivated with medium alone (medium), IFN-γ at 10 ng/ml (IFN), TNF-α at 5 ng/ml (TNF), or combined IFN-γ and TNF-α (IFN/TNF) in culture

    Techniques Used: Translocation Assay

    22) Product Images from "Leukadherin-1 ameliorates endothelial barrier damage mediated by neutrophils from critically ill patients"

    Article Title: Leukadherin-1 ameliorates endothelial barrier damage mediated by neutrophils from critically ill patients

    Journal: Journal of Intensive Care

    doi: 10.1186/s40560-018-0289-5

    LA-1 inhibits migration of neutrophils on endothelial monolayers. HUVEC monolayers were grown on delta T dishes coated with 40 μg/mL type I collagen and TNF-α activated. Neutrophils were either TNF-α-activated and given no treatment, 15 μM LA-1, or DMSO vehicle control. Cell interactions were allowed to adhere at 37 °C and neutrophil migration dynamics were imaged. × 20 bright-field images were captured every 30 s for 20 min. Neutrophil migration paths were tracked in ImageJ and analyzed using ibidi’s Chemotaxis and Migration Tool ( a ). LA-1 treatment reduced neutrophil path length ( b ), displacement ( c ), and speed ( d ) regardless of donor. Two-way ANOVA with post hoc Tukey analysis, * p
    Figure Legend Snippet: LA-1 inhibits migration of neutrophils on endothelial monolayers. HUVEC monolayers were grown on delta T dishes coated with 40 μg/mL type I collagen and TNF-α activated. Neutrophils were either TNF-α-activated and given no treatment, 15 μM LA-1, or DMSO vehicle control. Cell interactions were allowed to adhere at 37 °C and neutrophil migration dynamics were imaged. × 20 bright-field images were captured every 30 s for 20 min. Neutrophil migration paths were tracked in ImageJ and analyzed using ibidi’s Chemotaxis and Migration Tool ( a ). LA-1 treatment reduced neutrophil path length ( b ), displacement ( c ), and speed ( d ) regardless of donor. Two-way ANOVA with post hoc Tukey analysis, * p

    Techniques Used: Migration, Chemotaxis Assay

    23) Product Images from "Therapeutic effects of bee venom and its major component, melittin, on atopic dermatitis in vivo and in vitro) Therapeutic effects of bee venom and its major component, melittin, on atopic dermatitis in vivo and in vitro"

    Article Title: Therapeutic effects of bee venom and its major component, melittin, on atopic dermatitis in vivo and in vitro) Therapeutic effects of bee venom and its major component, melittin, on atopic dermatitis in vivo and in vitro

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.14487

    Effects of melittin on activation of the (A) JAK2, STAT1 and STAT3 signalling pathways in TNF‐α/IFN‐γ‐stimulated HaCaT cells. (B) Immunofluorescence staining for p‐STAT1 and p‐STAT3 (labelled with Alexa Fluor 555, red), and F‐actin (labelled with Alexa Fluor 488, green). Cells were counterstained with Hoechst 33342 (blue). Effects of melittin on activation of the (C) NF‐κB signalling pathway in TNF‐α/IFN‐γ‐stimulated HaCaT cells. (D) NF‐κB DNA binding activity in the nuclear extract was measured by EMSA. Representative images from each group. Scale bar = 50 μm. NC, normal control; T+I, TNF‐α/IFN‐γ‐stimulated; Mel1, TNF‐α/IFN‐γ‐stimulated +1 μg·mL −1 Mel.
    Figure Legend Snippet: Effects of melittin on activation of the (A) JAK2, STAT1 and STAT3 signalling pathways in TNF‐α/IFN‐γ‐stimulated HaCaT cells. (B) Immunofluorescence staining for p‐STAT1 and p‐STAT3 (labelled with Alexa Fluor 555, red), and F‐actin (labelled with Alexa Fluor 488, green). Cells were counterstained with Hoechst 33342 (blue). Effects of melittin on activation of the (C) NF‐κB signalling pathway in TNF‐α/IFN‐γ‐stimulated HaCaT cells. (D) NF‐κB DNA binding activity in the nuclear extract was measured by EMSA. Representative images from each group. Scale bar = 50 μm. NC, normal control; T+I, TNF‐α/IFN‐γ‐stimulated; Mel1, TNF‐α/IFN‐γ‐stimulated +1 μg·mL −1 Mel.

    Techniques Used: Activation Assay, Immunofluorescence, Staining, Binding Assay, Activity Assay

    Cytotoxic effects of (A) melittin on HaCaT cells. Cell viability was determined by CCK‐8 assay ( n = 5). Effects of melittin on the chemokine expressions of cytokines (B) and chemokines ( n = 5) (C) in HaCaT cells that were stimulated by TNF‐α/IFN‐γ. T+I, TNF‐α/IFN‐γ‐stimulated; * P
    Figure Legend Snippet: Cytotoxic effects of (A) melittin on HaCaT cells. Cell viability was determined by CCK‐8 assay ( n = 5). Effects of melittin on the chemokine expressions of cytokines (B) and chemokines ( n = 5) (C) in HaCaT cells that were stimulated by TNF‐α/IFN‐γ. T+I, TNF‐α/IFN‐γ‐stimulated; * P

    Techniques Used: CCK-8 Assay

    24) Product Images from "Commensal bacteria modulate cullin-dependent signaling via generation of reactive oxygen species"

    Article Title: Commensal bacteria modulate cullin-dependent signaling via generation of reactive oxygen species

    Journal:

    doi: 10.1038/sj.emboj.7601867

    Oxidant modulation of E3-SCF βTrCP mediated signaling. ( A ) H 2 O 2 prevents TNF-α-induced p65 nuclear translocation. Immunofluorescent staining of p65 in HeLa cells preincubated with 1 mM H 2 O 2 for 45 min and treated with TNF-α for
    Figure Legend Snippet: Oxidant modulation of E3-SCF βTrCP mediated signaling. ( A ) H 2 O 2 prevents TNF-α-induced p65 nuclear translocation. Immunofluorescent staining of p65 in HeLa cells preincubated with 1 mM H 2 O 2 for 45 min and treated with TNF-α for

    Techniques Used: Translocation Assay, Staining

    25) Product Images from "The Unfolded Protein Response is a Major Mechanism by which LRP1 Regulates Schwann Cell Survival After Injury"

    Article Title: The Unfolded Protein Response is a Major Mechanism by which LRP1 Regulates Schwann Cell Survival After Injury

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.2850-11.2011

    LRP1 ligands block TNF-α mediated apoptosis
    Figure Legend Snippet: LRP1 ligands block TNF-α mediated apoptosis

    Techniques Used: Blocking Assay

    26) Product Images from "IL-17 regulates gene expression and protein synthesis of the complement system, C3 and factor B, in skin fibroblasts"

    Article Title: IL-17 regulates gene expression and protein synthesis of the complement system, C3 and factor B, in skin fibroblasts

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.2000.01199.x

    Effect of simultaneous incubation with hIL-17 and tumour necrosis factor-alpha (TNF-α) on C3 and factor B (Bf) synthesis. Fibroblast monolayers were incubated for 24 h in medium/bovine serum albumin (BSA) (lane C) or with 10 ng/ml hIL-17 (IL-17), 1 ng/ml TNF-α (TNF), or both (TNF + IL-17). Cells were subsequently subjected to metabolic labelling with 35 S-methionine, and C3 protein (a) and Bf protein (b) were immunoprecipitated from cell lysates by specific antibodies and subjected to SDS–PAGE followed by impregnating with autofluor and exposure to x-ray film (upper inserts). Incorporation of 35 S-methionine into specific immunoprecipitated protein was determined in gel slices after digestion and counting with scintillation fluid in a β-counter. Data are expressed as the percent ratio of specific protein to total TCA-precipitable protein in the same condition. The expected additive effects (calculated from counts/TCA from IL-17 alone + TNF-α alone – control, hatched bars) were compared with the actual effects (▪).
    Figure Legend Snippet: Effect of simultaneous incubation with hIL-17 and tumour necrosis factor-alpha (TNF-α) on C3 and factor B (Bf) synthesis. Fibroblast monolayers were incubated for 24 h in medium/bovine serum albumin (BSA) (lane C) or with 10 ng/ml hIL-17 (IL-17), 1 ng/ml TNF-α (TNF), or both (TNF + IL-17). Cells were subsequently subjected to metabolic labelling with 35 S-methionine, and C3 protein (a) and Bf protein (b) were immunoprecipitated from cell lysates by specific antibodies and subjected to SDS–PAGE followed by impregnating with autofluor and exposure to x-ray film (upper inserts). Incorporation of 35 S-methionine into specific immunoprecipitated protein was determined in gel slices after digestion and counting with scintillation fluid in a β-counter. Data are expressed as the percent ratio of specific protein to total TCA-precipitable protein in the same condition. The expected additive effects (calculated from counts/TCA from IL-17 alone + TNF-α alone – control, hatched bars) were compared with the actual effects (▪).

    Techniques Used: Incubation, Metabolic Labelling, Immunoprecipitation, SDS Page

    27) Product Images from "Interferon-Beta Induces Distinct Gene Expression Response Patterns in Human Monocytes versus T cells"

    Article Title: Interferon-Beta Induces Distinct Gene Expression Response Patterns in Human Monocytes versus T cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0062366

    CD38 protein expression is increased in monocytes, but not in T cells, in response to IFN-β. Flow cytometry analysis of CD38 expression in monocytes and T cells incubated with TNF-α for 2 hours and then treated with IFN-β for 40 hours. Representative results of CD38 expression with and without IFN-β treatment in A. CD14+ monocytes and B. CD3+ T cells. N≥3 for each cell type.
    Figure Legend Snippet: CD38 protein expression is increased in monocytes, but not in T cells, in response to IFN-β. Flow cytometry analysis of CD38 expression in monocytes and T cells incubated with TNF-α for 2 hours and then treated with IFN-β for 40 hours. Representative results of CD38 expression with and without IFN-β treatment in A. CD14+ monocytes and B. CD3+ T cells. N≥3 for each cell type.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Incubation

    RT-PCR confirms differential expression between monocytes and T cells in response to IFN-β. Monocytes, T cells, and PBMCs from healthy donors were included in the validation of eight DEGs selected from the microarray data. Triangles represent monocytes, circles- T cells, and diamonds- PBMCs. Full and empty symbols represent cells pre-incubated or not with TNF-α. The horizontal bars mark the median values. The Y axis depicts the changes in expression levels in response to IFN-β as fold change (2 −ΔΔCT ); *p-values
    Figure Legend Snippet: RT-PCR confirms differential expression between monocytes and T cells in response to IFN-β. Monocytes, T cells, and PBMCs from healthy donors were included in the validation of eight DEGs selected from the microarray data. Triangles represent monocytes, circles- T cells, and diamonds- PBMCs. Full and empty symbols represent cells pre-incubated or not with TNF-α. The horizontal bars mark the median values. The Y axis depicts the changes in expression levels in response to IFN-β as fold change (2 −ΔΔCT ); *p-values

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Microarray, Incubation

    28) Product Images from "Impaired Immune Tolerance to Porphyromonas gingivalis Lipopolysaccharide Promotes Neutrophil Migration and Decreased Apoptosis ▿"

    Article Title: Impaired Immune Tolerance to Porphyromonas gingivalis Lipopolysaccharide Promotes Neutrophil Migration and Decreased Apoptosis ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00600-10

    Production of IL-8 and TNF-α by naïve and tolerized THP-1 cells after 4 h of treatment with 1 μg/ml P. gingivalis LPS. Cells were tolerized by 24 h pretreatment with the same LPS. Control cells were incubated in medium alone. Values
    Figure Legend Snippet: Production of IL-8 and TNF-α by naïve and tolerized THP-1 cells after 4 h of treatment with 1 μg/ml P. gingivalis LPS. Cells were tolerized by 24 h pretreatment with the same LPS. Control cells were incubated in medium alone. Values

    Techniques Used: Incubation

    Production of IL-8 and TNF-α by naïve and tolerized THP-1 cells after 4 h of treatment with 1 μg/ml E. coli LPS. Cells were tolerized by 24 h pretreatment with the same LPS. Control cells were incubated in medium alone. Values
    Figure Legend Snippet: Production of IL-8 and TNF-α by naïve and tolerized THP-1 cells after 4 h of treatment with 1 μg/ml E. coli LPS. Cells were tolerized by 24 h pretreatment with the same LPS. Control cells were incubated in medium alone. Values

    Techniques Used: Incubation

    Effect of endotoxin tolerance on neutrophil caspase-3 activity. Freshly isolated neutrophils were incubated for 3 h in nontreated THP-1 supernatant (control), medium containing 10 ng/ml TNF-α (positive control), medium containing 1 μg/ml
    Figure Legend Snippet: Effect of endotoxin tolerance on neutrophil caspase-3 activity. Freshly isolated neutrophils were incubated for 3 h in nontreated THP-1 supernatant (control), medium containing 10 ng/ml TNF-α (positive control), medium containing 1 μg/ml

    Techniques Used: Activity Assay, Isolation, Incubation, Positive Control

    Effects of endotoxin tolerance on neutrophil mitochondrial transmembrane potential. Mitochondrial transmembrane potential was analyzed in neutrophils incubated for 3 h in supernatant from nontreated THP-1 cells (a), recombinant human TNF-α (10
    Figure Legend Snippet: Effects of endotoxin tolerance on neutrophil mitochondrial transmembrane potential. Mitochondrial transmembrane potential was analyzed in neutrophils incubated for 3 h in supernatant from nontreated THP-1 cells (a), recombinant human TNF-α (10

    Techniques Used: Incubation, Recombinant

    29) Product Images from "Cervical squamous carcinoma cells are resistant to the combined action of tumor necrosis factor-? and histamine whereas normal keratinocytes undergo cytolysis"

    Article Title: Cervical squamous carcinoma cells are resistant to the combined action of tumor necrosis factor-? and histamine whereas normal keratinocytes undergo cytolysis

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-8-46

    The cytotoxic effect of histamine and/or TNF-α on keratinocytes (a), SiHa cells (b) and ME-180 cells (c). The cells were cultivated in incomplete or complete medium and treated with indicated concentrations of histamine and/or TNF-α. The results are expressed as the percentage of the absorbance in the control wells. Each bar represents the mean of four individual cultures ± SE.
    Figure Legend Snippet: The cytotoxic effect of histamine and/or TNF-α on keratinocytes (a), SiHa cells (b) and ME-180 cells (c). The cells were cultivated in incomplete or complete medium and treated with indicated concentrations of histamine and/or TNF-α. The results are expressed as the percentage of the absorbance in the control wells. Each bar represents the mean of four individual cultures ± SE. "*" denotes p

    Techniques Used:

    The effect of histamine and/or TNF-α on SiHa cell migration in the transwell assay. TNF-α significantly increased the cell migration but addition of histamine could not produce any additional effect over that by TNF-α alone. The results are expressed as the percentage of migrated cells compared to those in control wells. (n = 3; mean ± SD). The control is indicated as 100%.
    Figure Legend Snippet: The effect of histamine and/or TNF-α on SiHa cell migration in the transwell assay. TNF-α significantly increased the cell migration but addition of histamine could not produce any additional effect over that by TNF-α alone. The results are expressed as the percentage of migrated cells compared to those in control wells. (n = 3; mean ± SD). The control is indicated as 100%. "*" denotes p

    Techniques Used: Migration, Transwell Assay

    TNF-α positive cells in the cervix carcinoma – immunohistochemical staining of SCC specimens. Note: strong immunopositivity of the epithelial cells of control sample (a); TNF-α positive tumor cells and peritumoral stroma in cervix carcinoma (b).
    Figure Legend Snippet: TNF-α positive cells in the cervix carcinoma – immunohistochemical staining of SCC specimens. Note: strong immunopositivity of the epithelial cells of control sample (a); TNF-α positive tumor cells and peritumoral stroma in cervix carcinoma (b).

    Techniques Used: Immunohistochemistry, Staining

    The effect of histamine and/or TNF-α on the growth of keratinocyte epithelium in a representative 3-day culture by using 10% fetal calf serum and DMEM as the culture medium. The following agents were added to the wells: well 1, diluent control; well 2, 0.1 mM histamine; well 3, 10 ng mL -1 TNF-α; well 4, 50 ng mL -1 TNF-α; well 5, 0.1 mM histamine + 10 ng mL -1 TNF-α; well 6, 0.1 mM histamine + 50 ng mL -1 TNF-α. Histamine (0.1 mM) or TNF-α (10 or 50 ng/ml) alone inhibits the growth of epithelium and the growth-inhibitory effect is increased when both agents are added to the same well.
    Figure Legend Snippet: The effect of histamine and/or TNF-α on the growth of keratinocyte epithelium in a representative 3-day culture by using 10% fetal calf serum and DMEM as the culture medium. The following agents were added to the wells: well 1, diluent control; well 2, 0.1 mM histamine; well 3, 10 ng mL -1 TNF-α; well 4, 50 ng mL -1 TNF-α; well 5, 0.1 mM histamine + 10 ng mL -1 TNF-α; well 6, 0.1 mM histamine + 50 ng mL -1 TNF-α. Histamine (0.1 mM) or TNF-α (10 or 50 ng/ml) alone inhibits the growth of epithelium and the growth-inhibitory effect is increased when both agents are added to the same well.

    Techniques Used:

    The effect of TNF-α on the DNA content of SiHa cells. The cells were grown in incomplete and complete MEM and treated with indicated concentrations of TNF-α. The data are expressed as the percentage of the fluorescence in the control wells. Each bar represents the mean of individual quadruplicate cultures ± SE (n = 4).
    Figure Legend Snippet: The effect of TNF-α on the DNA content of SiHa cells. The cells were grown in incomplete and complete MEM and treated with indicated concentrations of TNF-α. The data are expressed as the percentage of the fluorescence in the control wells. Each bar represents the mean of individual quadruplicate cultures ± SE (n = 4). "*" denotes p

    Techniques Used: Fluorescence

    The effect of histamine (1 mM) and/or TNF-α (10 and 50 ng/ml) on the DNA content of normal keratinocytes. The cells were cultivated in incomplete or complete Keratinocyte-SFM medium and treated with indicated concentrations of histamine and/or TNF-α. The results are expressed as the percentage of the fluorescence in the control wells. Each bar represents the mean of four individual quadruplicate cultures ± SE.
    Figure Legend Snippet: The effect of histamine (1 mM) and/or TNF-α (10 and 50 ng/ml) on the DNA content of normal keratinocytes. The cells were cultivated in incomplete or complete Keratinocyte-SFM medium and treated with indicated concentrations of histamine and/or TNF-α. The results are expressed as the percentage of the fluorescence in the control wells. Each bar represents the mean of four individual quadruplicate cultures ± SE. "*" denotes p

    Techniques Used: Fluorescence

    The effect of histamine and/or TNF-α on the DNA content of SiHa and ME-180 cells. SiHa cells were cultured with 0.1 mM histamine and/or TNF-α (a) or with 1 mM histamine and/or TNF-α (b) using incomplete or complete MEM. ME-180 cells were cultured with 1 mM histamine and/or TNF-α (c) using incomplete or complete McCoy's 5a medium. The results are expressed as the percentage of the fluorescence in the control wells. Each bar represents the mean of four individual quadruplicate cultures ± SE.
    Figure Legend Snippet: The effect of histamine and/or TNF-α on the DNA content of SiHa and ME-180 cells. SiHa cells were cultured with 0.1 mM histamine and/or TNF-α (a) or with 1 mM histamine and/or TNF-α (b) using incomplete or complete MEM. ME-180 cells were cultured with 1 mM histamine and/or TNF-α (c) using incomplete or complete McCoy's 5a medium. The results are expressed as the percentage of the fluorescence in the control wells. Each bar represents the mean of four individual quadruplicate cultures ± SE. "*" denotes p

    Techniques Used: Cell Culture, Fluorescence

    The effect of histamine and/or TNF-α on SiHa cells with and without inhibition of protein synthesis. The cells were pretreated with emetine and then cultivated and treated with indicated concentrations of histamine and/or TNF-α. The results are expressed as the percentage of the absorbance in the control wells. Each bar represents the mean of three individual cultures ± SD.
    Figure Legend Snippet: The effect of histamine and/or TNF-α on SiHa cells with and without inhibition of protein synthesis. The cells were pretreated with emetine and then cultivated and treated with indicated concentrations of histamine and/or TNF-α. The results are expressed as the percentage of the absorbance in the control wells. Each bar represents the mean of three individual cultures ± SD. "*" denotes p

    Techniques Used: Inhibition

    30) Product Images from "Membrane Type 1–Matrix Metalloproteinase/Akt Signaling Axis Modulates TNF-α-Induced Procoagulant Activity and Apoptosis in Endothelial Cells"

    Article Title: Membrane Type 1–Matrix Metalloproteinase/Akt Signaling Axis Modulates TNF-α-Induced Procoagulant Activity and Apoptosis in Endothelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105697

    Suppressive effects of MT1-MMP siRNA on TNF-α-induced endothelial apoptosis mediated via the phosphorylation of FoxO1. ( A–D ) Effect of knockdown of MT1-MMP by siRNA on TNF-α-induced endothelial cell apoptosis. ECs were incubated with or without TNF-α for 24 hours. Cell apoptosis was determined by TUNNEL staining. Photomicrographs are from an experiment representative of 4 independent experiments. Scale bars indicate 50 µm (lens x10). ( E ) Quantitative image analysis of cell apoptosis was performed by ImageJ. Data are expressed as means ± SD (n = 3, each group). ** P
    Figure Legend Snippet: Suppressive effects of MT1-MMP siRNA on TNF-α-induced endothelial apoptosis mediated via the phosphorylation of FoxO1. ( A–D ) Effect of knockdown of MT1-MMP by siRNA on TNF-α-induced endothelial cell apoptosis. ECs were incubated with or without TNF-α for 24 hours. Cell apoptosis was determined by TUNNEL staining. Photomicrographs are from an experiment representative of 4 independent experiments. Scale bars indicate 50 µm (lens x10). ( E ) Quantitative image analysis of cell apoptosis was performed by ImageJ. Data are expressed as means ± SD (n = 3, each group). ** P

    Techniques Used: Incubation, Staining

    Enhancement of molecular interaction of MT1-MMP and Akt in response to TNF-α. ( A and B ) Formation of a complex of MT1-MMP and phosphorylated Akt as determined by immunoprecipitation. The bands of 60 kDa phospho- and total- Akt were detected in the MT1-MMP-associated immunoprecipitates. Immunoblotting shows that phospho- and total- Akt in membrane and cytosol fractions of ECs was detected in the MT1-MMP-immunoprecipitates in TNF-α stimulation, whereas little or no expression was detected by normal mouse IgG as a negative control antibody. ( C ) Markers for membrane fraction, cytosol and total lysates were determined by Western blotting. Pan-Cadherin and LDH indicate protein markers of membrane fraction, cytosol fraction and total lysates and β-actin, respectively. ( D–I ) Association of MT1-MMP and Akt according to fluorescent immunohistochemistry. ECs treated with or without TNF-α were reacted with the antibodies to phospho-Akt and MT1-MMP, and the stained cells were analyzed by fluorescence microscope. Merged image indicates that MT1-MMP is colocalized with Akt in TNF-α-stimulated ECs. Photomicrographs are from an experiment representative of 4 independent experiments. Scale bars indicate 50 µm (lens x20).
    Figure Legend Snippet: Enhancement of molecular interaction of MT1-MMP and Akt in response to TNF-α. ( A and B ) Formation of a complex of MT1-MMP and phosphorylated Akt as determined by immunoprecipitation. The bands of 60 kDa phospho- and total- Akt were detected in the MT1-MMP-associated immunoprecipitates. Immunoblotting shows that phospho- and total- Akt in membrane and cytosol fractions of ECs was detected in the MT1-MMP-immunoprecipitates in TNF-α stimulation, whereas little or no expression was detected by normal mouse IgG as a negative control antibody. ( C ) Markers for membrane fraction, cytosol and total lysates were determined by Western blotting. Pan-Cadherin and LDH indicate protein markers of membrane fraction, cytosol fraction and total lysates and β-actin, respectively. ( D–I ) Association of MT1-MMP and Akt according to fluorescent immunohistochemistry. ECs treated with or without TNF-α were reacted with the antibodies to phospho-Akt and MT1-MMP, and the stained cells were analyzed by fluorescence microscope. Merged image indicates that MT1-MMP is colocalized with Akt in TNF-α-stimulated ECs. Photomicrographs are from an experiment representative of 4 independent experiments. Scale bars indicate 50 µm (lens x20).

    Techniques Used: Immunoprecipitation, Expressing, Negative Control, Western Blot, Immunohistochemistry, Staining, Fluorescence, Microscopy

    Schematic diagram describing the mechanisms of MT1-MMP/Akt signaling axis in TNF-α-dependent procoagulant activity and apoptosis of ECs. A crucial role of MT1-MMP in Akt-dependednt signaling pathways in TNF-α stimulation. MT1-MMP in the cytoplasm of ECs forms a complex with Akt in the intracellular signaling pathways in TNF-α-stimulated ECs. The interaction between MT1-MMP and Akt regulates TNF-α-induced changes in TF and TM expression in ECs and contributes to endothelial apoptosis through FoxO1 phosphorylation as well as caspase-3 activation.
    Figure Legend Snippet: Schematic diagram describing the mechanisms of MT1-MMP/Akt signaling axis in TNF-α-dependent procoagulant activity and apoptosis of ECs. A crucial role of MT1-MMP in Akt-dependednt signaling pathways in TNF-α stimulation. MT1-MMP in the cytoplasm of ECs forms a complex with Akt in the intracellular signaling pathways in TNF-α-stimulated ECs. The interaction between MT1-MMP and Akt regulates TNF-α-induced changes in TF and TM expression in ECs and contributes to endothelial apoptosis through FoxO1 phosphorylation as well as caspase-3 activation.

    Techniques Used: Activity Assay, Expressing, Activation Assay

    Effects of siRNA-mediated knockdown of MT1-MMP on the changes in TF and TM expression and MT1-MMP activity in response to TNF-α. (A and B) Effects of knockdown of MT1-MMP by siRNA on the MT1-MMP protein and mRNA levels. ECs were transfected with siRNA to MT1-MMP. siRNA-mediated knockdown of MT1-MMP significantly reduced MT1-MMP protein levels and mRNA as compared to the scrambled negative control. Bars are the means±SD of quantitative densitometric analyses from 4 separate experiments. * P
    Figure Legend Snippet: Effects of siRNA-mediated knockdown of MT1-MMP on the changes in TF and TM expression and MT1-MMP activity in response to TNF-α. (A and B) Effects of knockdown of MT1-MMP by siRNA on the MT1-MMP protein and mRNA levels. ECs were transfected with siRNA to MT1-MMP. siRNA-mediated knockdown of MT1-MMP significantly reduced MT1-MMP protein levels and mRNA as compared to the scrambled negative control. Bars are the means±SD of quantitative densitometric analyses from 4 separate experiments. * P

    Techniques Used: Expressing, Activity Assay, Transfection, Negative Control

    Suppressive effects of TIMP-2 and MT1-MMP siRNA on Akt and NF-κB phosphorylation in response to TNF-α. ( A ) TNF-α-induced Akt phosphorylation in a time dependent manner. ECs were treated with 10 ng/mL TNF-α for 120 minutes, followed by Western blotting. Akt phosphorylation was reduced by approximately 36% within 60 minutes in ECs stimulated by TNF-α. Bars are the means±SD of quantitative densitometric analyses from 4 separate experiments. * P
    Figure Legend Snippet: Suppressive effects of TIMP-2 and MT1-MMP siRNA on Akt and NF-κB phosphorylation in response to TNF-α. ( A ) TNF-α-induced Akt phosphorylation in a time dependent manner. ECs were treated with 10 ng/mL TNF-α for 120 minutes, followed by Western blotting. Akt phosphorylation was reduced by approximately 36% within 60 minutes in ECs stimulated by TNF-α. Bars are the means±SD of quantitative densitometric analyses from 4 separate experiments. * P

    Techniques Used: Western Blot

    Effects of Akt inhibition on the expression and activation of TF and TM and MT1-MMP activity in response to TNF-α. (A and B) Effects of knockdown of Akt by siRNA on the Akt protein and mRNA levels. ECs were transfected with siRNA to Akt. The siRNA-mediated knockdown of Akt reduced Akt protein and mRNA as compared to the scrambled negative control. Bars are the means±SD of quantitative densitometric analyses from 4 separate experiments. * P
    Figure Legend Snippet: Effects of Akt inhibition on the expression and activation of TF and TM and MT1-MMP activity in response to TNF-α. (A and B) Effects of knockdown of Akt by siRNA on the Akt protein and mRNA levels. ECs were transfected with siRNA to Akt. The siRNA-mediated knockdown of Akt reduced Akt protein and mRNA as compared to the scrambled negative control. Bars are the means±SD of quantitative densitometric analyses from 4 separate experiments. * P

    Techniques Used: Inhibition, Expressing, Activation Assay, Activity Assay, Transfection, Negative Control

    Suppressive effects of TIMP-2 on the changes in TF and TM expression and MT1-MMP activity in response to TNF-α. (A and B) TNF-α-induced TF and TM expression in a dose dependent manner. ECs were treated with TNF-α (1 to 50 ng/mL) for 18 hours. Immunoblots are from an experiment representative of 3 similar experiments. (C) Effect of TNF-α on MT1-MMP activity. ECs were pretreated with 40 nmol/L TIMP-2 for 60 min and then stimulated by TNF-α (10 ng/mL), and thereafter MT1-MMP activity was determined as described in the Methods section. Results are expressed as means±SD of 4 separate experiments. * P
    Figure Legend Snippet: Suppressive effects of TIMP-2 on the changes in TF and TM expression and MT1-MMP activity in response to TNF-α. (A and B) TNF-α-induced TF and TM expression in a dose dependent manner. ECs were treated with TNF-α (1 to 50 ng/mL) for 18 hours. Immunoblots are from an experiment representative of 3 similar experiments. (C) Effect of TNF-α on MT1-MMP activity. ECs were pretreated with 40 nmol/L TIMP-2 for 60 min and then stimulated by TNF-α (10 ng/mL), and thereafter MT1-MMP activity was determined as described in the Methods section. Results are expressed as means±SD of 4 separate experiments. * P

    Techniques Used: Expressing, Activity Assay, Western Blot

    31) Product Images from "Differential Responses of Bovine Macrophages to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium"

    Article Title: Differential Responses of Bovine Macrophages to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium

    Journal: Infection and Immunity

    doi: 10.1128/IAI.70.10.5556-5561.2002

    TNF-α bioactivity produced by bovine macrophages 6 h after addition of medium only (negative control), IFN-γ and LPS (positive control), M. avium subsp. paratuberculosis ( M. a. ptb ) organisms, or M. avium subsp. avium ( M. a. a ) organisms. Data a represent the mean ± standard deviation of two separate experiments on each of three cows. All tests were conducted in triplicate, and results were averaged. a, statistically different from positive control ( P
    Figure Legend Snippet: TNF-α bioactivity produced by bovine macrophages 6 h after addition of medium only (negative control), IFN-γ and LPS (positive control), M. avium subsp. paratuberculosis ( M. a. ptb ) organisms, or M. avium subsp. avium ( M. a. a ) organisms. Data a represent the mean ± standard deviation of two separate experiments on each of three cows. All tests were conducted in triplicate, and results were averaged. a, statistically different from positive control ( P

    Techniques Used: Produced, Negative Control, Positive Control, Standard Deviation

    32) Product Images from "Tks5-dependent formation of circumferential podosomes/invadopodia mediates cell-cell fusion"

    Article Title: Tks5-dependent formation of circumferential podosomes/invadopodia mediates cell-cell fusion

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201111116

    Circumferential podosomes/invadopodia mediate invasive and fusion activities. (A) B16F0 murine melanoma cells transfected with control (Ctr) or Tks5 siRNAs were cultured in the presence of 10 ng/ml RANKL alone or together with 5 ng/ml TGF-β and 5 ng/ml TNF-α for 48 h. (left) The cells were then stained with rhodamine-phalloidin to visualize F-actin, with antibodies to Tks5, and with DAPI to visualize nuclei. Arrowheads indicate circumferential invadopodia. Pixel intensities on the traversing dashed line were measured using LAS AF software and shown in a boxed area (arbitrary units). Bars, 25 µm. (right) The percentage of cells with circumferential invadopodia among > 100 cells scored was also determined. Data are means ± SD from three independent experiments. *, P
    Figure Legend Snippet: Circumferential podosomes/invadopodia mediate invasive and fusion activities. (A) B16F0 murine melanoma cells transfected with control (Ctr) or Tks5 siRNAs were cultured in the presence of 10 ng/ml RANKL alone or together with 5 ng/ml TGF-β and 5 ng/ml TNF-α for 48 h. (left) The cells were then stained with rhodamine-phalloidin to visualize F-actin, with antibodies to Tks5, and with DAPI to visualize nuclei. Arrowheads indicate circumferential invadopodia. Pixel intensities on the traversing dashed line were measured using LAS AF software and shown in a boxed area (arbitrary units). Bars, 25 µm. (right) The percentage of cells with circumferential invadopodia among > 100 cells scored was also determined. Data are means ± SD from three independent experiments. *, P

    Techniques Used: Transfection, Cell Culture, Staining, Software

    33) Product Images from "Increased signaling through p62 in the marrow microenvironment increases myeloma cell growth and osteoclast formation"

    Article Title: Increased signaling through p62 in the marrow microenvironment increases myeloma cell growth and osteoclast formation

    Journal: Blood

    doi: 10.1182/blood-2008-08-173948

    VCAM-1 expression, IL-6 production, and downstream signaling of NF-κB and p38 MAPK by stromal cells from MM patients and healthy subjects. (A) Stromal cells from healthy and MM patients were cultured with 10 ng/mL TNF-α for 4 days, the
    Figure Legend Snippet: VCAM-1 expression, IL-6 production, and downstream signaling of NF-κB and p38 MAPK by stromal cells from MM patients and healthy subjects. (A) Stromal cells from healthy and MM patients were cultured with 10 ng/mL TNF-α for 4 days, the

    Techniques Used: Expressing, Cell Culture

    OCL formation in cocultures of normal OCL precursors with p62 −/− stromal cells treated with TNF-α. (A) CFU-GM cells (10 4 cells/well) from spleens of p62 +/− mice were cultured with stromal cells from p62 +/− and p62
    Figure Legend Snippet: OCL formation in cocultures of normal OCL precursors with p62 −/− stromal cells treated with TNF-α. (A) CFU-GM cells (10 4 cells/well) from spleens of p62 +/− mice were cultured with stromal cells from p62 +/− and p62

    Techniques Used: Mouse Assay, Cell Culture

    34) Product Images from "miR-98 and let-7g* protect the blood–brain barrier under neuroinflammatory conditions"

    Article Title: miR-98 and let-7g* protect the blood–brain barrier under neuroinflammatory conditions

    Journal: Journal of Cerebral Blood Flow & Metabolism

    doi: 10.1038/jcbfm.2015.154

    MicroRNA (miRNA) delivered into cortical microvessels (MVs) diminishes leukocyte adhesion to and migration across the cortical MVs and improves blood–brain barrier (BBB) tightness in vivo . ( A ) Experimental design. miRNA quantification by qPCR was performed in quadruplicate from several mice. ( B ) miR-98 and let-7g* are downregulated in cortical MVs in mice treated with tumor necrosis factor- α (TNF α ). ( C ) Liposome/exogenous miRNA complex was twice injected (intravenously, i.v.) into mice with mimic oligos of miR-98 or let-7g*. Mice were killed after 0, 48, or 72 hours. Cortical MVs were isolated and miRNA expression was quantified by qPCR. Control mice were injected with liposome reagent only (vehicle). ( D ) Representative images from videos of leukocytes labeled with DiI. Mimic oligos of miR-98, let-7g*, or noncoding (NC) sequence that were delivered into MVs in vivo decreased leukocyte adhesion to endothelium. Six days after implanting the cranial window, mice were injected with TNF α (intracerebral, IC) and 1 hour later were injected with DiI and videos were taken. Quantitative calculation of adherent leukocytes ( E ) and of migrated leukocytes ( F ). ( G ) Quantification of Na-F accumulation in the brain in lipopolysaccharide (LPS)-associated encephalitis. Experiments performed in triplicate with five mice in each group. The results are shown as mean adhesion±s.e.m. * P
    Figure Legend Snippet: MicroRNA (miRNA) delivered into cortical microvessels (MVs) diminishes leukocyte adhesion to and migration across the cortical MVs and improves blood–brain barrier (BBB) tightness in vivo . ( A ) Experimental design. miRNA quantification by qPCR was performed in quadruplicate from several mice. ( B ) miR-98 and let-7g* are downregulated in cortical MVs in mice treated with tumor necrosis factor- α (TNF α ). ( C ) Liposome/exogenous miRNA complex was twice injected (intravenously, i.v.) into mice with mimic oligos of miR-98 or let-7g*. Mice were killed after 0, 48, or 72 hours. Cortical MVs were isolated and miRNA expression was quantified by qPCR. Control mice were injected with liposome reagent only (vehicle). ( D ) Representative images from videos of leukocytes labeled with DiI. Mimic oligos of miR-98, let-7g*, or noncoding (NC) sequence that were delivered into MVs in vivo decreased leukocyte adhesion to endothelium. Six days after implanting the cranial window, mice were injected with TNF α (intracerebral, IC) and 1 hour later were injected with DiI and videos were taken. Quantitative calculation of adherent leukocytes ( E ) and of migrated leukocytes ( F ). ( G ) Quantification of Na-F accumulation in the brain in lipopolysaccharide (LPS)-associated encephalitis. Experiments performed in triplicate with five mice in each group. The results are shown as mean adhesion±s.e.m. * P

    Techniques Used: Migration, In Vivo, Real-time Polymerase Chain Reaction, Mouse Assay, Injection, Isolation, Expressing, Labeling, Sequencing

    Glycogen synthase kinase 3 β (GSK3 β ) inhibition reversed tumor necrosis factor- α (TNF α )-mediated downregulation of microRNA (miRNA) in human primary rain microvascular endothelial cells (BMVEC). GSK3 β inhibitors were able to reverse downregulation of miR-98 ( A ) and let-7g* ( B ) in BMVEC. miRNA quantification by qPCR performed in triplicate from four brain microvascular endothelial cells (BMVEC) donors. Data are shown as fold change in GSK3 β /TNF α -treated BMVEC (±s.e.m.) versus TNF α -treated BMVEC only. * P > 0.05. I3'M, 5-iodo-indirubin-3'-monoxime; AR, N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea [AR-A014418] SB2, 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione [SB 216763].
    Figure Legend Snippet: Glycogen synthase kinase 3 β (GSK3 β ) inhibition reversed tumor necrosis factor- α (TNF α )-mediated downregulation of microRNA (miRNA) in human primary rain microvascular endothelial cells (BMVEC). GSK3 β inhibitors were able to reverse downregulation of miR-98 ( A ) and let-7g* ( B ) in BMVEC. miRNA quantification by qPCR performed in triplicate from four brain microvascular endothelial cells (BMVEC) donors. Data are shown as fold change in GSK3 β /TNF α -treated BMVEC (±s.e.m.) versus TNF α -treated BMVEC only. * P > 0.05. I3'M, 5-iodo-indirubin-3'-monoxime; AR, N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea [AR-A014418] SB2, 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione [SB 216763].

    Techniques Used: Inhibition, Real-time Polymerase Chain Reaction

    Schematic shows effects of miR-98 and let-7g* on endothelial function. BBB, blood brain barrier; GSK3 β , glycogen synthase kinase 3 β ; miRNAs, microRNAs; TNF α , tumor necrosis factor- α .
    Figure Legend Snippet: Schematic shows effects of miR-98 and let-7g* on endothelial function. BBB, blood brain barrier; GSK3 β , glycogen synthase kinase 3 β ; miRNAs, microRNAs; TNF α , tumor necrosis factor- α .

    Techniques Used:

    35) Product Images from "TNF-? Promotes an Odontoblastic Phenotype in Dental Pulp Cells"

    Article Title: TNF-? Promotes an Odontoblastic Phenotype in Dental Pulp Cells

    Journal:

    doi: 10.1177/0022034509334070

    Expression of mineralization-associated proteins and mineralized nodule formation after treatment with TNF-α. ( A ) Dental pulp and PDL cells were cultured for 6 and 24 hrs in the absence or presence of 10 ng/mL of TNF-α, and expression
    Figure Legend Snippet: Expression of mineralization-associated proteins and mineralized nodule formation after treatment with TNF-α. ( A ) Dental pulp and PDL cells were cultured for 6 and 24 hrs in the absence or presence of 10 ng/mL of TNF-α, and expression

    Techniques Used: Expressing, Cell Culture

    Time-course effects of TNF-α on MMP-1 (55 kDa) and MMP-13 (60 kDa) secretion by dental pulp and PDL cells. Western immunoblots are representative of conditioned medium samples from cells treated with 10 ng/mL of TNF-α for 3, 6, 12, 24,
    Figure Legend Snippet: Time-course effects of TNF-α on MMP-1 (55 kDa) and MMP-13 (60 kDa) secretion by dental pulp and PDL cells. Western immunoblots are representative of conditioned medium samples from cells treated with 10 ng/mL of TNF-α for 3, 6, 12, 24,

    Techniques Used: Western Blot

    p38 MAPK siRNA effects on TNF-α-mediated DPP/DSP and MMP-1 expression. Efficacy of p38 siRNA inhibition on total p38 protein expression was quantified after GAPDH normalization, and values are shown above the panels ( A ). The effects of p38 MAPK
    Figure Legend Snippet: p38 MAPK siRNA effects on TNF-α-mediated DPP/DSP and MMP-1 expression. Efficacy of p38 siRNA inhibition on total p38 protein expression was quantified after GAPDH normalization, and values are shown above the panels ( A ). The effects of p38 MAPK

    Techniques Used: Expressing, Inhibition

    Effects of NFκB and MAPK inhibitors on TNF-α-mediated MMP-1 and MMP-13 expression in dental pulp ( A, left) and PDL ( A, right) cells. Cells were pre-treated with BMS345541 (IKK phosphorylation inhibitor; 5 µM; 1.27 mg/mL), U0126
    Figure Legend Snippet: Effects of NFκB and MAPK inhibitors on TNF-α-mediated MMP-1 and MMP-13 expression in dental pulp ( A, left) and PDL ( A, right) cells. Cells were pre-treated with BMS345541 (IKK phosphorylation inhibitor; 5 µM; 1.27 mg/mL), U0126

    Techniques Used: Expressing

    36) Product Images from "HDAC6 inhibition prevents TNF-α-induced caspase 3 activation in lung endothelial cell and maintains cell-cell junctions"

    Article Title: HDAC6 inhibition prevents TNF-α-induced caspase 3 activation in lung endothelial cell and maintains cell-cell junctions

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10591

    HDAC6 knockdown and HDAC6 inhibition by CAY10603 block TNF-α-induced caspase-3 activation in endothelial cells ( A ) HPAECs were transfected with HDAC6 siRNA or control siRNA for 48 h, then challenged with TNF-α (20 ng/ml) for 24 h. Cells were divided into 4 groups: Control (Con), TNF-α alone (TNFα), siRNA alone (siRNA), and TNF-α+siRNA(TNFα+siRNA). ( B ) HPAECs were pre-treated with CAY10603 (CAY, 0.1 μM) for 6 h, then challenged with TNFα (20 ng/ml) for 18 h. Cells were divided into 4 groups: Control (Con), TNF-α alone (TNFα), CAY10603 alone (CAY), and TNF-α+CAY10603 (TNFα+CAY). Representative blots and densitometry analysis of cleaved caspase-3. * P
    Figure Legend Snippet: HDAC6 knockdown and HDAC6 inhibition by CAY10603 block TNF-α-induced caspase-3 activation in endothelial cells ( A ) HPAECs were transfected with HDAC6 siRNA or control siRNA for 48 h, then challenged with TNF-α (20 ng/ml) for 24 h. Cells were divided into 4 groups: Control (Con), TNF-α alone (TNFα), siRNA alone (siRNA), and TNF-α+siRNA(TNFα+siRNA). ( B ) HPAECs were pre-treated with CAY10603 (CAY, 0.1 μM) for 6 h, then challenged with TNFα (20 ng/ml) for 18 h. Cells were divided into 4 groups: Control (Con), TNF-α alone (TNFα), CAY10603 alone (CAY), and TNF-α+CAY10603 (TNFα+CAY). Representative blots and densitometry analysis of cleaved caspase-3. * P

    Techniques Used: Inhibition, Blocking Assay, Activation Assay, Transfection

    HDAC6 inhibition prevents TNF-α-induced ZO-1 disassembly in endothelial cells HPAECs and HLMVECs were pre-treated with Tubastatin A (TubA, 3 μM) for 6 h, then challenged with TNF-α (20 ng/ml) for 18 h. Cells were divided into 4 groups: Control (Con), TNF-α alone (TNFα), Tubastatin A alone (TubA), and TNF-α+Tubastatin A (TNFα+TubA). Immunofluorescence staining of ZO-1 in HPAECs ( A ) and HLMVECs ( B ). Images are representatives of three to six independent experiments.
    Figure Legend Snippet: HDAC6 inhibition prevents TNF-α-induced ZO-1 disassembly in endothelial cells HPAECs and HLMVECs were pre-treated with Tubastatin A (TubA, 3 μM) for 6 h, then challenged with TNF-α (20 ng/ml) for 18 h. Cells were divided into 4 groups: Control (Con), TNF-α alone (TNFα), Tubastatin A alone (TubA), and TNF-α+Tubastatin A (TNFα+TubA). Immunofluorescence staining of ZO-1 in HPAECs ( A ) and HLMVECs ( B ). Images are representatives of three to six independent experiments.

    Techniques Used: Inhibition, Immunofluorescence, Staining

    HDAC6 inhibition alleviates TNF-α-induced endothelial hyper-permeability and lung edema in endotoxemia ( A ) HPAECs were pre-treated with CAY10603 (CAY, 0.1 μM) for 6 h, then challenged with TNFα (20 ng/ml) for 18 h. Cells were divided into 4 groups: Control (Con), TNF-α alone (TNFα), CAY10603 alone (CAY), and TNF-α+CAY10603 (TNFα+CAY). TNF-α-induced endothelial permeability to FITC-Dextran was measured ( n = 4). * P
    Figure Legend Snippet: HDAC6 inhibition alleviates TNF-α-induced endothelial hyper-permeability and lung edema in endotoxemia ( A ) HPAECs were pre-treated with CAY10603 (CAY, 0.1 μM) for 6 h, then challenged with TNFα (20 ng/ml) for 18 h. Cells were divided into 4 groups: Control (Con), TNF-α alone (TNFα), CAY10603 alone (CAY), and TNF-α+CAY10603 (TNFα+CAY). TNF-α-induced endothelial permeability to FITC-Dextran was measured ( n = 4). * P

    Techniques Used: Inhibition, Permeability

    HDAC6 inhibition by Tubastatin A attenuates caspase-3 activation in endothelial cells HPAECs and HLMVECs were pre-treated with Tubastatin A (TubA, 3 μM) for 6 h, then challenged with TNF-α (20 ng/ml) for 18 h. Cells were divided into 4 groups: Control (Con), TNF-α alone (TNFα), Tubastatin A alone (TubA), and TNF-α+Tubastatin A (TNFα+TubA). Representative blots and densitometry analysis of cleaved-caspase-3 in HPAECs ( A ) and HLMVECs ( B ). * P
    Figure Legend Snippet: HDAC6 inhibition by Tubastatin A attenuates caspase-3 activation in endothelial cells HPAECs and HLMVECs were pre-treated with Tubastatin A (TubA, 3 μM) for 6 h, then challenged with TNF-α (20 ng/ml) for 18 h. Cells were divided into 4 groups: Control (Con), TNF-α alone (TNFα), Tubastatin A alone (TubA), and TNF-α+Tubastatin A (TNFα+TubA). Representative blots and densitometry analysis of cleaved-caspase-3 in HPAECs ( A ) and HLMVECs ( B ). * P

    Techniques Used: Inhibition, Activation Assay

    37) Product Images from "Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2"

    Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

    Journal: Nature Communications

    doi: 10.1038/ncomms8679

    Phosphorylation of pS-EphA2 is induced by RSK. ( a ) HeLa cells were stimulated with TNF-α for the indicated periods. Whole-cell lysates were immunoblotted with anti-pS-EphA2, EphA2, pRSK, RSK1, RSK2 and α-tubulin antibodies. ( b , c ) Whole-cell lysates from HeLa cells pre-treated with LY294002 (10 μM), SB203580 (10 μM), U0126 (5 μM) or BI-D1870 (10 μM) for 30 min and then stimulated with TNF-α for 20 min were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 and pS-EphA2 antibodies ( b ), or by normal SDS–PAGE and immunoblotted with anti-pS-EphA2, EphA2, pT-EGFR, pS-EGFR, EGFR, pRSK, RSK1, RSK2 and α-tubulin antibodies ( c ). ( d ) HeLa cells were pre-treated with LY294002 or BI-D1870 for 30 min and then stimulated with NaCl (0.3 M), TPA (100 ng ml −1 ) or EGF (10 ng ml −1 ) for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pAKT and α-tubulin. ( e ) T98G and U-87 MG cells starved in FCS-free medium for 24 h were treated with LY294002, MK-2206, U0126 and BI-D1870 for 30 min and then stimulated with 10% FCS for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pAKT, pERK and α-tubulin.
    Figure Legend Snippet: Phosphorylation of pS-EphA2 is induced by RSK. ( a ) HeLa cells were stimulated with TNF-α for the indicated periods. Whole-cell lysates were immunoblotted with anti-pS-EphA2, EphA2, pRSK, RSK1, RSK2 and α-tubulin antibodies. ( b , c ) Whole-cell lysates from HeLa cells pre-treated with LY294002 (10 μM), SB203580 (10 μM), U0126 (5 μM) or BI-D1870 (10 μM) for 30 min and then stimulated with TNF-α for 20 min were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 and pS-EphA2 antibodies ( b ), or by normal SDS–PAGE and immunoblotted with anti-pS-EphA2, EphA2, pT-EGFR, pS-EGFR, EGFR, pRSK, RSK1, RSK2 and α-tubulin antibodies ( c ). ( d ) HeLa cells were pre-treated with LY294002 or BI-D1870 for 30 min and then stimulated with NaCl (0.3 M), TPA (100 ng ml −1 ) or EGF (10 ng ml −1 ) for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pAKT and α-tubulin. ( e ) T98G and U-87 MG cells starved in FCS-free medium for 24 h were treated with LY294002, MK-2206, U0126 and BI-D1870 for 30 min and then stimulated with 10% FCS for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pAKT, pERK and α-tubulin.

    Techniques Used: SDS Page

    The phosphorylation of EphA2 at Ser-897 is induced by TAK1, but not by Akt. ( a , b ) HeLa ( a ) or T98G ( b left) cells were pre-treated with LY294002 (10 μM) or MK-2206 (10 μM) for 30 min and then stimulated with TNF-α for 20 min. T98G cells were starved using FCS-free medium for 24 h, treated with LY294002 for 30 min and then treated with 10% FCS for 10 min ( b , right). ( c ) MDA-MB-231 and Panc-1 cells were treated with LY294002 for 30 min. ( d ) HeLa cells stably transfected shRNA expression vectors against luciferase and TAK1 were stimulated with TNF-α for 20 min. ( e ) HeLa cells were transfected with siRNAs against TAK1 or negative control. At 72 h post transfection, cells were treated with TNF-α for 20 min. Whole-cell lysates were immunoblotted with anti-pS-EphA2, EphA2, pAKT, pRSK, RSK1, RSK2, TAK1, β-actin and α-tubulin antibodies.
    Figure Legend Snippet: The phosphorylation of EphA2 at Ser-897 is induced by TAK1, but not by Akt. ( a , b ) HeLa ( a ) or T98G ( b left) cells were pre-treated with LY294002 (10 μM) or MK-2206 (10 μM) for 30 min and then stimulated with TNF-α for 20 min. T98G cells were starved using FCS-free medium for 24 h, treated with LY294002 for 30 min and then treated with 10% FCS for 10 min ( b , right). ( c ) MDA-MB-231 and Panc-1 cells were treated with LY294002 for 30 min. ( d ) HeLa cells stably transfected shRNA expression vectors against luciferase and TAK1 were stimulated with TNF-α for 20 min. ( e ) HeLa cells were transfected with siRNAs against TAK1 or negative control. At 72 h post transfection, cells were treated with TNF-α for 20 min. Whole-cell lysates were immunoblotted with anti-pS-EphA2, EphA2, pAKT, pRSK, RSK1, RSK2, TAK1, β-actin and α-tubulin antibodies.

    Techniques Used: Multiple Displacement Amplification, Stable Transfection, Transfection, shRNA, Expressing, Luciferase, Negative Control

    The RSK–EphA2 axis controls cell motility. ( a ) Whole-cell lysates from HeLa cells treated with TNF-α for 20 min or untreated MDA-MB-231 cells were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 antibody. ( b – f ) MDA-MB-231 cells were pre-treated with BI-D1870 (10 μM) for 30 min and then scratched with a pipette tip. After 48 h of incubation, whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2 and β-actin ( b ) Migrated cells were counted manually under a microscope ( c ) Data are the means±s.d. of at least three fields. Similar results were obtained in at least three independent experiments. * P
    Figure Legend Snippet: The RSK–EphA2 axis controls cell motility. ( a ) Whole-cell lysates from HeLa cells treated with TNF-α for 20 min or untreated MDA-MB-231 cells were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 antibody. ( b – f ) MDA-MB-231 cells were pre-treated with BI-D1870 (10 μM) for 30 min and then scratched with a pipette tip. After 48 h of incubation, whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2 and β-actin ( b ) Migrated cells were counted manually under a microscope ( c ) Data are the means±s.d. of at least three fields. Similar results were obtained in at least three independent experiments. * P

    Techniques Used: Multiple Displacement Amplification, SDS Page, Transferring, Incubation, Microscopy

    EphA2 at Ser-897 is phosphorylated by RSK1/2. ( a , b ) HEK293 cells were transfected with expression vectors for EphA2, RSK1 and its substitution mutants. At 24 h post transfection, whole-cell lysates were immunoblotted with anti-pS-EphA2, pY-EphA2, EphA2, pRSK, RSK1 and α-tubulin antibodies. ( c ) HeLa cells were transfected with siRNAs against RSK1, RSK2 or negative control. At 72 h post transfection, cells were stimulated with TNF-α for 20 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2 and α-tubulin. ( d ) Recombinant human GST-EphA2 was incubated with recombinant human active GST-RSK1 or RSK2 in the absence or presence of BI-D1870 (0.1 μM) at 30 °C for 30 min. The reaction mixtures were analysed by immunoblotting with anti-pS-EphA2, EphA2, RSK1 and RSK2 antibodies.
    Figure Legend Snippet: EphA2 at Ser-897 is phosphorylated by RSK1/2. ( a , b ) HEK293 cells were transfected with expression vectors for EphA2, RSK1 and its substitution mutants. At 24 h post transfection, whole-cell lysates were immunoblotted with anti-pS-EphA2, pY-EphA2, EphA2, pRSK, RSK1 and α-tubulin antibodies. ( c ) HeLa cells were transfected with siRNAs against RSK1, RSK2 or negative control. At 72 h post transfection, cells were stimulated with TNF-α for 20 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2 and α-tubulin. ( d ) Recombinant human GST-EphA2 was incubated with recombinant human active GST-RSK1 or RSK2 in the absence or presence of BI-D1870 (0.1 μM) at 30 °C for 30 min. The reaction mixtures were analysed by immunoblotting with anti-pS-EphA2, EphA2, RSK1 and RSK2 antibodies.

    Techniques Used: Transfection, Expressing, Negative Control, Recombinant, Incubation

    Phosphorylation of EphA2 at Ser-897 is induced by TNF-α stimulation. ( a ) Whole-cell lysates from HeLa cells treated with TNF-α (20 ng ml −1 ) for 10, 20 and 60 min were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 and EGFR antibodies. ( b ) Whole-cell lysates from HeLa cells treated with TNF-α for 20 min were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2, pS-EphA2 and pY-EphA2. ( c ) Whole-cell lysates from HeLa cells treated with ephrin-A1 (100 ng ml −1 ) for 10 min or TNF-α for 20 min were separated by normal SDS–PAGE and immunoblotted with anti-pS-EphA2, pY-EphA2, EphA2 and α-tubulin antibodies. ( d ) HeLa cells were stimulated with TNF-α for the indicated periods. Whole-cell lysates were electrophoresed and probed with primary antibodies against pS-EphA2, pY-EphA2, EphA2, pT-EGFR, pS-EGFR, EGFR and α-tubulin. ( e ) HeLa cells were stimulated with TNF-α for 20 and 60 min. After fixation and permeabilization, cells were immunofluorescently stained with pS-EphA2, EphA2 or EGFR (clone LA1). Scale bar, 20 μm. Shown are representative images from three independent experiments.
    Figure Legend Snippet: Phosphorylation of EphA2 at Ser-897 is induced by TNF-α stimulation. ( a ) Whole-cell lysates from HeLa cells treated with TNF-α (20 ng ml −1 ) for 10, 20 and 60 min were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 and EGFR antibodies. ( b ) Whole-cell lysates from HeLa cells treated with TNF-α for 20 min were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2, pS-EphA2 and pY-EphA2. ( c ) Whole-cell lysates from HeLa cells treated with ephrin-A1 (100 ng ml −1 ) for 10 min or TNF-α for 20 min were separated by normal SDS–PAGE and immunoblotted with anti-pS-EphA2, pY-EphA2, EphA2 and α-tubulin antibodies. ( d ) HeLa cells were stimulated with TNF-α for the indicated periods. Whole-cell lysates were electrophoresed and probed with primary antibodies against pS-EphA2, pY-EphA2, EphA2, pT-EGFR, pS-EGFR, EGFR and α-tubulin. ( e ) HeLa cells were stimulated with TNF-α for 20 and 60 min. After fixation and permeabilization, cells were immunofluorescently stained with pS-EphA2, EphA2 or EGFR (clone LA1). Scale bar, 20 μm. Shown are representative images from three independent experiments.

    Techniques Used: SDS Page, Staining

    38) Product Images from "Changes in the Molecular and Functional Phenotype of Bovine Monocytes during Theileria parva Infection"

    Article Title: Changes in the Molecular and Functional Phenotype of Bovine Monocytes during Theileria parva Infection

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00703-19

    Differential response of monocytes from T. parva lethally and nonlethally infected cattle to T. parva infected cells and E. coli LPS. (A) Intracellular staining for the T. parva polymorphic immunodominant molecule in a representative schizont-infected cell line. (B) Intracellular staining for IFN-γ in a representative schizont-infected cell line. (C and D) Expression of mRNA for TNF-α (C) and IL-10 (D) in a representative schizont-infected cell line at 4 and 16 h after Histopaque centrifugation. (E) Levels of soluble IL-1β in CD172a + cell supernatant following stimulation of monocytes from uninfected ( n = 6), T. parva lethally infected ( n = 6), and T. parva nonlethally infected ( n = 4) cattle with T. parva schizont-infected cells (using a transwell format) or E. coli LPS (1 μg/ml). Amounts of soluble IL-1β were compared using ANOVA and Tukey’s post hoc test. (F) Expression of IL-10 mRNA in CD172a + cells from T. parva lethally infected ( n = 6) and nonlethally infected ( n = 4) cattle following stimulation with T. parva schizont-infected cells (using a transwell format) or E. coli LPS (1 μg/ml). IL-10 mRNA expression was compared using ANOVA and Tukey’s post hoc test. *, P
    Figure Legend Snippet: Differential response of monocytes from T. parva lethally and nonlethally infected cattle to T. parva infected cells and E. coli LPS. (A) Intracellular staining for the T. parva polymorphic immunodominant molecule in a representative schizont-infected cell line. (B) Intracellular staining for IFN-γ in a representative schizont-infected cell line. (C and D) Expression of mRNA for TNF-α (C) and IL-10 (D) in a representative schizont-infected cell line at 4 and 16 h after Histopaque centrifugation. (E) Levels of soluble IL-1β in CD172a + cell supernatant following stimulation of monocytes from uninfected ( n = 6), T. parva lethally infected ( n = 6), and T. parva nonlethally infected ( n = 4) cattle with T. parva schizont-infected cells (using a transwell format) or E. coli LPS (1 μg/ml). Amounts of soluble IL-1β were compared using ANOVA and Tukey’s post hoc test. (F) Expression of IL-10 mRNA in CD172a + cells from T. parva lethally infected ( n = 6) and nonlethally infected ( n = 4) cattle following stimulation with T. parva schizont-infected cells (using a transwell format) or E. coli LPS (1 μg/ml). IL-10 mRNA expression was compared using ANOVA and Tukey’s post hoc test. *, P

    Techniques Used: Infection, Staining, Expressing, Centrifugation

    T. parva upregulates the production of inflammatory mediators by monocytes during lethal infection. (A) Percentage of CD172a + cells in PBMC of an uninfected, representative steer. (B) Positive selection of CD172a + cells from PBMC of an uninfected, representative steer. (C) CD172a + cells include the classical (CD14 ++ CD16 − ), intermediate (CD14 ++ CD16 + ), and nonclassical (CD14 + CD16 + ) monocyte subsets. (D and E) Expression of mRNA for IL-1β (D) and TNF-α (E) in CD172a + cells from T. parva lethally infected ( n = 6) and nonlethally infected ( n = 4) cattle. Gene expression levels were compared using Student's t test. (F) Production of nitric oxide (NO) in CD172a + cells from T. parva lethally infected ( n = 6) and nonlethally infected ( n = 4) cattle following 96 h of exposure to exogenous recombinant bovine IFN-γ (50 U/ml; Ciba-Geigy) plus recombinant human TNF-α (2,500 U/ml; R D Systems). NO production was compared using ANOVA and Tukey’s post hoc test. (G) T. parva peripheral blood parasite load, determined by quantitative PCR, in T. parva lethally ( n = 6) and nonlethally ( n = 4) infected cattle. Parasite loads were compared using Student's t test. *, P
    Figure Legend Snippet: T. parva upregulates the production of inflammatory mediators by monocytes during lethal infection. (A) Percentage of CD172a + cells in PBMC of an uninfected, representative steer. (B) Positive selection of CD172a + cells from PBMC of an uninfected, representative steer. (C) CD172a + cells include the classical (CD14 ++ CD16 − ), intermediate (CD14 ++ CD16 + ), and nonclassical (CD14 + CD16 + ) monocyte subsets. (D and E) Expression of mRNA for IL-1β (D) and TNF-α (E) in CD172a + cells from T. parva lethally infected ( n = 6) and nonlethally infected ( n = 4) cattle. Gene expression levels were compared using Student's t test. (F) Production of nitric oxide (NO) in CD172a + cells from T. parva lethally infected ( n = 6) and nonlethally infected ( n = 4) cattle following 96 h of exposure to exogenous recombinant bovine IFN-γ (50 U/ml; Ciba-Geigy) plus recombinant human TNF-α (2,500 U/ml; R D Systems). NO production was compared using ANOVA and Tukey’s post hoc test. (G) T. parva peripheral blood parasite load, determined by quantitative PCR, in T. parva lethally ( n = 6) and nonlethally ( n = 4) infected cattle. Parasite loads were compared using Student's t test. *, P

    Techniques Used: Infection, Selection, Expressing, Recombinant, Real-time Polymerase Chain Reaction

    39) Product Images from "An NF-?B p65-cIAP2 Link is Necessary for Mediating Apoptotic Resistance to TNF-? in Gliomas"

    Article Title: An NF-?B p65-cIAP2 Link is Necessary for Mediating Apoptotic Resistance to TNF-? in Gliomas

    Journal: Journal of neuro-oncology

    doi: 10.1007/s11060-010-0346-y

    TNF-α Impairs Cell Growth and Induces Apoptosis in the Absence of NF-κB p65
    Figure Legend Snippet: TNF-α Impairs Cell Growth and Induces Apoptosis in the Absence of NF-κB p65

    Techniques Used:

    NF-κB p65 and cIAP2 are Key in Determining the Outcome of TNF-α Signaling in Glioma Cells
    Figure Legend Snippet: NF-κB p65 and cIAP2 are Key in Determining the Outcome of TNF-α Signaling in Glioma Cells

    Techniques Used:

    TNF-α Induced cIAP2 Expression is Dependent on NF-κB p65
    Figure Legend Snippet: TNF-α Induced cIAP2 Expression is Dependent on NF-κB p65

    Techniques Used: Expressing

    RIP1 is Hypo-Ubiquitinated in the Presence of TNF-α and the Absence of NF-κB p65
    Figure Legend Snippet: RIP1 is Hypo-Ubiquitinated in the Presence of TNF-α and the Absence of NF-κB p65

    Techniques Used:

    TNF-α Induces Apoptosis in the Absence of cIAP2
    Figure Legend Snippet: TNF-α Induces Apoptosis in the Absence of cIAP2

    Techniques Used:

    40) Product Images from "Oncolytic Adenoviruses Armed with Tumor Necrosis Factor Alpha and Interleukin-2 Enable Successful Adoptive Cell Therapy"

    Article Title: Oncolytic Adenoviruses Armed with Tumor Necrosis Factor Alpha and Interleukin-2 Enable Successful Adoptive Cell Therapy

    Journal: Molecular Therapy Oncolytics

    doi: 10.1016/j.omto.2016.12.004

    Biologically Active Cytokines Are Produced from Human and Hamster Cell Lines and Expressed Locally In Vivo (A) Human and hamster cell lines were incubated for 72 hr with 1,000 or 5,000 VPs per cell, respectively. Cytokine concentrations were measured from cell culture supernatants with a cytometric bead array. (B) Indicator cell line L929 was used in the cell-killing assay to confirm the biological activity of TNF-α, while mouse T cell line CTLL-2 was utilized in the IL-2-induced cell proliferation assay. The samples were derived from virus-infected HapT1 supernatants. Means ± SD are shown. (C) HapT1 tumors were injected with 1 × 10 8 VPs and collected together with blood after 48 hr. Cytokine concentrations were measured with a cytometric bead array and normalized against total protein concentration of the sample. Horizontal lines indicate mean values. OAd, Ad5/3-E2F-d24; OAd.TNFa, Ad5/3-E2F-d24-hTNFa; OAd.IL2, Ad5/3-E2F-d24-hIL2; OAd.TNFa-IL2, Ad5/3-E2F-d24-hTNFa-IRES-hIL2.
    Figure Legend Snippet: Biologically Active Cytokines Are Produced from Human and Hamster Cell Lines and Expressed Locally In Vivo (A) Human and hamster cell lines were incubated for 72 hr with 1,000 or 5,000 VPs per cell, respectively. Cytokine concentrations were measured from cell culture supernatants with a cytometric bead array. (B) Indicator cell line L929 was used in the cell-killing assay to confirm the biological activity of TNF-α, while mouse T cell line CTLL-2 was utilized in the IL-2-induced cell proliferation assay. The samples were derived from virus-infected HapT1 supernatants. Means ± SD are shown. (C) HapT1 tumors were injected with 1 × 10 8 VPs and collected together with blood after 48 hr. Cytokine concentrations were measured with a cytometric bead array and normalized against total protein concentration of the sample. Horizontal lines indicate mean values. OAd, Ad5/3-E2F-d24; OAd.TNFa, Ad5/3-E2F-d24-hTNFa; OAd.IL2, Ad5/3-E2F-d24-hIL2; OAd.TNFa-IL2, Ad5/3-E2F-d24-hTNFa-IRES-hIL2.

    Techniques Used: Produced, In Vivo, Incubation, Cell Culture, Activity Assay, Proliferation Assay, Derivative Assay, Infection, Injection, Protein Concentration

    Oncolytic Activity of Adenoviral Vectors in Human and Hamster Cancer Cell Lines (A) A schematic presentation of chimeric oncolytic adenovirus with E2F promoter; 24-base-pair deletion in E1A ; human TNF-α, IL-2, or TNF-α-IRES-IL2 inserted in the E3 region; and an Ad3 serotype knob in the Ad5 fiber. (B) Oncolytic activity of the viruses was shown in human lung adenocarcinoma (A549) and luciferase-expressing ovarian carcinoma (SKOV3-Luc) as well as in hamster pancreatic cancer (HapT1) and leiomyosarcoma (DDT1-MF2). The cells were incubated with the viruses for 3 days (A549 and DDT1-MF2), 5 days (SKOV3-Luc), or 6 days (HapT1) before determining cell viability. (C) Cell-killing efficacy was enhanced when combining viruses with HapT1-specific TILs. The cells were incubated 72 hr with 5,000 VPs and 24 hr with TILs. Means ± SEM are shown (n = 8). Statistical differences were evaluated with one-way ANOVA. ****p ≤ 0.0001. Ad.Luc1, replication-deficient Ad5/3-Luc1; OAd, Ad5/3-E2F-d24; OAd.TNFa, Ad5/3-E2F-d24-hTNFa; OAd.IL2, Ad5/3-E2F-d24-hIL2; OAd.TNFa-IL2, Ad5/3-E2F-d24-hTNFa-IRES-hIL2.
    Figure Legend Snippet: Oncolytic Activity of Adenoviral Vectors in Human and Hamster Cancer Cell Lines (A) A schematic presentation of chimeric oncolytic adenovirus with E2F promoter; 24-base-pair deletion in E1A ; human TNF-α, IL-2, or TNF-α-IRES-IL2 inserted in the E3 region; and an Ad3 serotype knob in the Ad5 fiber. (B) Oncolytic activity of the viruses was shown in human lung adenocarcinoma (A549) and luciferase-expressing ovarian carcinoma (SKOV3-Luc) as well as in hamster pancreatic cancer (HapT1) and leiomyosarcoma (DDT1-MF2). The cells were incubated with the viruses for 3 days (A549 and DDT1-MF2), 5 days (SKOV3-Luc), or 6 days (HapT1) before determining cell viability. (C) Cell-killing efficacy was enhanced when combining viruses with HapT1-specific TILs. The cells were incubated 72 hr with 5,000 VPs and 24 hr with TILs. Means ± SEM are shown (n = 8). Statistical differences were evaluated with one-way ANOVA. ****p ≤ 0.0001. Ad.Luc1, replication-deficient Ad5/3-Luc1; OAd, Ad5/3-E2F-d24; OAd.TNFa, Ad5/3-E2F-d24-hTNFa; OAd.IL2, Ad5/3-E2F-d24-hIL2; OAd.TNFa-IL2, Ad5/3-E2F-d24-hTNFa-IRES-hIL2.

    Techniques Used: Activity Assay, Luciferase, Expressing, Incubation

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    Article Snippet: .. Recombinant human tumor necrosis factor (TNF)-α was purchased from R & D Systems Europe Ltd. ..

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    Article Snippet: .. Recombinant human TNF-α was obtained from R & D Systems (Minneapolis, MN). .. Human umbilical vein endothelial cells (HUVEC), trypsin, and endothelial growth medium (EGM-2), containing SingleQuots supplements®, were purchased from Lonza (Walkersville, MD).

    Article Title: Transcriptional Repression of the Transforming Growth Factor β (TGF-β) Pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) by Nuclear Factor κB (NF-κB) p50 Enhances TGF-β Signaling in Hepatic Stellate Cells *
    Article Snippet: .. LPS (Sigma; Escherichia coli serotype 055:B5), recombinant human TNF-α (R & D Systems), and trichostatin A (TSA) (catalog no. T8552, Sigma) were used in this study. .. The antibodies used for the ChIP assay, Western blot analysis, and coimmunoprecipitation are p50 (catalog no. sc-8414x), HDAC1 (catalog no. sc-7872x), BAMBI (catalog no. sc-100681), and P-Smad2/3 (catalog no. sc-11769), all purchased from Santa Cruz Biotechnology, Inc. siRNA p50 (catalog no. sc-29407), siRNA HDAC1 (catalog no. sc-29343), siRNA p65, siRNA BAMBI (catalog no. sc-60243), and control siRNA (catalog no. sc-36869) were also purchased from Santa Cruz Biotechnology, Inc. Lipofectamine 2000 transfection regent was purchased from Invitrogen.

    Article Title: Largazole, a class I histone deacetylase inhibitor, enhances TNF-α to induce ICAM-1 and VCAM-1 expression in Rheumatoid Arthritis Synovial Fibroblasts
    Article Snippet: .. Recombinant human TNF-α, goat polyclonal antibodies against human ICAM-1 and VCAM-1 were purchased from R & D Systems (Minneapolis, MN). .. Rabbit polyclonal antibodies against phosphorylated ERK1/2, JNK/SAPK, and p38, and anti-rabbit and anti-mouse horseradish peroxide-linked secondary antibodies were purchased from Cell Signaling Technologies (Beverly, MA).

    Article Title: Kinetics and Role of Plasma Matrix Metalloproteinase-9 Expression in Acute Lung Injury and the Acute Respiratory Distress Syndrome
    Article Snippet: .. Human recombinant TNF-α was obtained from R & D Systems (Minneapolis, MN). .. Human recombinant GM-CSF was obtained from Genzyme (Cambridge, MA).

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    R&D Systems rhtnf α like activities
    <t>rhTNF-α-like</t> activity in supernatants of synovial explant cultures exposed to high-passage untreated or UV light-treated B. burgdorferi (experiments 1 and 2). In each test plate, the effects of culture supernatants on a TNF-sensitive cell line (WEHI 164.S13) were compared to a standard curve obtained with a serial dilution of rhTNF-α. Means and pooled SE from experiments 1 and 2 are shown.
    Rhtnf α Like Activities, supplied by R&D Systems, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rhtnf α like activities/product/R&D Systems
    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rhtnf α like activities - by Bioz Stars, 2020-11
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    99
    R&D Systems materials recombinant human tnf α
    <t>TNF-α</t> stimulates p300/ATF2/c-Jun/c-Fos complex formation. (A) Cells were transfected with scrambled or ATF2 siRNA, and then incubated with TNF-α for 24 h. The protein levels of ATF2 and cPLA 2 were determined. (B) Cells were incubated with TNF-α for the indicated time intervals. The levels of c-Fos, c-Jun, phospho-c-Jun, and phospho-ATF2 were determined. (C) Cells were pretreated with PD98059, SB202190, or SP600125, and then incubated with TNF-α for 90 min or 15 min. The levels of phospho-ATF2 and phospho-c-Jun were determined. (D) Cells were incubated with TNF-α for the indicated time intervals. The cell lysates were subjected to immunoprecipitation using an anti-p300 antibody, and then the immunoprecipitates were analyzed by Western blot using an anti-c-Fos, anti-c-Jun, anti-ATF2, or anti-p300 antibody. (E) Cells were treated with TNF-α for the indicated time intervals, and then ChIP assay was performed. Chromatin was immunoprecipitated using an anti-p300, anti-ATF2, anti-c-Fos, or anti-c-Jun antibody. One percent of the precipitated chromatin was assayed to verify equal loading (Input). Data are expressed as mean±S.E.M. of three independent experiments. # P
    Materials Recombinant Human Tnf α, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 374 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/materials recombinant human tnf α/product/R&D Systems
    Average 99 stars, based on 374 article reviews
    Price from $9.99 to $1999.99
    materials recombinant human tnf α - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

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    rhTNF-α-like activity in supernatants of synovial explant cultures exposed to high-passage untreated or UV light-treated B. burgdorferi (experiments 1 and 2). In each test plate, the effects of culture supernatants on a TNF-sensitive cell line (WEHI 164.S13) were compared to a standard curve obtained with a serial dilution of rhTNF-α. Means and pooled SE from experiments 1 and 2 are shown.

    Journal: Infection and Immunity

    Article Title: Borrelia burgdorferi Induces the Production and Release of Proinflammatory Cytokines in Canine Synovial Explant Cultures

    doi:

    Figure Lengend Snippet: rhTNF-α-like activity in supernatants of synovial explant cultures exposed to high-passage untreated or UV light-treated B. burgdorferi (experiments 1 and 2). In each test plate, the effects of culture supernatants on a TNF-sensitive cell line (WEHI 164.S13) were compared to a standard curve obtained with a serial dilution of rhTNF-α. Means and pooled SE from experiments 1 and 2 are shown.

    Article Snippet: To demonstrate the specificity of this assay for TNF-α, samples with confirmed rhTNF-α-like activities were incubated with the same volume of phosphate-buffered saline (PBS) containing a 10-μg/ml concentration of polyclonal antibodies raised against rhTNF-α (R & D Systems).

    Techniques: Activity Assay, Serial Dilution

    Effects of various numbers of B. burgdorferi . rhTNF-α-like, rhIL-1-like, and rcIL-8-like activities (chemotactic activities) in supernatants of synovial explant cultures challenged with 10 7 , 10 5 , and 10 3 untreated spirochetes for 24 h (experiment 6) are shown. Note the dose-independent up-regulation of TNF activity, the unresponsiveness of synovial tissues to low numbers of B. burgdorferi in the case of IL-1, and the dose-dependent induction of chemotactic activities.

    Journal: Infection and Immunity

    Article Title: Borrelia burgdorferi Induces the Production and Release of Proinflammatory Cytokines in Canine Synovial Explant Cultures

    doi:

    Figure Lengend Snippet: Effects of various numbers of B. burgdorferi . rhTNF-α-like, rhIL-1-like, and rcIL-8-like activities (chemotactic activities) in supernatants of synovial explant cultures challenged with 10 7 , 10 5 , and 10 3 untreated spirochetes for 24 h (experiment 6) are shown. Note the dose-independent up-regulation of TNF activity, the unresponsiveness of synovial tissues to low numbers of B. burgdorferi in the case of IL-1, and the dose-dependent induction of chemotactic activities.

    Article Snippet: To demonstrate the specificity of this assay for TNF-α, samples with confirmed rhTNF-α-like activities were incubated with the same volume of phosphate-buffered saline (PBS) containing a 10-μg/ml concentration of polyclonal antibodies raised against rhTNF-α (R & D Systems).

    Techniques: Activity Assay

    TNF-α stimulates p300/ATF2/c-Jun/c-Fos complex formation. (A) Cells were transfected with scrambled or ATF2 siRNA, and then incubated with TNF-α for 24 h. The protein levels of ATF2 and cPLA 2 were determined. (B) Cells were incubated with TNF-α for the indicated time intervals. The levels of c-Fos, c-Jun, phospho-c-Jun, and phospho-ATF2 were determined. (C) Cells were pretreated with PD98059, SB202190, or SP600125, and then incubated with TNF-α for 90 min or 15 min. The levels of phospho-ATF2 and phospho-c-Jun were determined. (D) Cells were incubated with TNF-α for the indicated time intervals. The cell lysates were subjected to immunoprecipitation using an anti-p300 antibody, and then the immunoprecipitates were analyzed by Western blot using an anti-c-Fos, anti-c-Jun, anti-ATF2, or anti-p300 antibody. (E) Cells were treated with TNF-α for the indicated time intervals, and then ChIP assay was performed. Chromatin was immunoprecipitated using an anti-p300, anti-ATF2, anti-c-Fos, or anti-c-Jun antibody. One percent of the precipitated chromatin was assayed to verify equal loading (Input). Data are expressed as mean±S.E.M. of three independent experiments. # P

    Journal: PLoS ONE

    Article Title: TNF-? Induces Cytosolic Phospholipase A2 Expression in Human Lung Epithelial Cells via JNK1/2- and p38 MAPK-Dependent AP-1 Activation

    doi: 10.1371/journal.pone.0072783

    Figure Lengend Snippet: TNF-α stimulates p300/ATF2/c-Jun/c-Fos complex formation. (A) Cells were transfected with scrambled or ATF2 siRNA, and then incubated with TNF-α for 24 h. The protein levels of ATF2 and cPLA 2 were determined. (B) Cells were incubated with TNF-α for the indicated time intervals. The levels of c-Fos, c-Jun, phospho-c-Jun, and phospho-ATF2 were determined. (C) Cells were pretreated with PD98059, SB202190, or SP600125, and then incubated with TNF-α for 90 min or 15 min. The levels of phospho-ATF2 and phospho-c-Jun were determined. (D) Cells were incubated with TNF-α for the indicated time intervals. The cell lysates were subjected to immunoprecipitation using an anti-p300 antibody, and then the immunoprecipitates were analyzed by Western blot using an anti-c-Fos, anti-c-Jun, anti-ATF2, or anti-p300 antibody. (E) Cells were treated with TNF-α for the indicated time intervals, and then ChIP assay was performed. Chromatin was immunoprecipitated using an anti-p300, anti-ATF2, anti-c-Fos, or anti-c-Jun antibody. One percent of the precipitated chromatin was assayed to verify equal loading (Input). Data are expressed as mean±S.E.M. of three independent experiments. # P

    Article Snippet: Materials Recombinant human TNF-α was from R & D System (Minneapolis, MN).

    Techniques: Transfection, Incubation, Immunoprecipitation, Western Blot, Chromatin Immunoprecipitation

    TNF-α induces leukocyte accumulation in BAL and cPLA 2 mRNA expression in mice via MAPKs and AP-1. (A) Mice were i.p. given one dose of PD98059, SB202190, SP600125, or Tanshinone IIA (2 mg/kg) for 1 h before TNF-α treatment, and sacrificed after 24 h. Lung tissues were homogenized to extract mRNA. The levels of cPLA 2 mRNA were determined by real-time PCR. (B, C) Mice were i.p. given one dose of PD98059, SB202190, SP600125, Tanshinone IIA, AACOCF 3 , AH 6809, SC-19220, or GW627368X (2 mg/kg) for 1 h before TNF-α treatment, and sacrificed after 24 h. BAL fluid was acquired and leukocyte count was determined by a hemocytometer. (D) Schematic representation of the signaling pathways involved in the TNF-α-induced cPLA 2 expression in HPAEpiCs. TNF-α-induced cPLA 2 expression and PGE 2 release are mediated through p38 MAPK- and JNK1/2-dependent p300/c-Fos/c-Jun/ATF2 complex formation in HPAEpiCs.

    Journal: PLoS ONE

    Article Title: TNF-? Induces Cytosolic Phospholipase A2 Expression in Human Lung Epithelial Cells via JNK1/2- and p38 MAPK-Dependent AP-1 Activation

    doi: 10.1371/journal.pone.0072783

    Figure Lengend Snippet: TNF-α induces leukocyte accumulation in BAL and cPLA 2 mRNA expression in mice via MAPKs and AP-1. (A) Mice were i.p. given one dose of PD98059, SB202190, SP600125, or Tanshinone IIA (2 mg/kg) for 1 h before TNF-α treatment, and sacrificed after 24 h. Lung tissues were homogenized to extract mRNA. The levels of cPLA 2 mRNA were determined by real-time PCR. (B, C) Mice were i.p. given one dose of PD98059, SB202190, SP600125, Tanshinone IIA, AACOCF 3 , AH 6809, SC-19220, or GW627368X (2 mg/kg) for 1 h before TNF-α treatment, and sacrificed after 24 h. BAL fluid was acquired and leukocyte count was determined by a hemocytometer. (D) Schematic representation of the signaling pathways involved in the TNF-α-induced cPLA 2 expression in HPAEpiCs. TNF-α-induced cPLA 2 expression and PGE 2 release are mediated through p38 MAPK- and JNK1/2-dependent p300/c-Fos/c-Jun/ATF2 complex formation in HPAEpiCs.

    Article Snippet: Materials Recombinant human TNF-α was from R & D System (Minneapolis, MN).

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    AP-1 is involved in TNF-α-induced cPLA 2 expression. (A) Cells were pretreated with Tanshinone IIA (TSIIA) for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA 2 were determined by Western blot. (B) Cells were pretreated with Tanshinone IIA (TSIIA), and then incubated with TNF-α for 6 h. cPLA 2 mRNA levels and promoter activity were determined. (C) Cells were transfected with scrambled, c-Jun, or c-Fos siRNA, and then incubated with TNF-α for 24 h. The protein levels of c-Jun, c-Fos, and cPLA 2 were determined. (D) Cells were transfected with pGL3-empty, wild-type cPLA 2 promoter, or AP-1-mutated cPLA 2 promoter, and then incubated with TNF-α for 6 h. The promoter activity of cPLA 2 was determined in the cell lysates. (E) Cells were pretreated with PD98059 (10 µM), SB202190 (10 µM), SP600125 (10 µM), or Tanshinone IIA (TSIIA; 10 µM) for 1 h, and then incubated with TNF-α for 24 h. The media were collected and analyzed for PGE 2 release. Data are expressed as mean±S.E.M. of three independent experiments. # P

    Journal: PLoS ONE

    Article Title: TNF-? Induces Cytosolic Phospholipase A2 Expression in Human Lung Epithelial Cells via JNK1/2- and p38 MAPK-Dependent AP-1 Activation

    doi: 10.1371/journal.pone.0072783

    Figure Lengend Snippet: AP-1 is involved in TNF-α-induced cPLA 2 expression. (A) Cells were pretreated with Tanshinone IIA (TSIIA) for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA 2 were determined by Western blot. (B) Cells were pretreated with Tanshinone IIA (TSIIA), and then incubated with TNF-α for 6 h. cPLA 2 mRNA levels and promoter activity were determined. (C) Cells were transfected with scrambled, c-Jun, or c-Fos siRNA, and then incubated with TNF-α for 24 h. The protein levels of c-Jun, c-Fos, and cPLA 2 were determined. (D) Cells were transfected with pGL3-empty, wild-type cPLA 2 promoter, or AP-1-mutated cPLA 2 promoter, and then incubated with TNF-α for 6 h. The promoter activity of cPLA 2 was determined in the cell lysates. (E) Cells were pretreated with PD98059 (10 µM), SB202190 (10 µM), SP600125 (10 µM), or Tanshinone IIA (TSIIA; 10 µM) for 1 h, and then incubated with TNF-α for 24 h. The media were collected and analyzed for PGE 2 release. Data are expressed as mean±S.E.M. of three independent experiments. # P

    Article Snippet: Materials Recombinant human TNF-α was from R & D System (Minneapolis, MN).

    Techniques: Expressing, Incubation, Western Blot, Activity Assay, Transfection

    p42/p44 MAPK is involved in TNF-α-induced cPLA 2 expression. (A) Cells were pretreated with PD98059 for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA 2 were determined by Western blot. (B) Cells were pretreated with PD98059 (10 µM) for 1 h, and then incubated with TNF-α for 6 h. cPLA 2 mRNA levels and promoter activity were determined. (C) Cells were transfected with scrambled or p42 siRNA, and then incubated with TNF-α for 24 h. The protein levels of p42 and cPLA 2 were determined. (D) Cells were pretreated with or without PD98059 (10 µM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-p42/p44 MAPK were determined. Data are expressed as mean±S.E.M. of three independent experiments. * P

    Journal: PLoS ONE

    Article Title: TNF-? Induces Cytosolic Phospholipase A2 Expression in Human Lung Epithelial Cells via JNK1/2- and p38 MAPK-Dependent AP-1 Activation

    doi: 10.1371/journal.pone.0072783

    Figure Lengend Snippet: p42/p44 MAPK is involved in TNF-α-induced cPLA 2 expression. (A) Cells were pretreated with PD98059 for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA 2 were determined by Western blot. (B) Cells were pretreated with PD98059 (10 µM) for 1 h, and then incubated with TNF-α for 6 h. cPLA 2 mRNA levels and promoter activity were determined. (C) Cells were transfected with scrambled or p42 siRNA, and then incubated with TNF-α for 24 h. The protein levels of p42 and cPLA 2 were determined. (D) Cells were pretreated with or without PD98059 (10 µM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-p42/p44 MAPK were determined. Data are expressed as mean±S.E.M. of three independent experiments. * P

    Article Snippet: Materials Recombinant human TNF-α was from R & D System (Minneapolis, MN).

    Techniques: Expressing, Incubation, Western Blot, Activity Assay, Transfection

    TNF-α induces cPLA 2 expression via TNFR1 in HPAEpiCs. (A) Cells were pretreated with Act. D or CHI for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA 2 were determined by Western blot. (B) Cells were pretreated with Act. D (1 µM) or CHI (1 µM) for 1 h, and then incubated with TNF-α for 6 h (for cPLA 2 mRNA levels) or 24 h (for PGE 2 release). cPLA 2 mRNA levels were determined by real-time PCR. The media were collected and analyzed for PGE 2 release. (C) Cells were transfected with scrambled or TNFR1 siRNA, and then incubated with TNF-α for 24 h. The protein expression of TNFR1 and cPLA 2 were determined. Data are expressed as mean±S.E.M. of three independent experiments. # P

    Journal: PLoS ONE

    Article Title: TNF-? Induces Cytosolic Phospholipase A2 Expression in Human Lung Epithelial Cells via JNK1/2- and p38 MAPK-Dependent AP-1 Activation

    doi: 10.1371/journal.pone.0072783

    Figure Lengend Snippet: TNF-α induces cPLA 2 expression via TNFR1 in HPAEpiCs. (A) Cells were pretreated with Act. D or CHI for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA 2 were determined by Western blot. (B) Cells were pretreated with Act. D (1 µM) or CHI (1 µM) for 1 h, and then incubated with TNF-α for 6 h (for cPLA 2 mRNA levels) or 24 h (for PGE 2 release). cPLA 2 mRNA levels were determined by real-time PCR. The media were collected and analyzed for PGE 2 release. (C) Cells were transfected with scrambled or TNFR1 siRNA, and then incubated with TNF-α for 24 h. The protein expression of TNFR1 and cPLA 2 were determined. Data are expressed as mean±S.E.M. of three independent experiments. # P

    Article Snippet: Materials Recombinant human TNF-α was from R & D System (Minneapolis, MN).

    Techniques: Expressing, Activated Clotting Time Assay, Incubation, Western Blot, Real-time Polymerase Chain Reaction, Transfection

    JNK1/2 is involved in TNF-α-induced cPLA 2 expression. (A) Cells were pretreated with SP600125 for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA 2 were determined by Western blot. (B) Cells were pretreated with SP600125 (10 µM) for 1 h, and then incubated with TNF-α for 6 h. cPLA 2 mRNA levels and promoter activity were determined. (C) Cells were transfected with scrambled or JNK2 siRNA, and then incubated with TNF-α for 24 h. The protein levels of JNK2 and cPLA 2 were determined. (D) Cells were pretreated with or without SP600125 (10 µM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-JNK1/2 were determined. Data are expressed as mean±S.E.M. of three independent experiments. # P

    Journal: PLoS ONE

    Article Title: TNF-? Induces Cytosolic Phospholipase A2 Expression in Human Lung Epithelial Cells via JNK1/2- and p38 MAPK-Dependent AP-1 Activation

    doi: 10.1371/journal.pone.0072783

    Figure Lengend Snippet: JNK1/2 is involved in TNF-α-induced cPLA 2 expression. (A) Cells were pretreated with SP600125 for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA 2 were determined by Western blot. (B) Cells were pretreated with SP600125 (10 µM) for 1 h, and then incubated with TNF-α for 6 h. cPLA 2 mRNA levels and promoter activity were determined. (C) Cells were transfected with scrambled or JNK2 siRNA, and then incubated with TNF-α for 24 h. The protein levels of JNK2 and cPLA 2 were determined. (D) Cells were pretreated with or without SP600125 (10 µM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-JNK1/2 were determined. Data are expressed as mean±S.E.M. of three independent experiments. # P

    Article Snippet: Materials Recombinant human TNF-α was from R & D System (Minneapolis, MN).

    Techniques: Expressing, Incubation, Western Blot, Activity Assay, Transfection

    p38 MAPK is involved in TNF-α-induced cPLA 2 expression. (A) Cells were pretreated with SB202190 for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA 2 were determined by Western blot. (B) Cells were pretreated with SB202190 (10 µM) for 1 h, and then incubated with TNF-α for 6 h. cPLA 2 mRNA levels and promoter activity were determined. (C) Cells were transfected with scrambled or p38 siRNA, and then incubated with TNF-α for 24 h. The protein levels of p38 and cPLA 2 were determined. (D) Cells were pretreated with or without SB202190 (10 µM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-p38 MAPK were determined. Data are expressed as mean±S.E.M. of three independent experiments. # P

    Journal: PLoS ONE

    Article Title: TNF-? Induces Cytosolic Phospholipase A2 Expression in Human Lung Epithelial Cells via JNK1/2- and p38 MAPK-Dependent AP-1 Activation

    doi: 10.1371/journal.pone.0072783

    Figure Lengend Snippet: p38 MAPK is involved in TNF-α-induced cPLA 2 expression. (A) Cells were pretreated with SB202190 for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA 2 were determined by Western blot. (B) Cells were pretreated with SB202190 (10 µM) for 1 h, and then incubated with TNF-α for 6 h. cPLA 2 mRNA levels and promoter activity were determined. (C) Cells were transfected with scrambled or p38 siRNA, and then incubated with TNF-α for 24 h. The protein levels of p38 and cPLA 2 were determined. (D) Cells were pretreated with or without SB202190 (10 µM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-p38 MAPK were determined. Data are expressed as mean±S.E.M. of three independent experiments. # P

    Article Snippet: Materials Recombinant human TNF-α was from R & D System (Minneapolis, MN).

    Techniques: Expressing, Incubation, Western Blot, Activity Assay, Transfection

    TNF-α induces cPLA 2 protein and mRNA expression. Cells were incubated with TNF-α for the indicated time intervals. (A) The protein levels of cPLA 2 were determined by Western blot, (B) the mRNA levels of cPLA 2 were determined by real-time PCR, and the promoter activity of cPLA 2 was determined in the cell lysates. (C) Cells were incubated with TNF-α (30 ng/ml) for the indicated time intervals. The media were collected and analyzed for PGE 2 release. Data are expressed as mean±S.E.M. of three independent experiments. * P

    Journal: PLoS ONE

    Article Title: TNF-? Induces Cytosolic Phospholipase A2 Expression in Human Lung Epithelial Cells via JNK1/2- and p38 MAPK-Dependent AP-1 Activation

    doi: 10.1371/journal.pone.0072783

    Figure Lengend Snippet: TNF-α induces cPLA 2 protein and mRNA expression. Cells were incubated with TNF-α for the indicated time intervals. (A) The protein levels of cPLA 2 were determined by Western blot, (B) the mRNA levels of cPLA 2 were determined by real-time PCR, and the promoter activity of cPLA 2 was determined in the cell lysates. (C) Cells were incubated with TNF-α (30 ng/ml) for the indicated time intervals. The media were collected and analyzed for PGE 2 release. Data are expressed as mean±S.E.M. of three independent experiments. * P

    Article Snippet: Materials Recombinant human TNF-α was from R & D System (Minneapolis, MN).

    Techniques: Expressing, Incubation, Western Blot, Real-time Polymerase Chain Reaction, Activity Assay

    LA-1 inhibits migration of neutrophils on endothelial monolayers. HUVEC monolayers were grown on delta T dishes coated with 40 μg/mL type I collagen and TNF-α activated. Neutrophils were either TNF-α-activated and given no treatment, 15 μM LA-1, or DMSO vehicle control. Cell interactions were allowed to adhere at 37 °C and neutrophil migration dynamics were imaged. × 20 bright-field images were captured every 30 s for 20 min. Neutrophil migration paths were tracked in ImageJ and analyzed using ibidi’s Chemotaxis and Migration Tool ( a ). LA-1 treatment reduced neutrophil path length ( b ), displacement ( c ), and speed ( d ) regardless of donor. Two-way ANOVA with post hoc Tukey analysis, * p

    Journal: Journal of Intensive Care

    Article Title: Leukadherin-1 ameliorates endothelial barrier damage mediated by neutrophils from critically ill patients

    doi: 10.1186/s40560-018-0289-5

    Figure Lengend Snippet: LA-1 inhibits migration of neutrophils on endothelial monolayers. HUVEC monolayers were grown on delta T dishes coated with 40 μg/mL type I collagen and TNF-α activated. Neutrophils were either TNF-α-activated and given no treatment, 15 μM LA-1, or DMSO vehicle control. Cell interactions were allowed to adhere at 37 °C and neutrophil migration dynamics were imaged. × 20 bright-field images were captured every 30 s for 20 min. Neutrophil migration paths were tracked in ImageJ and analyzed using ibidi’s Chemotaxis and Migration Tool ( a ). LA-1 treatment reduced neutrophil path length ( b ), displacement ( c ), and speed ( d ) regardless of donor. Two-way ANOVA with post hoc Tukey analysis, * p

    Article Snippet: Recombinant human TNF-α was obtained from R & D Systems (Minneapolis, MN).

    Techniques: Migration, Chemotaxis Assay

    Effects of melittin on activation of the (A) JAK2, STAT1 and STAT3 signalling pathways in TNF‐α/IFN‐γ‐stimulated HaCaT cells. (B) Immunofluorescence staining for p‐STAT1 and p‐STAT3 (labelled with Alexa Fluor 555, red), and F‐actin (labelled with Alexa Fluor 488, green). Cells were counterstained with Hoechst 33342 (blue). Effects of melittin on activation of the (C) NF‐κB signalling pathway in TNF‐α/IFN‐γ‐stimulated HaCaT cells. (D) NF‐κB DNA binding activity in the nuclear extract was measured by EMSA. Representative images from each group. Scale bar = 50 μm. NC, normal control; T+I, TNF‐α/IFN‐γ‐stimulated; Mel1, TNF‐α/IFN‐γ‐stimulated +1 μg·mL −1 Mel.

    Journal: British Journal of Pharmacology

    Article Title: Therapeutic effects of bee venom and its major component, melittin, on atopic dermatitis in vivo and in vitro) Therapeutic effects of bee venom and its major component, melittin, on atopic dermatitis in vivo and in vitro

    doi: 10.1111/bph.14487

    Figure Lengend Snippet: Effects of melittin on activation of the (A) JAK2, STAT1 and STAT3 signalling pathways in TNF‐α/IFN‐γ‐stimulated HaCaT cells. (B) Immunofluorescence staining for p‐STAT1 and p‐STAT3 (labelled with Alexa Fluor 555, red), and F‐actin (labelled with Alexa Fluor 488, green). Cells were counterstained with Hoechst 33342 (blue). Effects of melittin on activation of the (C) NF‐κB signalling pathway in TNF‐α/IFN‐γ‐stimulated HaCaT cells. (D) NF‐κB DNA binding activity in the nuclear extract was measured by EMSA. Representative images from each group. Scale bar = 50 μm. NC, normal control; T+I, TNF‐α/IFN‐γ‐stimulated; Mel1, TNF‐α/IFN‐γ‐stimulated +1 μg·mL −1 Mel.

    Article Snippet: Recombinant human TNF‐α and IFN‐γ were purchased from R & D Systems (Minneapolis, MN, USA).

    Techniques: Activation Assay, Immunofluorescence, Staining, Binding Assay, Activity Assay

    Cytotoxic effects of (A) melittin on HaCaT cells. Cell viability was determined by CCK‐8 assay ( n = 5). Effects of melittin on the chemokine expressions of cytokines (B) and chemokines ( n = 5) (C) in HaCaT cells that were stimulated by TNF‐α/IFN‐γ. T+I, TNF‐α/IFN‐γ‐stimulated; * P

    Journal: British Journal of Pharmacology

    Article Title: Therapeutic effects of bee venom and its major component, melittin, on atopic dermatitis in vivo and in vitro) Therapeutic effects of bee venom and its major component, melittin, on atopic dermatitis in vivo and in vitro

    doi: 10.1111/bph.14487

    Figure Lengend Snippet: Cytotoxic effects of (A) melittin on HaCaT cells. Cell viability was determined by CCK‐8 assay ( n = 5). Effects of melittin on the chemokine expressions of cytokines (B) and chemokines ( n = 5) (C) in HaCaT cells that were stimulated by TNF‐α/IFN‐γ. T+I, TNF‐α/IFN‐γ‐stimulated; * P

    Article Snippet: Recombinant human TNF‐α and IFN‐γ were purchased from R & D Systems (Minneapolis, MN, USA).

    Techniques: CCK-8 Assay