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  • 99
    Thermo Fisher tnf α
    Assay of IL-1β (panel A) and <t>TNF-α</t> (panel B) in brain tissues from healthy, AD, and HIV-1-positive subjects. Data represent means ± SE from 5 healthy individuals, 5 AD subjects and 5 HIV-1-positive subjects *p
    Tnf α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22718 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tnf α
    Melatonin effects on HMSC stemness following inflammatory treatment. (a) RT-PCR analysis of biomarkers of HMSC stemness with different concentrations of <t>TNF-</t> α . (b) Colony formation assays and quantitative analysis of HMSCs with different treatments. (c) RT-PCR analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P3 HMSCs with different treatments. (d) RT-PCR analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P7 HMSCs with different treatments. (e) RT-PCR analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P10 HMSCs with different treatments. (f) Western blot analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P3 HMSCs with different treatments. (g) Western blot analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P7 HMSCs with different treatments. (h) Western blot analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P10 HMSCs with different treatments. (i) Alizarin red staining and quantification of osteogenic differentiation of HMSCs with different treatments. P3: primary cells expanded to the third generation; P7: primary cells expanded to the seventh generation; P10: primary cells expanded to the tenth generation. ∗ P
    Tnf α, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8876 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    R&D Systems tnf α
    (a) Dose-dependent secretion of IP-10 and Mig by gastric epithelial cells. Confluent monolayers of NCI cell lines were stimulated with increasing concentrations of either <t>TNF-α</t> or IFN-γ or both in combination (• IFN-γ; ○ TNF-α; ▾ TNF-α + IFN-γ). Supernatants were harvested after 18 h and chemokine-secretion was measured by ELISA. Similar results were obtained using AGS and NCI cell lines. Values are expressed as mean ± CI. Representative results from one of four independent experiments are shown. (b) Time-dependent secretion of IP-10 and Mig by gastric epithelial cells. Confluent monolayers of AGS and Kato III cells were stimulated for different times (as indicated) with either IFN-γ 50 ng/ml or TNF-α 50 ng/ml alone or both cytokines in combination. Supernatants were harvested after 6, 12 and 18 h and chemokine concentrations were measured by ELISA. Values are expressed as mean ± CI. Representative results from one of four experiments are shown.
    Tnf α, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 26310 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson tnf α
    Pedigree and experimental analysis. ( A ) Pedigree depicting the presence of the c.1049delG, p.(Gly350Glufs*15) IRAK4 variant. The arrow denotes the index patient. ( B ) Sanger sequencing of the IRAK4 gene depicting the single-base-pair deletion in the affected family but not seen in the healthy control (HC). ( C ) Immunoblot showing lack of IRAK4 protein expression compared to a HC, as well as the predicted molecular weight (MW) and position of the truncated IRAK4 due to the variant ( n = 3). ( D ) Simplified schematic overview of the TLR7 signaling pathway resulting in NF-κB activation and <t>TNF-α</t> transcription emphasizing the central role of the IRAK4 protein in signal transduction. ( E ) Cytometric bead array (CBA) showing no TNF-α response to imiquimod (IMQ) stimulation of TLR7 in the IRAK4-deficient cells compared to a HC. PMA/ionomycin serves as a positive control confirming cell viability. Data are represented as the mean ( n = 3), and error bars represent the standard error of the mean (SEM). P -value calculated by the two-tailed Student's t -test with a Bonferroni correction.
    Tnf α, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 95/100, based on 14084 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam tnf α
    Detection of mRNA levels of <t>TNF-α</t> and IL-6 in placenta tissues of pregnant rats using fluorescence quantitative PCR compared with those in the control group. NS, not significant. **P
    Tnf α, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 3955 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    PeproTech tnf α
    CQ resets tumor-associated M2 macrophages to M1 phenotype. a , b BMDM-M2 cells were treated with or without 10 μM CQ for 24 h. The expression of IFN-γ, IL-12p40, and <t>TNF-α</t> was measured by flow cytometry a ( n = 3). Quantification of CD80, CD86, and MHC-II in BMDM-M2 cells with or without CQ treatment b ( n = 3). c NO production in BMDM-M2 cells lysate with or without CQ treatment were measured ( n = 3). d Quantification of the number of CD206 + CD301 + cells in BMDM-M2 cells with or without CQ treatment ( n = 3). e The mRNA expression of NOS2 and Arg1 in BMDM-M2 cells with or without CQ treatment was analyzed by qPCR (left); the expression of Arg1 was analyzed by western blotting (center); the expression of iNOS was analyzed by flow cytometry (right). f Arginase1 + cells in IL-12p40 - IFN-γ − M2 macrophages with or without CQ treatment were analyzed by flow cytometry ( n = 3). g Representative flow cytometric analysis and quantification of iNOS in F4/80 + ascites fluid macrophages, isolated from mice with H22 hepatocarcinoma that had received PBS or CQ treatment ( n = 5) (left); the protein expression of iNOS and Arg1 in the same F4/80 + ascites macrophages were also analyzed by western blotting (right) ( n = 3). h Western blot analysis of iNOS and Arg1 in tumor-infiltrating macrophages after PBS or CQ treatment in mice bearing subcutaneous B16 melanoma ( n = 3). i The mRNA expression of IFN-γ , IL-12p40 , IL-12p35 , TNF-α , and IL-10 was analyzed by real-time qPCR in B16 tumor tissues in mice with or without CQ treatment ( n = 3). j , k Splenocytes from OT-I and OT-II TCR transgenic mice were labeled with carboxyfluorescein succinimidyl ester (CFSE) and then stimulated with OVA257-264 ( j ) and OVA323-339 ( k ) respectively in the presence of conditioned medium from PBS or CQ treated BMDM-M2 cells. Representative histograms of CD8 + or CD4 + T-cell proliferation (left panel) and quantification of CD8 + or CD4 + T-cell proliferation (right panel) were analyzed by flow cytometry after 72 h ( n = 3). Data shown are representative of three independent experiments and error bars represent mean ± SEM. * P
    Tnf α, supplied by PeproTech, used in various techniques. Bioz Stars score: 98/100, based on 5243 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc tnf α
    IL-1 receptor (IL-1R) activation suppresses <t>TNF-α</t> expression and attenuates regulated necrosis. A : receptor-interacting protein (RIP)1, RIP3, and cytokine mRNA levels were quantitated in kidneys from IL-1R wild-type (WT) mice with selective deletion of polycystin-1 in the kidney and IL-1R knockout (KO) KPKD1 control mice. Compared with IL-1R WT KPKD1 kidneys, TNF-α protein was upregulated in IL-1R KO KPKD1 kidneys [typical Western blot bands ( B ) and protein abundance analysis ( C )]. n.s., not significant.
    Tnf α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1810 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    R&D Systems recombinant human tnf α
    The RPE secreted higher levels of GM-CSF after stimulation with HNE and <t>TNF-α.</t> 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
    Recombinant Human Tnf α, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 1509 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore tnfα
    Secretion of pro-inflammatory cytokines such as ( A ) <t>TNFα,</t> ( B ) IL-1β and ( C ) IL-12 in cell culture supernatant as markers of inflammation following nano-WC–Co exposure. Notes: * P
    Tnfα, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 3663 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore tumor necrosis factor tnf α
    Probiotic Lactobacillus spp. alleviated gastric inflammation in mice. ( A ) Mice were fed with Lactobacillus spp. (GMNL-74 and GMNL-185) for 24 days followed by intragastric gavage with H. pylori 26695 once every 2 days for a total of six administrations. Arrows show the days of H. pylori inoculation. ( B ) Mice were euthanized and gastric tissues were subjected to hematoxylin–eosin (H E) and immunohistochemical (IHC) staining with specific antibodies against cyclooxygenase-2 (COX-2) and tumor necrosis factor <t>(TNF)-α,</t> respectively (original magnification: 200×). The magnified images are shown in the lower panel of each cropped area. Severe infiltration of inflammatory cells in the gastric epithelium (H E) and pronounced expression of COX-2 and TNF-α in gastric tissues are indicated by red arrows (IHC).
    Tumor Necrosis Factor Tnf α, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 396 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson tumor necrosis factor tnf α
    Functional analysis of GD2/CAR-T cells in vitro. a Cytotoxic activity of GD2/CAR-T cells. We used an LDH release assay to evaluate the cytotoxic activity of GD2.BBζ CAR-T cells and non-transduced T cells. Target cells were melanoma lines with varying GD2 expression levels. The figure illustrates the mean and SD of LDH release from 9 T cell lines after 4 h of incubation. A significant difference was detected in GD2-specific antitumor activity at each E:T ratio between GD2.BBζ CAR-T cells and control non-transduced CAR-T cells. In contrast, GD2.BBζ CAR-T cells had little antitumor activity against the GD2-negative tumor cell lines 293T, A875 (3.1% GD2 expression), and MMYC-7 (10.2% GD2 expression). b Th1 cytokine release of GD2/CAR-T cells. Non-transduced T cells and GD2.BBζ CAR-T cells were co-cultured (ratio of T lymphocytes:tumor cells of 20:1) with four different cell lines that were GD2-negative (293T) or were 27.4% GD2-positive (GAK), were 47.3% GD2-positive (HMV-II) or exhibited high (WM-266-4) levels of GD2-positive cells. Culture supernatant was collected 24 h later, and the production of IL-2, <t>TNF-α,</t> and IFN-γ were measured using a CBA assay. A substantial amount of IL2, TNF-α, and IFN-γ was released by GD2.BBζ CAR-T cells, and their releases were associated with the level of GD2 expression in the melanoma cells. In contrast, the release of IL2, TNF-α, and IFN-γ remained unchanged in non-transduced T cells or 293 T cells. The results are presented as the mean and SD from experiments that were performed in triplicate
    Tumor Necrosis Factor Tnf α, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 680 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PeproTech recombinant human tnf α
    <t>TNF-α</t> and IFN-γ promote Dsg2 intracellular and extracellular cleavage. a Model of Dsg2 with mapped epitopes for AH12.2 (extracellular [N-term] specific) and 4B2 (intracellular [C-term] specific) monoclonal antibodies. EC extracellular cadherin domain, EA extracellular anchor, TM transmembrane domain, IA intracellular anchor, ICS intracellular cadherin-typical sequence, IPL intracellular proline-rich linker domain, RUD repeated unit domain, DTD desmoglein-specific terminal domain. b Top. Western blot using an antibody against Dsg2 C-term of cell lysates of T-84s treated with TNF-α and IFN-γ for 24 h. Bottom. Densitometry of blots. Density for all bands in each lane was collected. The ratio of the density of the ~55 kDa Dsg2 ICF band to the density of all bands for a given lane was then calculated. The data in the graph is the average of these ratios within each experimental group. * p = 0.0067; # p = 0.0003, n . c . d Top. Western blot using an antibody against Dsg2 C-term of cell lysates of T-84s treated with TNF-α and IFN-γ in the presence or absence of GI254023X for 24 h. Bottom. Densitometry of blots. Densitometry collected and analyzed as for Fig. 1b. n ≥ 4 per group. e Western blot using an antibody against Dsg2 N-term of cell culture supernatants of T-84s treated as in Fig. 1d. f Western blot using an antibody against Dsg2 C-term of cell lysates of T-84s treated with TNF-α and IFN-γ for 6, 12, or 24 h. g Western blot using an antibody against Dsg2 N-term of T-84 cell culture supernatants of cells treated as in Fig. 1f. All blots are representative of at least three independent experiments
    Recombinant Human Tnf α, supplied by PeproTech, used in various techniques. Bioz Stars score: 99/100, based on 663 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology tnf α
    Effects of gelatin hydrogel on pro-inflammatory cytokine. (A) Immunohistochemical staining of IL-1β around the lesion. (B) The area percentage of IL-1β. (C) Immunohistochemical staining of <t>TNF-α</t> around the lesion. (D) The area percentage of TNF-α. Scale bar = 50 μm. *Represents the hydrogel. Data are mean ± SD, n = 4 mice per group, *** P
    Tnf α, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 2573 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems human tnf α
    Effect of miR-4717 mimics or miR-4717 inhibitor on the production of tumor necrosis factor <t>(TNF)-α</t> and interferon (IFN)-γ by lymphocytes from chronic HBV patients with rs10204525 genotypes AA and GG ( A ) TNF-α. ( B ) IFN-γ. The values were log transformed to obtain normal distribution and then analyzed by using ANOVA and Post Hoc test.
    Human Tnf α, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 682 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    R&D Systems mouse tnf α
    Apoptosis, ROS, and <t>TNF-α</t> production are reduced in silica exposed BMMC cultured from SR-AI/II KO, MARCO KO, or SR-A/MARCO double KO mice. ( A ) DNA fragmentation was measured in BMMC obtained from scavenger receptor KO mice and exposed to 50 μg/cm
    Mouse Tnf α, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 593 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti tnf α
    NOX2 activation leads to a surge of proinflammatory mediators in NAFLD-Kidney A. mRNA expression analysis of IL-1β, IFN-γ, <t>TNF-α,</t> CD4, and CD8 genes in kidney tissue of NAFLD, NAFLD + BDCM, and P47phox KO mice fed with high-fat diet. All mRNA expression had been assessed by quantitative real-time PCR (qRTPCR) and expressions were normalized against NAFLD group (*P
    Anti Tnf α, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 828 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeproTech tnfα
    Induction of ‘bystander’ senescence in B16 tumour cells. (A) Senescence-associated β-galactosidase activity in B16 cells cultured for 4 days in the medium from DTX-treated cells (SM), IFNγ + <t>TNFα-treated</t> cells or proliferating cell medium (TM). (B) The size and granularity of control and senescent B16 cells was determined by forward and side scatter flow cytometry analysis. (C) Expression of p21 in B16 cells cultured for 4 days in different media (reverse transcription-quantitative polymerase chain reaction). (D) Immunoblotting detection of mouse p21 in B16 cells harvested on day 4 after cultivation in different media. GAPDH was used as a loading control. Data are presented as the mean ± standard deviation. ** P
    Tnfα, supplied by PeproTech, used in various techniques. Bioz Stars score: 99/100, based on 2292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mouse tnf α
    Effects of Banhasasim-tang (BHSST) on production of chemokines in HaCaT cells. Production of RANTES (a), TARC (b), MDC (c), and IL-8 (d) were measured using the culture supernatant. Cells were cotreated with BHSST (125, 250, or 500 μ g/mL), and <t>TNF-</t> α and IFN- γ (TI, each 10 ng/mL) for 24 h. Silymarin (6.25, 12.5, or 25 μ g/mL) was used as a positive control. Values are expressed as mean ± SEM of three independent experiments. ## P
    Mouse Tnf α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 482 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad tnf α
    Influence of <t>TNF-α</t> on the activity of glutamate transporters from wild-type or hSOD1 G93A cortical astrocytes. Glutamate transporter activity was evaluated by measuring velocity of d -[ 3 H]-aspartate uptake (50 nmol/L) in wild-type and hSOD1 G93A astrocytes treated or not with TNF-α (20 ng/mL) for 72 h. (A) shows total uptake while (B) and (C) illustrate GLT-1- and GLAST-dependent uptake, respectively evaluated in presence of the selective inhibitors WAY-213613 (100 µmol/L) and UCPH-101 (10 µmol/L). Shown are mean with SEM from five independent experiments realized in quintuplicate. # p
    Tnf α, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 3315 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tumor necrosis factor α tnf α
    TM5441 pre-treatment and siPAI-1 improved <t>TNF-α-induced</t> mitochondrial dysfunction in HepG2 cells HepG2 cells were pretreated with 20 μM TM5441 before the exposure of 50 ng/ml TNF-α for 24 hours. mRNA expressions of ( A ) PAI-1 and ( B) mitochondria biogenesis-related genes, such as PGC-1α, mtDNA, TFAM, NRF1, and NRF2 were measured by RT-qPCR. Then, PAI-1 is downregulated with siRNA in which confirmed by immunoblot analysis. ( C ) 20 nM siPAI-1 was transfected 24 hours before the exposure of 50 ng/ml TNF-α for 24 hour. The mRNA gene expression of ( D ) PAI-1 and ( E ) the mitochondrial function-related genes were measured by RT-qPCR. Data are presented as mean±SE of 4-5 experiments. * p
    Tumor Necrosis Factor α Tnf α, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 538 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Assay of IL-1β (panel A) and TNF-α (panel B) in brain tissues from healthy, AD, and HIV-1-positive subjects. Data represent means ± SE from 5 healthy individuals, 5 AD subjects and 5 HIV-1-positive subjects *p

    Journal: BBA Clinical

    Article Title: Analysis of glutathione levels in the brain tissue samples from HIV-1-positive individuals and subject with Alzheimer's disease and its implication in the pathophysiology of the disease process

    doi: 10.1016/j.bbacli.2016.05.006

    Figure Lengend Snippet: Assay of IL-1β (panel A) and TNF-α (panel B) in brain tissues from healthy, AD, and HIV-1-positive subjects. Data represent means ± SE from 5 healthy individuals, 5 AD subjects and 5 HIV-1-positive subjects *p

    Article Snippet: The cytokines that were measured in the brain tissue lysates isolated from the subjects IL-1β and TNF-α (eBioscience ELISA Ready-Set-Go: IL-1β cat # 88-7010, TNF-α cat # 88-7346).

    Techniques:

    Cytokine/chemokine levels in lung homogenates of aMPV/C- and sham-inoculated mice. Six mice from both aMPV/C- and sham-inoculated groups were sacrificed on days 1, 2, 3, 4, 5, 6, 7, 10, 14, and 21 post-inoculation, and 100 μl of lung homogenates was used to quantify IL-1β (A), IFN-γ (B), MCP-1 (C), MIP-1α (D), RANTES (E), and TNF-α (F) by enzyme-linked immunosorbent assay. Significant difference is expressed as p

    Journal: PLoS ONE

    Article Title: Viral Replication and Lung Lesions in BALB/c Mice Experimentally Inoculated with Avian Metapneumovirus Subgroup C Isolated from Chickens

    doi: 10.1371/journal.pone.0092136

    Figure Lengend Snippet: Cytokine/chemokine levels in lung homogenates of aMPV/C- and sham-inoculated mice. Six mice from both aMPV/C- and sham-inoculated groups were sacrificed on days 1, 2, 3, 4, 5, 6, 7, 10, 14, and 21 post-inoculation, and 100 μl of lung homogenates was used to quantify IL-1β (A), IFN-γ (B), MCP-1 (C), MIP-1α (D), RANTES (E), and TNF-α (F) by enzyme-linked immunosorbent assay. Significant difference is expressed as p

    Article Snippet: Pulmonary cytokine levels Cytokine levels, including monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), RANTES, IL-1β, interferon (IFN)-α, IFN-γ, tumor necrosis factor (TNF)-α were measured by respective mouse enzyme-linked immunosorbent assay (ELISA) kit obtained from Invitrogen according to the protocols of the manufacturer.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    The frequency of responding SVV-specific T cells. The frequency of SVV-specific T cells in (A - D) BAL and (E and F) PBMC producing IFNγ, TNFα and IFNγ/TNFα was measured by intracellular cytokine staining following stimulation with either (A , B , E and F) SVV lysate or (C and D) overlapping peptide pool (SVV ORFs 4, 31, 61 and 63), average ± SEM. SVV BAC (white bar) and WT SVV (black bar).

    Journal: Virology Journal

    Article Title: Bacterial artificial chromosome derived simian varicella virus is pathogenic in vivo

    doi: 10.1186/1743-422X-10-278

    Figure Lengend Snippet: The frequency of responding SVV-specific T cells. The frequency of SVV-specific T cells in (A - D) BAL and (E and F) PBMC producing IFNγ, TNFα and IFNγ/TNFα was measured by intracellular cytokine staining following stimulation with either (A , B , E and F) SVV lysate or (C and D) overlapping peptide pool (SVV ORFs 4, 31, 61 and 63), average ± SEM. SVV BAC (white bar) and WT SVV (black bar).

    Article Snippet: Samples were fixed, permeabilized (BioLegend) and dual-stained using antibodies against IFNγ (eBioscience) and TNFα (eBioscience).

    Techniques: Staining, BAC Assay

    Attenuation of the level of reactive oxygen species (ROS) by genistein. (A) Keratinocytes were stimulated respectively with a proinflammatory “cytokine mix” corresponding to a concentration of 2 ng/mL (ACT 2) or 5 ng/mL (ACT 5) in each compound of the mix and incubated with 10 mM N-acetyl-cysteine (NAC) (ACT 2 + NAC), 100 μM genistein (ACT 2 + GEN), 10 ng/mL TNF-α or 1 μg/mL LPS alone, and with 10 ng/mL TNF-α or 1 μg/mL LPS incubated with 100 μM genistein (TNF-α + GEN or LPS + GEN). DMSO-treated, unstimulated cells were used as the control (NACT). Additional control was used in the form of unstimulated cells treated with 100 μM genistein (GEN). Intracellular ROS levels were examined by a CellROX Deep Red Reagent and confocal fluorescence microscopy. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Results representative of three independent experiments (with scale bars 25 μm) are shown. (B) Analysis of ROS were additionally performed by fluorescent cell analyzer. The data are presented as the means ± standard deviation (SD) from three independent experiments. Significant differences ( p ≤ 0.05) between cell populations not expressing intracellular ROS (ROS [–]) and expressing intracellular ROS (ROS [+]) were observed for all tested conditions, except for LPS where cell numbers of ROS (-) and ROS (+) were comparable. Most important, statistically significant differences of p ≤ 0.05 within ROS groups are indicated with * for samples of TNF-α, TNF-α + GEN, and LPS versus NACT, with † for sample TNF-α + GEN with respect to TNF-α, while with ‡ for sample LPS + GEN referred to LPS. Statistical analysis was performed using ANOVA with Tukey’s HSD test.

    Journal: PLoS ONE

    Article Title: Molecular action of isoflavone genistein in the human epithelial cell line HaCaT

    doi: 10.1371/journal.pone.0192297

    Figure Lengend Snippet: Attenuation of the level of reactive oxygen species (ROS) by genistein. (A) Keratinocytes were stimulated respectively with a proinflammatory “cytokine mix” corresponding to a concentration of 2 ng/mL (ACT 2) or 5 ng/mL (ACT 5) in each compound of the mix and incubated with 10 mM N-acetyl-cysteine (NAC) (ACT 2 + NAC), 100 μM genistein (ACT 2 + GEN), 10 ng/mL TNF-α or 1 μg/mL LPS alone, and with 10 ng/mL TNF-α or 1 μg/mL LPS incubated with 100 μM genistein (TNF-α + GEN or LPS + GEN). DMSO-treated, unstimulated cells were used as the control (NACT). Additional control was used in the form of unstimulated cells treated with 100 μM genistein (GEN). Intracellular ROS levels were examined by a CellROX Deep Red Reagent and confocal fluorescence microscopy. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Results representative of three independent experiments (with scale bars 25 μm) are shown. (B) Analysis of ROS were additionally performed by fluorescent cell analyzer. The data are presented as the means ± standard deviation (SD) from three independent experiments. Significant differences ( p ≤ 0.05) between cell populations not expressing intracellular ROS (ROS [–]) and expressing intracellular ROS (ROS [+]) were observed for all tested conditions, except for LPS where cell numbers of ROS (-) and ROS (+) were comparable. Most important, statistically significant differences of p ≤ 0.05 within ROS groups are indicated with * for samples of TNF-α, TNF-α + GEN, and LPS versus NACT, with † for sample TNF-α + GEN with respect to TNF-α, while with ‡ for sample LPS + GEN referred to LPS. Statistical analysis was performed using ANOVA with Tukey’s HSD test.

    Article Snippet: For further experiments, cells were stimulated with a combination of a proinflammatory “cytokine mix”: IL-1A, IL-17A, IL-22, oncostatin M (OSM), and tumor necrosis factor-α (TNF-α) (Gibco, Thermo Fisher Scientific, CA, USA) 2 ng/mL each or lipopolysaccharide (LPS, from Escherichia coli 055 :B5 , Sigma-Aldrich, St. Louis, USA) 1 μg/mL in the presence or absence of genistein for 24 hours [ ].

    Techniques: Concentration Assay, Activated Clotting Time Assay, Incubation, Fluorescence, Microscopy, Staining, Standard Deviation, Expressing

    A , Western blot analysis. Effect of high ophiopogonin D (OP-D-H) on protein expression of interleukin (IL)-6 ( B ), tumor necrosis factor α (TNF-α) ( C ), and nuclear factor (NF)-κB ( D ). Data are reported as mean±SEM (n=8) (ANOVA followed by Tukey's post hoc analysis). Glic: Gliclazide.

    Journal: Brazilian Journal of Medical and Biological Research

    Article Title: Ophiopogonin D of Ophiopogon japonicus ameliorates renal function by suppressing oxidative stress and inflammatory response in streptozotocin-induced diabetic nephropathy rats

    doi: 10.1590/1414-431X20209628

    Figure Lengend Snippet: A , Western blot analysis. Effect of high ophiopogonin D (OP-D-H) on protein expression of interleukin (IL)-6 ( B ), tumor necrosis factor α (TNF-α) ( C ), and nuclear factor (NF)-κB ( D ). Data are reported as mean±SEM (n=8) (ANOVA followed by Tukey's post hoc analysis). Glic: Gliclazide.

    Article Snippet: Blotted membranes were incubated with the following primary antibodies overnight at 4°C: anti-IL-6 (1:200; Invitrogen, USA), anti-TNF-α (1:200; Invitrogen), anti-NF-κB (1:200; Invitrogen), and GAPDH (1:200; Invitrogen).

    Techniques: Western Blot, Expressing

    Melatonin effects on HMSC stemness following inflammatory treatment. (a) RT-PCR analysis of biomarkers of HMSC stemness with different concentrations of TNF- α . (b) Colony formation assays and quantitative analysis of HMSCs with different treatments. (c) RT-PCR analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P3 HMSCs with different treatments. (d) RT-PCR analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P7 HMSCs with different treatments. (e) RT-PCR analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P10 HMSCs with different treatments. (f) Western blot analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P3 HMSCs with different treatments. (g) Western blot analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P7 HMSCs with different treatments. (h) Western blot analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P10 HMSCs with different treatments. (i) Alizarin red staining and quantification of osteogenic differentiation of HMSCs with different treatments. P3: primary cells expanded to the third generation; P7: primary cells expanded to the seventh generation; P10: primary cells expanded to the tenth generation. ∗ P

    Journal: Stem Cells International

    Article Title: Melatonin Reverses the Loss of Stemness Induced by TNF-α in Human Bone Marrow Mesenchymal Stem Cells through Upregulation of YAP Expression

    doi: 10.1155/2019/6568394

    Figure Lengend Snippet: Melatonin effects on HMSC stemness following inflammatory treatment. (a) RT-PCR analysis of biomarkers of HMSC stemness with different concentrations of TNF- α . (b) Colony formation assays and quantitative analysis of HMSCs with different treatments. (c) RT-PCR analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P3 HMSCs with different treatments. (d) RT-PCR analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P7 HMSCs with different treatments. (e) RT-PCR analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P10 HMSCs with different treatments. (f) Western blot analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P3 HMSCs with different treatments. (g) Western blot analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P7 HMSCs with different treatments. (h) Western blot analysis of biomarkers of HMSC stemness, osteogenic differentiation, and adipose differentiation of P10 HMSCs with different treatments. (i) Alizarin red staining and quantification of osteogenic differentiation of HMSCs with different treatments. P3: primary cells expanded to the third generation; P7: primary cells expanded to the seventh generation; P10: primary cells expanded to the tenth generation. ∗ P

    Article Snippet: Melatonin, TNF-α , Luz, and VP were obtained from Sigma-Aldrich (USA).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining

    (a) Dose-dependent secretion of IP-10 and Mig by gastric epithelial cells. Confluent monolayers of NCI cell lines were stimulated with increasing concentrations of either TNF-α or IFN-γ or both in combination (• IFN-γ; ○ TNF-α; ▾ TNF-α + IFN-γ). Supernatants were harvested after 18 h and chemokine-secretion was measured by ELISA. Similar results were obtained using AGS and NCI cell lines. Values are expressed as mean ± CI. Representative results from one of four independent experiments are shown. (b) Time-dependent secretion of IP-10 and Mig by gastric epithelial cells. Confluent monolayers of AGS and Kato III cells were stimulated for different times (as indicated) with either IFN-γ 50 ng/ml or TNF-α 50 ng/ml alone or both cytokines in combination. Supernatants were harvested after 6, 12 and 18 h and chemokine concentrations were measured by ELISA. Values are expressed as mean ± CI. Representative results from one of four experiments are shown.

    Journal: Clinical and Experimental Immunology

    Article Title: IFN-? synergizes with TNF-? but not with viable H. pylori in up-regulating CXC chemokine secretion in gastric epithelial cells

    doi: 10.1046/j.1365-2249.2001.01634.x

    Figure Lengend Snippet: (a) Dose-dependent secretion of IP-10 and Mig by gastric epithelial cells. Confluent monolayers of NCI cell lines were stimulated with increasing concentrations of either TNF-α or IFN-γ or both in combination (• IFN-γ; ○ TNF-α; ▾ TNF-α + IFN-γ). Supernatants were harvested after 18 h and chemokine-secretion was measured by ELISA. Similar results were obtained using AGS and NCI cell lines. Values are expressed as mean ± CI. Representative results from one of four independent experiments are shown. (b) Time-dependent secretion of IP-10 and Mig by gastric epithelial cells. Confluent monolayers of AGS and Kato III cells were stimulated for different times (as indicated) with either IFN-γ 50 ng/ml or TNF-α 50 ng/ml alone or both cytokines in combination. Supernatants were harvested after 6, 12 and 18 h and chemokine concentrations were measured by ELISA. Values are expressed as mean ± CI. Representative results from one of four experiments are shown.

    Article Snippet: The recombinant human (rh) IFN-γ and TNF-α, anti-IP-10 (1 µg/µl), anti-Mig (1 µg/µl), as well as biotinylated antibodies against IP-10 and Mig used in the ELISA protocol and rh IP-10 (20 ng/µl) and rh Mig (20 ng/µl) were obtained from R & D Systems (Wiesbaden, Germany).

    Techniques: Enzyme-linked Immunosorbent Assay

    Pedigree and experimental analysis. ( A ) Pedigree depicting the presence of the c.1049delG, p.(Gly350Glufs*15) IRAK4 variant. The arrow denotes the index patient. ( B ) Sanger sequencing of the IRAK4 gene depicting the single-base-pair deletion in the affected family but not seen in the healthy control (HC). ( C ) Immunoblot showing lack of IRAK4 protein expression compared to a HC, as well as the predicted molecular weight (MW) and position of the truncated IRAK4 due to the variant ( n = 3). ( D ) Simplified schematic overview of the TLR7 signaling pathway resulting in NF-κB activation and TNF-α transcription emphasizing the central role of the IRAK4 protein in signal transduction. ( E ) Cytometric bead array (CBA) showing no TNF-α response to imiquimod (IMQ) stimulation of TLR7 in the IRAK4-deficient cells compared to a HC. PMA/ionomycin serves as a positive control confirming cell viability. Data are represented as the mean ( n = 3), and error bars represent the standard error of the mean (SEM). P -value calculated by the two-tailed Student's t -test with a Bonferroni correction.

    Journal: Cold Spring Harbor Molecular Case Studies

    Article Title: Clinical IRAK4 deficiency caused by homozygosity for the novel IRAK4 (c.1049delG, p.Gly350Glufs*15) variant

    doi: 10.1101/mcs.a005298

    Figure Lengend Snippet: Pedigree and experimental analysis. ( A ) Pedigree depicting the presence of the c.1049delG, p.(Gly350Glufs*15) IRAK4 variant. The arrow denotes the index patient. ( B ) Sanger sequencing of the IRAK4 gene depicting the single-base-pair deletion in the affected family but not seen in the healthy control (HC). ( C ) Immunoblot showing lack of IRAK4 protein expression compared to a HC, as well as the predicted molecular weight (MW) and position of the truncated IRAK4 due to the variant ( n = 3). ( D ) Simplified schematic overview of the TLR7 signaling pathway resulting in NF-κB activation and TNF-α transcription emphasizing the central role of the IRAK4 protein in signal transduction. ( E ) Cytometric bead array (CBA) showing no TNF-α response to imiquimod (IMQ) stimulation of TLR7 in the IRAK4-deficient cells compared to a HC. PMA/ionomycin serves as a positive control confirming cell viability. Data are represented as the mean ( n = 3), and error bars represent the standard error of the mean (SEM). P -value calculated by the two-tailed Student's t -test with a Bonferroni correction.

    Article Snippet: A CBA was used to measure the concentration of TNF-α (BD Biosciences), performed as previously described ( ).

    Techniques: Variant Assay, Sequencing, Expressing, Molecular Weight, Activation Assay, Transduction, Crocin Bleaching Assay, Positive Control, Two Tailed Test

    PBMC from normal pregnant women produce more TNFα and IL-6 in response to pSTBM than non pregnant women's PBMC. 10 6 /ml PBMC from non pregnant (light bars) or pregnant women (black bars) (n = 10) were treated with 50 µg/ml pSTBM for 20 hours at 37°C. Supernatants were harvested and cytokine production assessed by ELISA for TNFα, IL-6, IL-8, IL-1α, MIP-1α and IL-1β (pg/ml). Values calculated as the mean of value from cells treated with 50 ug/ml pSTBM minus background cytokine production.

    Journal: PLoS ONE

    Article Title: The Immunomodulatory Role of Syncytiotrophoblast Microvesicles

    doi: 10.1371/journal.pone.0020245

    Figure Lengend Snippet: PBMC from normal pregnant women produce more TNFα and IL-6 in response to pSTBM than non pregnant women's PBMC. 10 6 /ml PBMC from non pregnant (light bars) or pregnant women (black bars) (n = 10) were treated with 50 µg/ml pSTBM for 20 hours at 37°C. Supernatants were harvested and cytokine production assessed by ELISA for TNFα, IL-6, IL-8, IL-1α, MIP-1α and IL-1β (pg/ml). Values calculated as the mean of value from cells treated with 50 ug/ml pSTBM minus background cytokine production.

    Article Snippet: Cells were then washed and treated with Permeabilization buffer (eBioscience) following manufacturers instructions prior to incubation for 20 minutes at 4°C with FITC conjugated antibodies towards IL-6, IL-8 and IL-1β or PE conjugated antibody towards TNFα (BD Bioscience) or appropriate isotype control.

    Techniques: Enzyme-linked Immunosorbent Assay

    pSTBM alter cytokine production by PBMC from non and normal pregnant women. 10 6 /ml PBMC from 10 non pregnant (light bars) or 10 pregnant women (black bars) were treated with varying concentrations of pSTBM for 20 hours at 37°C. Supernatants were harvested and cytokine production assessed by ELISA for A) TNFα, B) IL-8, C) MIP-1α, D) IP-10, E) IL-6, F) IL-1α, and G) IL-1β (pg/ml). H) Cytokines and chemokines expressed by the pSTBM alone were determined by cytokine array, cytokines present above background levels in the human serum media were 1-RANTES, 2-MIF, 3-sICAM-1 and 4-Serpin E1.

    Journal: PLoS ONE

    Article Title: The Immunomodulatory Role of Syncytiotrophoblast Microvesicles

    doi: 10.1371/journal.pone.0020245

    Figure Lengend Snippet: pSTBM alter cytokine production by PBMC from non and normal pregnant women. 10 6 /ml PBMC from 10 non pregnant (light bars) or 10 pregnant women (black bars) were treated with varying concentrations of pSTBM for 20 hours at 37°C. Supernatants were harvested and cytokine production assessed by ELISA for A) TNFα, B) IL-8, C) MIP-1α, D) IP-10, E) IL-6, F) IL-1α, and G) IL-1β (pg/ml). H) Cytokines and chemokines expressed by the pSTBM alone were determined by cytokine array, cytokines present above background levels in the human serum media were 1-RANTES, 2-MIF, 3-sICAM-1 and 4-Serpin E1.

    Article Snippet: Cells were then washed and treated with Permeabilization buffer (eBioscience) following manufacturers instructions prior to incubation for 20 minutes at 4°C with FITC conjugated antibodies towards IL-6, IL-8 and IL-1β or PE conjugated antibody towards TNFα (BD Bioscience) or appropriate isotype control.

    Techniques: Enzyme-linked Immunosorbent Assay

    Monocytes, not B cells, produce the cytokines IL-6, IL-8, TNFα and IL-1β and downregulate HLA-DR in response to pSTBM. A) 10 6 /ml PBMC from non-pregnant donors (n = 3) were stimulated with 50 µg/ml pSTBM, or left untreated, for 20 hours in the presence of Brefeldin A. Intracellular cytokine analysis was performed to detect production of TNFα, IL-6, IL-8 and IL-1β from either B cells (grey bars) or monocytes (black bars). Data shown is the increase in proportion of cells expressing each cytokine above cytokine production in untreated samples, mean (+/− S.D.). B) pSTBM binding to monocytes, but not B cells, caused down-regulation of HLA-DR, shown by median fluorescence intensity of HLA-DR antibody staining (C), (n = 3; mean +/− S.D.).

    Journal: PLoS ONE

    Article Title: The Immunomodulatory Role of Syncytiotrophoblast Microvesicles

    doi: 10.1371/journal.pone.0020245

    Figure Lengend Snippet: Monocytes, not B cells, produce the cytokines IL-6, IL-8, TNFα and IL-1β and downregulate HLA-DR in response to pSTBM. A) 10 6 /ml PBMC from non-pregnant donors (n = 3) were stimulated with 50 µg/ml pSTBM, or left untreated, for 20 hours in the presence of Brefeldin A. Intracellular cytokine analysis was performed to detect production of TNFα, IL-6, IL-8 and IL-1β from either B cells (grey bars) or monocytes (black bars). Data shown is the increase in proportion of cells expressing each cytokine above cytokine production in untreated samples, mean (+/− S.D.). B) pSTBM binding to monocytes, but not B cells, caused down-regulation of HLA-DR, shown by median fluorescence intensity of HLA-DR antibody staining (C), (n = 3; mean +/− S.D.).

    Article Snippet: Cells were then washed and treated with Permeabilization buffer (eBioscience) following manufacturers instructions prior to incubation for 20 minutes at 4°C with FITC conjugated antibodies towards IL-6, IL-8 and IL-1β or PE conjugated antibody towards TNFα (BD Bioscience) or appropriate isotype control.

    Techniques: Expressing, Binding Assay, Fluorescence, Staining

    Detection of mRNA levels of TNF-α and IL-6 in placenta tissues of pregnant rats using fluorescence quantitative PCR compared with those in the control group. NS, not significant. **P

    Journal: Experimental and Therapeutic Medicine

    Article Title: The relationship between inflammatory factor expression and blood pressure and urinary protein in the placenta of gestational hypertension rats

    doi: 10.3892/etm.2018.6668

    Figure Lengend Snippet: Detection of mRNA levels of TNF-α and IL-6 in placenta tissues of pregnant rats using fluorescence quantitative PCR compared with those in the control group. NS, not significant. **P

    Article Snippet: The total protein membrane was transferred using polyvinylidene fluoride (PVDF), and the bands were incubated using the primary antibodies and anti-rabbit secondary antibodies of IL-6 (1:800; Abcam, Cambridge, UK; cat. no. ab6672) and TNF-α (1:600; Abcam, cat. no. ab6671).

    Techniques: Fluorescence, Real-time Polymerase Chain Reaction

    Correlation analyses of TNF-α level in placenta tissues with blood pressure and urine proteins. Placental TNF-α level of the hypoxia-induced PIH rat model is positively correlated with blood pressure (A) and urine proteins (B). R 2 values are 0.655 and 0.736, respectively, and Pearson's correlation analysis was used.

    Journal: Experimental and Therapeutic Medicine

    Article Title: The relationship between inflammatory factor expression and blood pressure and urinary protein in the placenta of gestational hypertension rats

    doi: 10.3892/etm.2018.6668

    Figure Lengend Snippet: Correlation analyses of TNF-α level in placenta tissues with blood pressure and urine proteins. Placental TNF-α level of the hypoxia-induced PIH rat model is positively correlated with blood pressure (A) and urine proteins (B). R 2 values are 0.655 and 0.736, respectively, and Pearson's correlation analysis was used.

    Article Snippet: The total protein membrane was transferred using polyvinylidene fluoride (PVDF), and the bands were incubated using the primary antibodies and anti-rabbit secondary antibodies of IL-6 (1:800; Abcam, Cambridge, UK; cat. no. ab6672) and TNF-α (1:600; Abcam, cat. no. ab6671).

    Techniques:

    Detection of protein levels of IL-6 and TNF-α in placenta tissues of pregnant rats using western blot analysis. NS, not significant. **P

    Journal: Experimental and Therapeutic Medicine

    Article Title: The relationship between inflammatory factor expression and blood pressure and urinary protein in the placenta of gestational hypertension rats

    doi: 10.3892/etm.2018.6668

    Figure Lengend Snippet: Detection of protein levels of IL-6 and TNF-α in placenta tissues of pregnant rats using western blot analysis. NS, not significant. **P

    Article Snippet: The total protein membrane was transferred using polyvinylidene fluoride (PVDF), and the bands were incubated using the primary antibodies and anti-rabbit secondary antibodies of IL-6 (1:800; Abcam, Cambridge, UK; cat. no. ab6672) and TNF-α (1:600; Abcam, cat. no. ab6671).

    Techniques: Western Blot

    CQ resets tumor-associated M2 macrophages to M1 phenotype. a , b BMDM-M2 cells were treated with or without 10 μM CQ for 24 h. The expression of IFN-γ, IL-12p40, and TNF-α was measured by flow cytometry a ( n = 3). Quantification of CD80, CD86, and MHC-II in BMDM-M2 cells with or without CQ treatment b ( n = 3). c NO production in BMDM-M2 cells lysate with or without CQ treatment were measured ( n = 3). d Quantification of the number of CD206 + CD301 + cells in BMDM-M2 cells with or without CQ treatment ( n = 3). e The mRNA expression of NOS2 and Arg1 in BMDM-M2 cells with or without CQ treatment was analyzed by qPCR (left); the expression of Arg1 was analyzed by western blotting (center); the expression of iNOS was analyzed by flow cytometry (right). f Arginase1 + cells in IL-12p40 - IFN-γ − M2 macrophages with or without CQ treatment were analyzed by flow cytometry ( n = 3). g Representative flow cytometric analysis and quantification of iNOS in F4/80 + ascites fluid macrophages, isolated from mice with H22 hepatocarcinoma that had received PBS or CQ treatment ( n = 5) (left); the protein expression of iNOS and Arg1 in the same F4/80 + ascites macrophages were also analyzed by western blotting (right) ( n = 3). h Western blot analysis of iNOS and Arg1 in tumor-infiltrating macrophages after PBS or CQ treatment in mice bearing subcutaneous B16 melanoma ( n = 3). i The mRNA expression of IFN-γ , IL-12p40 , IL-12p35 , TNF-α , and IL-10 was analyzed by real-time qPCR in B16 tumor tissues in mice with or without CQ treatment ( n = 3). j , k Splenocytes from OT-I and OT-II TCR transgenic mice were labeled with carboxyfluorescein succinimidyl ester (CFSE) and then stimulated with OVA257-264 ( j ) and OVA323-339 ( k ) respectively in the presence of conditioned medium from PBS or CQ treated BMDM-M2 cells. Representative histograms of CD8 + or CD4 + T-cell proliferation (left panel) and quantification of CD8 + or CD4 + T-cell proliferation (right panel) were analyzed by flow cytometry after 72 h ( n = 3). Data shown are representative of three independent experiments and error bars represent mean ± SEM. * P

    Journal: Nature Communications

    Article Title: Chloroquine modulates antitumor immune response by resetting tumor-associated macrophages toward M1 phenotype

    doi: 10.1038/s41467-018-03225-9

    Figure Lengend Snippet: CQ resets tumor-associated M2 macrophages to M1 phenotype. a , b BMDM-M2 cells were treated with or without 10 μM CQ for 24 h. The expression of IFN-γ, IL-12p40, and TNF-α was measured by flow cytometry a ( n = 3). Quantification of CD80, CD86, and MHC-II in BMDM-M2 cells with or without CQ treatment b ( n = 3). c NO production in BMDM-M2 cells lysate with or without CQ treatment were measured ( n = 3). d Quantification of the number of CD206 + CD301 + cells in BMDM-M2 cells with or without CQ treatment ( n = 3). e The mRNA expression of NOS2 and Arg1 in BMDM-M2 cells with or without CQ treatment was analyzed by qPCR (left); the expression of Arg1 was analyzed by western blotting (center); the expression of iNOS was analyzed by flow cytometry (right). f Arginase1 + cells in IL-12p40 - IFN-γ − M2 macrophages with or without CQ treatment were analyzed by flow cytometry ( n = 3). g Representative flow cytometric analysis and quantification of iNOS in F4/80 + ascites fluid macrophages, isolated from mice with H22 hepatocarcinoma that had received PBS or CQ treatment ( n = 5) (left); the protein expression of iNOS and Arg1 in the same F4/80 + ascites macrophages were also analyzed by western blotting (right) ( n = 3). h Western blot analysis of iNOS and Arg1 in tumor-infiltrating macrophages after PBS or CQ treatment in mice bearing subcutaneous B16 melanoma ( n = 3). i The mRNA expression of IFN-γ , IL-12p40 , IL-12p35 , TNF-α , and IL-10 was analyzed by real-time qPCR in B16 tumor tissues in mice with or without CQ treatment ( n = 3). j , k Splenocytes from OT-I and OT-II TCR transgenic mice were labeled with carboxyfluorescein succinimidyl ester (CFSE) and then stimulated with OVA257-264 ( j ) and OVA323-339 ( k ) respectively in the presence of conditioned medium from PBS or CQ treated BMDM-M2 cells. Representative histograms of CD8 + or CD4 + T-cell proliferation (left panel) and quantification of CD8 + or CD4 + T-cell proliferation (right panel) were analyzed by flow cytometry after 72 h ( n = 3). Data shown are representative of three independent experiments and error bars represent mean ± SEM. * P

    Article Snippet: Human and mouse IFN-γ, TNF-α, and IL-10 ELISA kits were purchased from PeproTech.

    Techniques: Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Western Blot, Isolation, Mouse Assay, Transgenic Assay, Labeling

    IL-1 receptor (IL-1R) activation suppresses TNF-α expression and attenuates regulated necrosis. A : receptor-interacting protein (RIP)1, RIP3, and cytokine mRNA levels were quantitated in kidneys from IL-1R wild-type (WT) mice with selective deletion of polycystin-1 in the kidney and IL-1R knockout (KO) KPKD1 control mice. Compared with IL-1R WT KPKD1 kidneys, TNF-α protein was upregulated in IL-1R KO KPKD1 kidneys [typical Western blot bands ( B ) and protein abundance analysis ( C )]. n.s., not significant.

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Interleukin-1 receptor activation aggravates autosomal dominant polycystic kidney disease by modulating regulated necrosis

    doi: 10.1152/ajprenal.00104.2019

    Figure Lengend Snippet: IL-1 receptor (IL-1R) activation suppresses TNF-α expression and attenuates regulated necrosis. A : receptor-interacting protein (RIP)1, RIP3, and cytokine mRNA levels were quantitated in kidneys from IL-1R wild-type (WT) mice with selective deletion of polycystin-1 in the kidney and IL-1R knockout (KO) KPKD1 control mice. Compared with IL-1R WT KPKD1 kidneys, TNF-α protein was upregulated in IL-1R KO KPKD1 kidneys [typical Western blot bands ( B ) and protein abundance analysis ( C )]. n.s., not significant.

    Article Snippet: The primary antibodies used were as follows: phospho-receptor-interacting protein 3 (RIP3; mouse, catalog no. ab195117, Abcam), phospho-MLKL (mouse, catalog no. ab196436, Abcam), phospho-RIP3 (human, catalog no. CST 93654, Cell Signaling Technology), TNF-α (mouse, catalog no. CST 11948s, Cell Signaling Technology), and phospho-MLKL (human, catalog no. ab187091, Abcam).

    Techniques: Activation Assay, Expressing, Mouse Assay, Knock-Out, Western Blot

    Necroptosis is upregulated in multiple autosomal dominant polycystic kidney disease (ADPKD) tissues. A : phosphorylation of receptor-interacting protein 3 (RIP3) and mixed lineage kinase domain-like pseudokinase (MLKL) was upregulated in human ADPKD tissues by Western blot analysis. B : immunohistochemical stains detected phosphorylated (p-)MLKL in epithelial cells lining cysts in human ADPKD sections. C and D : mRNA levels of RIP3 ( C ) and MLKL ( D ) were increased in kidneys from mice with selective deletion of polycystin-1 ( pkd1 ) in the kidney (KPKD1 mice). E : phosphorylation of RIP3 and MLKL was upregulated in KPKD1 kidneys. Typical Western blot bands and protein abundance analysis are shown. F : in primary epithelial (EPI) cells, pkd1 knockout (KO) induced ~1.5-fold RIP3 and MLKL mRNA upregulation. G and H : TNF-α mRNA levels were induced in KPKD1 tissues ( G ) and primary KPDK1 tubular epithelial cells ( H ).

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Interleukin-1 receptor activation aggravates autosomal dominant polycystic kidney disease by modulating regulated necrosis

    doi: 10.1152/ajprenal.00104.2019

    Figure Lengend Snippet: Necroptosis is upregulated in multiple autosomal dominant polycystic kidney disease (ADPKD) tissues. A : phosphorylation of receptor-interacting protein 3 (RIP3) and mixed lineage kinase domain-like pseudokinase (MLKL) was upregulated in human ADPKD tissues by Western blot analysis. B : immunohistochemical stains detected phosphorylated (p-)MLKL in epithelial cells lining cysts in human ADPKD sections. C and D : mRNA levels of RIP3 ( C ) and MLKL ( D ) were increased in kidneys from mice with selective deletion of polycystin-1 ( pkd1 ) in the kidney (KPKD1 mice). E : phosphorylation of RIP3 and MLKL was upregulated in KPKD1 kidneys. Typical Western blot bands and protein abundance analysis are shown. F : in primary epithelial (EPI) cells, pkd1 knockout (KO) induced ~1.5-fold RIP3 and MLKL mRNA upregulation. G and H : TNF-α mRNA levels were induced in KPKD1 tissues ( G ) and primary KPDK1 tubular epithelial cells ( H ).

    Article Snippet: The primary antibodies used were as follows: phospho-receptor-interacting protein 3 (RIP3; mouse, catalog no. ab195117, Abcam), phospho-MLKL (mouse, catalog no. ab196436, Abcam), phospho-RIP3 (human, catalog no. CST 93654, Cell Signaling Technology), TNF-α (mouse, catalog no. CST 11948s, Cell Signaling Technology), and phospho-MLKL (human, catalog no. ab187091, Abcam).

    Techniques: Western Blot, Immunohistochemistry, Mouse Assay, Knock-Out

    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

    Journal: Molecular Vision

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

    doi:

    Figure Lengend 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

    Article Snippet: Then, 200 µl of 1× phenol-free minimum essential media (MEM)/F12 medium (Life Technologies) containing either HNE (Millipore, Etobicoke, Canada) at 10 µM or recombinant human TNF-α (R & D Systems, Minneapolis, MN) at 20 ng/ml was added into each corresponding well and incubated for 6 h. C3a or C5a (R & D Systems) at 5 µg/ml was used to incubate the cell culture for 24 h. After incubation, the resulting supernatants or the cell lysate were collected, centrifuged, and stored at −80 °C for the suspension assay or qPCR.

    Techniques:

    Secretion of pro-inflammatory cytokines such as ( A ) TNFα, ( B ) IL-1β and ( C ) IL-12 in cell culture supernatant as markers of inflammation following nano-WC–Co exposure. Notes: * P

    Journal: International Journal of Nanomedicine

    Article Title: In vitro inflammatory effects of hard metal (WC–Co) nanoparticle exposure

    doi: 10.2147/IJN.S121141

    Figure Lengend Snippet: Secretion of pro-inflammatory cytokines such as ( A ) TNFα, ( B ) IL-1β and ( C ) IL-12 in cell culture supernatant as markers of inflammation following nano-WC–Co exposure. Notes: * P

    Article Snippet: Isopropanol, hydrochloric acid, Triton-X-100, thiazolyl blue tetrazolium bromide (MTT reagent), phorbol-12-mystirate-13-acetate (PMA), LPS and enzyme-linked immunosorbent assay (ELISA) kits for human IL-12 (#RAB0252), IL-10 (#RAB0244), IL-1β (#RAB0273) and TNFα (#RAB0476) were purchased from Sigma-Aldrich (St Louis, MO, USA).

    Techniques: Cell Culture

    Probiotic Lactobacillus spp. alleviated gastric inflammation in mice. ( A ) Mice were fed with Lactobacillus spp. (GMNL-74 and GMNL-185) for 24 days followed by intragastric gavage with H. pylori 26695 once every 2 days for a total of six administrations. Arrows show the days of H. pylori inoculation. ( B ) Mice were euthanized and gastric tissues were subjected to hematoxylin–eosin (H E) and immunohistochemical (IHC) staining with specific antibodies against cyclooxygenase-2 (COX-2) and tumor necrosis factor (TNF)-α, respectively (original magnification: 200×). The magnified images are shown in the lower panel of each cropped area. Severe infiltration of inflammatory cells in the gastric epithelium (H E) and pronounced expression of COX-2 and TNF-α in gastric tissues are indicated by red arrows (IHC).

    Journal: Journal of Clinical Medicine

    Article Title: Probiotic Lactobacillus spp. Act Against Helicobacter pylori-induced Inflammation

    doi: 10.3390/jcm8010090

    Figure Lengend Snippet: Probiotic Lactobacillus spp. alleviated gastric inflammation in mice. ( A ) Mice were fed with Lactobacillus spp. (GMNL-74 and GMNL-185) for 24 days followed by intragastric gavage with H. pylori 26695 once every 2 days for a total of six administrations. Arrows show the days of H. pylori inoculation. ( B ) Mice were euthanized and gastric tissues were subjected to hematoxylin–eosin (H E) and immunohistochemical (IHC) staining with specific antibodies against cyclooxygenase-2 (COX-2) and tumor necrosis factor (TNF)-α, respectively (original magnification: 200×). The magnified images are shown in the lower panel of each cropped area. Severe infiltration of inflammatory cells in the gastric epithelium (H E) and pronounced expression of COX-2 and TNF-α in gastric tissues are indicated by red arrows (IHC).

    Article Snippet: Antibodies specific to cyclooxygenase-2 (COX-2) and Tumor necrosis factor (TNF)-α were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Mouse Assay, Immunohistochemistry, Staining, Expressing

    Functional analysis of GD2/CAR-T cells in vitro. a Cytotoxic activity of GD2/CAR-T cells. We used an LDH release assay to evaluate the cytotoxic activity of GD2.BBζ CAR-T cells and non-transduced T cells. Target cells were melanoma lines with varying GD2 expression levels. The figure illustrates the mean and SD of LDH release from 9 T cell lines after 4 h of incubation. A significant difference was detected in GD2-specific antitumor activity at each E:T ratio between GD2.BBζ CAR-T cells and control non-transduced CAR-T cells. In contrast, GD2.BBζ CAR-T cells had little antitumor activity against the GD2-negative tumor cell lines 293T, A875 (3.1% GD2 expression), and MMYC-7 (10.2% GD2 expression). b Th1 cytokine release of GD2/CAR-T cells. Non-transduced T cells and GD2.BBζ CAR-T cells were co-cultured (ratio of T lymphocytes:tumor cells of 20:1) with four different cell lines that were GD2-negative (293T) or were 27.4% GD2-positive (GAK), were 47.3% GD2-positive (HMV-II) or exhibited high (WM-266-4) levels of GD2-positive cells. Culture supernatant was collected 24 h later, and the production of IL-2, TNF-α, and IFN-γ were measured using a CBA assay. A substantial amount of IL2, TNF-α, and IFN-γ was released by GD2.BBζ CAR-T cells, and their releases were associated with the level of GD2 expression in the melanoma cells. In contrast, the release of IL2, TNF-α, and IFN-γ remained unchanged in non-transduced T cells or 293 T cells. The results are presented as the mean and SD from experiments that were performed in triplicate

    Journal: Journal of Hematology & Oncology

    Article Title: Anti-GD2/4-1BB chimeric antigen receptor T cell therapy for the treatment of Chinese melanoma patients

    doi: 10.1186/s13045-017-0548-2

    Figure Lengend Snippet: Functional analysis of GD2/CAR-T cells in vitro. a Cytotoxic activity of GD2/CAR-T cells. We used an LDH release assay to evaluate the cytotoxic activity of GD2.BBζ CAR-T cells and non-transduced T cells. Target cells were melanoma lines with varying GD2 expression levels. The figure illustrates the mean and SD of LDH release from 9 T cell lines after 4 h of incubation. A significant difference was detected in GD2-specific antitumor activity at each E:T ratio between GD2.BBζ CAR-T cells and control non-transduced CAR-T cells. In contrast, GD2.BBζ CAR-T cells had little antitumor activity against the GD2-negative tumor cell lines 293T, A875 (3.1% GD2 expression), and MMYC-7 (10.2% GD2 expression). b Th1 cytokine release of GD2/CAR-T cells. Non-transduced T cells and GD2.BBζ CAR-T cells were co-cultured (ratio of T lymphocytes:tumor cells of 20:1) with four different cell lines that were GD2-negative (293T) or were 27.4% GD2-positive (GAK), were 47.3% GD2-positive (HMV-II) or exhibited high (WM-266-4) levels of GD2-positive cells. Culture supernatant was collected 24 h later, and the production of IL-2, TNF-α, and IFN-γ were measured using a CBA assay. A substantial amount of IL2, TNF-α, and IFN-γ was released by GD2.BBζ CAR-T cells, and their releases were associated with the level of GD2 expression in the melanoma cells. In contrast, the release of IL2, TNF-α, and IFN-γ remained unchanged in non-transduced T cells or 293 T cells. The results are presented as the mean and SD from experiments that were performed in triplicate

    Article Snippet: Interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-10 (IL-10), tumor necrosis factor (TNF-α), and interferon-γ (IFN-γ) cytokine release after 24 h of culture was measured using the cytometric bead array (CBA) human Th1/Th2 cytokine kit (BD Bioscience).

    Techniques: Functional Assay, In Vitro, Activity Assay, Lactate Dehydrogenase Assay, Expressing, Incubation, Cell Culture, Crocin Bleaching Assay

    TNF-α and IFN-γ promote Dsg2 intracellular and extracellular cleavage. a Model of Dsg2 with mapped epitopes for AH12.2 (extracellular [N-term] specific) and 4B2 (intracellular [C-term] specific) monoclonal antibodies. EC extracellular cadherin domain, EA extracellular anchor, TM transmembrane domain, IA intracellular anchor, ICS intracellular cadherin-typical sequence, IPL intracellular proline-rich linker domain, RUD repeated unit domain, DTD desmoglein-specific terminal domain. b Top. Western blot using an antibody against Dsg2 C-term of cell lysates of T-84s treated with TNF-α and IFN-γ for 24 h. Bottom. Densitometry of blots. Density for all bands in each lane was collected. The ratio of the density of the ~55 kDa Dsg2 ICF band to the density of all bands for a given lane was then calculated. The data in the graph is the average of these ratios within each experimental group. * p = 0.0067; # p = 0.0003, n . c . d Top. Western blot using an antibody against Dsg2 C-term of cell lysates of T-84s treated with TNF-α and IFN-γ in the presence or absence of GI254023X for 24 h. Bottom. Densitometry of blots. Densitometry collected and analyzed as for Fig. 1b. n ≥ 4 per group. e Western blot using an antibody against Dsg2 N-term of cell culture supernatants of T-84s treated as in Fig. 1d. f Western blot using an antibody against Dsg2 C-term of cell lysates of T-84s treated with TNF-α and IFN-γ for 6, 12, or 24 h. g Western blot using an antibody against Dsg2 N-term of T-84 cell culture supernatants of cells treated as in Fig. 1f. All blots are representative of at least three independent experiments

    Journal: Cell Death & Disease

    Article Title: Intracellular Desmoglein-2 cleavage sensitizes epithelial cells to apoptosis in response to pro-inflammatory cytokines

    doi: 10.1038/s41419-018-0380-9

    Figure Lengend Snippet: TNF-α and IFN-γ promote Dsg2 intracellular and extracellular cleavage. a Model of Dsg2 with mapped epitopes for AH12.2 (extracellular [N-term] specific) and 4B2 (intracellular [C-term] specific) monoclonal antibodies. EC extracellular cadherin domain, EA extracellular anchor, TM transmembrane domain, IA intracellular anchor, ICS intracellular cadherin-typical sequence, IPL intracellular proline-rich linker domain, RUD repeated unit domain, DTD desmoglein-specific terminal domain. b Top. Western blot using an antibody against Dsg2 C-term of cell lysates of T-84s treated with TNF-α and IFN-γ for 24 h. Bottom. Densitometry of blots. Density for all bands in each lane was collected. The ratio of the density of the ~55 kDa Dsg2 ICF band to the density of all bands for a given lane was then calculated. The data in the graph is the average of these ratios within each experimental group. * p = 0.0067; # p = 0.0003, n . c . d Top. Western blot using an antibody against Dsg2 C-term of cell lysates of T-84s treated with TNF-α and IFN-γ in the presence or absence of GI254023X for 24 h. Bottom. Densitometry of blots. Densitometry collected and analyzed as for Fig. 1b. n ≥ 4 per group. e Western blot using an antibody against Dsg2 N-term of cell culture supernatants of T-84s treated as in Fig. 1d. f Western blot using an antibody against Dsg2 C-term of cell lysates of T-84s treated with TNF-α and IFN-γ for 6, 12, or 24 h. g Western blot using an antibody against Dsg2 N-term of T-84 cell culture supernatants of cells treated as in Fig. 1f. All blots are representative of at least three independent experiments

    Article Snippet: Recombinant human TNF-α, IFN-γ, and TRAIL were purchased from PeproTech (Rocky Hill, NJ, USA).

    Techniques: IA, Sequencing, Western Blot, Cell Culture

    Effects of inhibition of candidate proteases on Dsg2 cleavage. a Western blot using an antibody against Dsg2 C-term and also for cleaved Notch of cell lysates of T-84s treated with TNF-α and IFN-γ for 24 h in the presence or absence of the γ-secretase inhibitor DAPT. b Western blot using an antibody against Dsg2 C-term of cell lysates of T-84s treated with TNF-α and IFN-γ in the presence or absence of increasing concentrations of calpeptin for 24 h. c Western blot using an antibody against Dsg2 N-term of cell culture supernatants of T-84s treated as in Fig. 2b. d Western blot for caspase-8 cell lysates of T-84s treated as in Fig. 2b. All blots are representative of at least three independent experiments

    Journal: Cell Death & Disease

    Article Title: Intracellular Desmoglein-2 cleavage sensitizes epithelial cells to apoptosis in response to pro-inflammatory cytokines

    doi: 10.1038/s41419-018-0380-9

    Figure Lengend Snippet: Effects of inhibition of candidate proteases on Dsg2 cleavage. a Western blot using an antibody against Dsg2 C-term and also for cleaved Notch of cell lysates of T-84s treated with TNF-α and IFN-γ for 24 h in the presence or absence of the γ-secretase inhibitor DAPT. b Western blot using an antibody against Dsg2 C-term of cell lysates of T-84s treated with TNF-α and IFN-γ in the presence or absence of increasing concentrations of calpeptin for 24 h. c Western blot using an antibody against Dsg2 N-term of cell culture supernatants of T-84s treated as in Fig. 2b. d Western blot for caspase-8 cell lysates of T-84s treated as in Fig. 2b. All blots are representative of at least three independent experiments

    Article Snippet: Recombinant human TNF-α, IFN-γ, and TRAIL were purchased from PeproTech (Rocky Hill, NJ, USA).

    Techniques: Inhibition, Western Blot, Cell Culture

    TRAIL treatment induces Dsg2 ECF and ICF generation similarly to TNF-α and IFN-γ. a Western blot using an antibody against Dsg2 C-term and against PARP of cell lysates of T-84s treated with TRAIL for 6, 12, or 24 h. b Western blot using an antibody against Dsg2 N-term of cell culture supernatants of T-84s treated as in Fig. 4a. All blots are representative of at least three independent experiments

    Journal: Cell Death & Disease

    Article Title: Intracellular Desmoglein-2 cleavage sensitizes epithelial cells to apoptosis in response to pro-inflammatory cytokines

    doi: 10.1038/s41419-018-0380-9

    Figure Lengend Snippet: TRAIL treatment induces Dsg2 ECF and ICF generation similarly to TNF-α and IFN-γ. a Western blot using an antibody against Dsg2 C-term and against PARP of cell lysates of T-84s treated with TRAIL for 6, 12, or 24 h. b Western blot using an antibody against Dsg2 N-term of cell culture supernatants of T-84s treated as in Fig. 4a. All blots are representative of at least three independent experiments

    Article Snippet: Recombinant human TNF-α, IFN-γ, and TRAIL were purchased from PeproTech (Rocky Hill, NJ, USA).

    Techniques: Western Blot, Cell Culture

    Caspase-8 is responsible for Dsg2 ICF generation. a Western blot using an antibody against Dsg2 C-term of cell lysates of T-84s treated with TNF-α and IFN-γ in the presence or absence of Z-VAD-fmk for 24 h. b Western blot using an antibody against Dsg2 N-term of cell culture supernatants of T-84s treated as in Fig. 3a. c Western blot using an antibody against Dsg2 C-term of cell lysates of T-84s treated with TNF-α and IFN-γ in the presence or absence of increasing concentrations of either Z-DEVD-fmk, Z-IETD-fmk, or Z-LEHD-fmk for 24 h. d Cos7 cells were transfected with expression plasmids encoding either WT Dsg2 [WT], or Dsg2 containing one of two putative caspase-8 cleavage consensus site mutations (D715A [715] or D675A [675]). Cells treated with Lipofectamine 3000 reagents alone with no DNA were used as controls [N/A]. Top. Western blot against the Myc tag (Top = Dsg2 full-length band; Middle = Dsg2 ICF band; Bottom = Tubulin). Bottom. Model of WT Dsg2, D715A, and D675A depicting region of Dsg2 containing sequences mutated in each highlighting the mutated residues in red. All blots are representative of at least three independent experiments

    Journal: Cell Death & Disease

    Article Title: Intracellular Desmoglein-2 cleavage sensitizes epithelial cells to apoptosis in response to pro-inflammatory cytokines

    doi: 10.1038/s41419-018-0380-9

    Figure Lengend Snippet: Caspase-8 is responsible for Dsg2 ICF generation. a Western blot using an antibody against Dsg2 C-term of cell lysates of T-84s treated with TNF-α and IFN-γ in the presence or absence of Z-VAD-fmk for 24 h. b Western blot using an antibody against Dsg2 N-term of cell culture supernatants of T-84s treated as in Fig. 3a. c Western blot using an antibody against Dsg2 C-term of cell lysates of T-84s treated with TNF-α and IFN-γ in the presence or absence of increasing concentrations of either Z-DEVD-fmk, Z-IETD-fmk, or Z-LEHD-fmk for 24 h. d Cos7 cells were transfected with expression plasmids encoding either WT Dsg2 [WT], or Dsg2 containing one of two putative caspase-8 cleavage consensus site mutations (D715A [715] or D675A [675]). Cells treated with Lipofectamine 3000 reagents alone with no DNA were used as controls [N/A]. Top. Western blot against the Myc tag (Top = Dsg2 full-length band; Middle = Dsg2 ICF band; Bottom = Tubulin). Bottom. Model of WT Dsg2, D715A, and D675A depicting region of Dsg2 containing sequences mutated in each highlighting the mutated residues in red. All blots are representative of at least three independent experiments

    Article Snippet: Recombinant human TNF-α, IFN-γ, and TRAIL were purchased from PeproTech (Rocky Hill, NJ, USA).

    Techniques: Western Blot, Cell Culture, Transfection, Expressing

    Effects of gelatin hydrogel on pro-inflammatory cytokine. (A) Immunohistochemical staining of IL-1β around the lesion. (B) The area percentage of IL-1β. (C) Immunohistochemical staining of TNF-α around the lesion. (D) The area percentage of TNF-α. Scale bar = 50 μm. *Represents the hydrogel. Data are mean ± SD, n = 4 mice per group, *** P

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Injectable Gelatin Hydrogel Suppresses Inflammation and Enhances Functional Recovery in a Mouse Model of Intracerebral Hemorrhage

    doi: 10.3389/fbioe.2020.00785

    Figure Lengend Snippet: Effects of gelatin hydrogel on pro-inflammatory cytokine. (A) Immunohistochemical staining of IL-1β around the lesion. (B) The area percentage of IL-1β. (C) Immunohistochemical staining of TNF-α around the lesion. (D) The area percentage of TNF-α. Scale bar = 50 μm. *Represents the hydrogel. Data are mean ± SD, n = 4 mice per group, *** P

    Article Snippet: Rabbit anti-IL-1β and TNF-α were obtained from Santa Cruz Biotechnology (United States).

    Techniques: Immunohistochemistry, Staining, Mouse Assay

    Effect of miR-4717 mimics or miR-4717 inhibitor on the production of tumor necrosis factor (TNF)-α and interferon (IFN)-γ by lymphocytes from chronic HBV patients with rs10204525 genotypes AA and GG ( A ) TNF-α. ( B ) IFN-γ. The values were log transformed to obtain normal distribution and then analyzed by using ANOVA and Post Hoc test.

    Journal: Oncotarget

    Article Title: microRNA-4717 differentially interacts with its polymorphic target in the PD1 3′ untranslated region: A mechanism for regulating PD-1 expression and function in HBV-associated liver diseases

    doi:

    Figure Lengend Snippet: Effect of miR-4717 mimics or miR-4717 inhibitor on the production of tumor necrosis factor (TNF)-α and interferon (IFN)-γ by lymphocytes from chronic HBV patients with rs10204525 genotypes AA and GG ( A ) TNF-α. ( B ) IFN-γ. The values were log transformed to obtain normal distribution and then analyzed by using ANOVA and Post Hoc test.

    Article Snippet: Determination of TNF-α and IFN-γ Levels of TNF-α and IFN-γ were determined using commercially available Human TNF-α and IFN-γ ELISA kits (R & D Systems, Inc., Minneapolis, MN, USA), respectively.

    Techniques: Transformation Assay

    Apoptosis, ROS, and TNF-α production are reduced in silica exposed BMMC cultured from SR-AI/II KO, MARCO KO, or SR-A/MARCO double KO mice. ( A ) DNA fragmentation was measured in BMMC obtained from scavenger receptor KO mice and exposed to 50 μg/cm

    Journal:

    Article Title: Silica-Directed Mast Cell Activation Is Enhanced by Scavenger Receptors

    doi: 10.1165/rcmb.2006-0197OC

    Figure Lengend Snippet: Apoptosis, ROS, and TNF-α production are reduced in silica exposed BMMC cultured from SR-AI/II KO, MARCO KO, or SR-A/MARCO double KO mice. ( A ) DNA fragmentation was measured in BMMC obtained from scavenger receptor KO mice and exposed to 50 μg/cm

    Article Snippet: Mouse TNF-α, IL-13, and monocyte chemotactic protein (MCP)-1 (CCL2) were measured using Quantikine ELISA kits (R & D Systems, Minneapolis, MN).

    Techniques: Cell Culture, Mouse Assay

    Treatment of silica exposed BMMC with Zileuton and Trolox reduces ROS formation and treatment with dexamethasone inhibits silica-directed TNF-α production. ( A ) Zileuton, a 5-lipoxygenase inhibitor, was added to BMMC at concentrations of 2, 10,

    Journal:

    Article Title: Silica-Directed Mast Cell Activation Is Enhanced by Scavenger Receptors

    doi: 10.1165/rcmb.2006-0197OC

    Figure Lengend Snippet: Treatment of silica exposed BMMC with Zileuton and Trolox reduces ROS formation and treatment with dexamethasone inhibits silica-directed TNF-α production. ( A ) Zileuton, a 5-lipoxygenase inhibitor, was added to BMMC at concentrations of 2, 10,

    Article Snippet: Mouse TNF-α, IL-13, and monocyte chemotactic protein (MCP)-1 (CCL2) were measured using Quantikine ELISA kits (R & D Systems, Minneapolis, MN).

    Techniques:

    Silica directs TNF-α, IL-13, and MCP-1 production and enhances FcεRI-mediated production of these mediators in BMMC. BMMC (2 × 10 5 /well) were exposed to 0, 6.25, 12.5, 25, or 50 μg/cm 2 silica for 24 h before the measurement

    Journal:

    Article Title: Silica-Directed Mast Cell Activation Is Enhanced by Scavenger Receptors

    doi: 10.1165/rcmb.2006-0197OC

    Figure Lengend Snippet: Silica directs TNF-α, IL-13, and MCP-1 production and enhances FcεRI-mediated production of these mediators in BMMC. BMMC (2 × 10 5 /well) were exposed to 0, 6.25, 12.5, 25, or 50 μg/cm 2 silica for 24 h before the measurement

    Article Snippet: Mouse TNF-α, IL-13, and monocyte chemotactic protein (MCP)-1 (CCL2) were measured using Quantikine ELISA kits (R & D Systems, Minneapolis, MN).

    Techniques:

    NOX2 activation leads to a surge of proinflammatory mediators in NAFLD-Kidney A. mRNA expression analysis of IL-1β, IFN-γ, TNF-α, CD4, and CD8 genes in kidney tissue of NAFLD, NAFLD + BDCM, and P47phox KO mice fed with high-fat diet. All mRNA expression had been assessed by quantitative real-time PCR (qRTPCR) and expressions were normalized against NAFLD group (*P

    Journal: Redox Biology

    Article Title: High circulatory leptin mediated NOX-2-peroxynitrite-miR21 axis activate mesangial cells and promotes renal inflammatory pathology in nonalcoholic fatty liver disease

    doi: 10.1016/j.redox.2018.04.002

    Figure Lengend Snippet: NOX2 activation leads to a surge of proinflammatory mediators in NAFLD-Kidney A. mRNA expression analysis of IL-1β, IFN-γ, TNF-α, CD4, and CD8 genes in kidney tissue of NAFLD, NAFLD + BDCM, and P47phox KO mice fed with high-fat diet. All mRNA expression had been assessed by quantitative real-time PCR (qRTPCR) and expressions were normalized against NAFLD group (*P

    Article Snippet: Anti–α-SMA, anti–IL-1β, anti–TNF-α, anti-TLR-4, Anti-3-Nitrotyrosine (3-NT), anti-P47phox and anti-GP91phox primary antibodies were purchased from Abcam (Cambridge, MA).

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

    Induction of ‘bystander’ senescence in B16 tumour cells. (A) Senescence-associated β-galactosidase activity in B16 cells cultured for 4 days in the medium from DTX-treated cells (SM), IFNγ + TNFα-treated cells or proliferating cell medium (TM). (B) The size and granularity of control and senescent B16 cells was determined by forward and side scatter flow cytometry analysis. (C) Expression of p21 in B16 cells cultured for 4 days in different media (reverse transcription-quantitative polymerase chain reaction). (D) Immunoblotting detection of mouse p21 in B16 cells harvested on day 4 after cultivation in different media. GAPDH was used as a loading control. Data are presented as the mean ± standard deviation. ** P

    Journal: International Journal of Oncology

    Article Title: Distinct phenotypes and ‘bystander’ effects of senescent tumour cells induced by docetaxel or immunomodulatory cytokines

    doi: 10.3892/ijo.2018.4553

    Figure Lengend Snippet: Induction of ‘bystander’ senescence in B16 tumour cells. (A) Senescence-associated β-galactosidase activity in B16 cells cultured for 4 days in the medium from DTX-treated cells (SM), IFNγ + TNFα-treated cells or proliferating cell medium (TM). (B) The size and granularity of control and senescent B16 cells was determined by forward and side scatter flow cytometry analysis. (C) Expression of p21 in B16 cells cultured for 4 days in different media (reverse transcription-quantitative polymerase chain reaction). (D) Immunoblotting detection of mouse p21 in B16 cells harvested on day 4 after cultivation in different media. GAPDH was used as a loading control. Data are presented as the mean ± standard deviation. ** P

    Article Snippet: Induction of ‘primary’ premature senescence TC-1 and B16 cells were cultured in fresh RPMI-1640 medium for 24 h, following which the medium was removed and replaced with medium containing either recombinant IFNγ (50 U/ml; R & D Systems, Inc., Minneapolis, MN, USA) and TNFα (5 ng/ml; PeproTech, Inc., Rocky Hill, NJ, USA) or 7.5 µ M DTX (Actavis Generics, Dublin, Ireland).

    Techniques: Activity Assay, Cell Culture, Flow Cytometry, Cytometry, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    DTX induces senescence in TC-1 cells. (A) Senescence-associated β-galactosidase activity in TC-1 cells treated with DTX or IFNγ + TNFα for 4 days. (B) The size and granularity of control or IFNγ + TNFα-treated senescent TC-1 cells was determined by forward and side scatter flow cytometry analysis. (C) Autofluorescence of the TC-1 control cells is presented in light grey, DTX-treated in black and IFNγ + TNFα-treated in grey. (D) Reverse transcription-quantitative polymerase chain reaction quantification of p21 in control, DTX- and IFNγ + TNFα-treated TC-1 cells. (E) Immunoblotting detection of mouse p21 in control, DTX- and IFNγ + TNFα-treated TC-1 cells harvested on day 4. GAPDH was used as a loading control. Representative results from at least three independent experiments are presented. Data are presented as the mean ± standard deviation. CTRL, control cells; DTX, docetaxel; IFNγ, interferon γ; TNFα, tumour necrosis factor α; FSC, forward scattering; SSC, side scattering; p21, p21 Waf1 .

    Journal: International Journal of Oncology

    Article Title: Distinct phenotypes and ‘bystander’ effects of senescent tumour cells induced by docetaxel or immunomodulatory cytokines

    doi: 10.3892/ijo.2018.4553

    Figure Lengend Snippet: DTX induces senescence in TC-1 cells. (A) Senescence-associated β-galactosidase activity in TC-1 cells treated with DTX or IFNγ + TNFα for 4 days. (B) The size and granularity of control or IFNγ + TNFα-treated senescent TC-1 cells was determined by forward and side scatter flow cytometry analysis. (C) Autofluorescence of the TC-1 control cells is presented in light grey, DTX-treated in black and IFNγ + TNFα-treated in grey. (D) Reverse transcription-quantitative polymerase chain reaction quantification of p21 in control, DTX- and IFNγ + TNFα-treated TC-1 cells. (E) Immunoblotting detection of mouse p21 in control, DTX- and IFNγ + TNFα-treated TC-1 cells harvested on day 4. GAPDH was used as a loading control. Representative results from at least three independent experiments are presented. Data are presented as the mean ± standard deviation. CTRL, control cells; DTX, docetaxel; IFNγ, interferon γ; TNFα, tumour necrosis factor α; FSC, forward scattering; SSC, side scattering; p21, p21 Waf1 .

    Article Snippet: Induction of ‘primary’ premature senescence TC-1 and B16 cells were cultured in fresh RPMI-1640 medium for 24 h, following which the medium was removed and replaced with medium containing either recombinant IFNγ (50 U/ml; R & D Systems, Inc., Minneapolis, MN, USA) and TNFα (5 ng/ml; PeproTech, Inc., Rocky Hill, NJ, USA) or 7.5 µ M DTX (Actavis Generics, Dublin, Ireland).

    Techniques: Activity Assay, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Standard Deviation

    DNA damage detection in TC-1 and B16 tumour cell lines. To detect DNA damage, control, DTX- or IFNγ + TNFα-treated (A) TC-1 and (B) B16 cells were stained with phosphoSer139 H2A histone family, member X antibody and mounted with Mowiol containing 4’,6-diamidine-2-phenylindole. Scale bar, 20 µ m. Percentage of cells with 1, 2, 3 or more micronuclei in (C) TC-1 and (D) B16 cells treated with DTX or IFNγ + TNFα was quantified. Data are presented as the mean ± standard deviation. * P

    Journal: International Journal of Oncology

    Article Title: Distinct phenotypes and ‘bystander’ effects of senescent tumour cells induced by docetaxel or immunomodulatory cytokines

    doi: 10.3892/ijo.2018.4553

    Figure Lengend Snippet: DNA damage detection in TC-1 and B16 tumour cell lines. To detect DNA damage, control, DTX- or IFNγ + TNFα-treated (A) TC-1 and (B) B16 cells were stained with phosphoSer139 H2A histone family, member X antibody and mounted with Mowiol containing 4’,6-diamidine-2-phenylindole. Scale bar, 20 µ m. Percentage of cells with 1, 2, 3 or more micronuclei in (C) TC-1 and (D) B16 cells treated with DTX or IFNγ + TNFα was quantified. Data are presented as the mean ± standard deviation. * P

    Article Snippet: Induction of ‘primary’ premature senescence TC-1 and B16 cells were cultured in fresh RPMI-1640 medium for 24 h, following which the medium was removed and replaced with medium containing either recombinant IFNγ (50 U/ml; R & D Systems, Inc., Minneapolis, MN, USA) and TNFα (5 ng/ml; PeproTech, Inc., Rocky Hill, NJ, USA) or 7.5 µ M DTX (Actavis Generics, Dublin, Ireland).

    Techniques: Staining, Standard Deviation

    Secretion of IL-6 and GROα by murine TC-1 and B16 tumour cell lines. Enzyme-linked immunosorbent assay of IL-6 and GROα in supernatants of (A) TC-1 and (B) B16 cells treated with DTX and IFNγ + TNFα. Supernatants were tested in triplicate and the results from three independent experiments are presented as the mean ± standard deviation. *** P

    Journal: International Journal of Oncology

    Article Title: Distinct phenotypes and ‘bystander’ effects of senescent tumour cells induced by docetaxel or immunomodulatory cytokines

    doi: 10.3892/ijo.2018.4553

    Figure Lengend Snippet: Secretion of IL-6 and GROα by murine TC-1 and B16 tumour cell lines. Enzyme-linked immunosorbent assay of IL-6 and GROα in supernatants of (A) TC-1 and (B) B16 cells treated with DTX and IFNγ + TNFα. Supernatants were tested in triplicate and the results from three independent experiments are presented as the mean ± standard deviation. *** P

    Article Snippet: Induction of ‘primary’ premature senescence TC-1 and B16 cells were cultured in fresh RPMI-1640 medium for 24 h, following which the medium was removed and replaced with medium containing either recombinant IFNγ (50 U/ml; R & D Systems, Inc., Minneapolis, MN, USA) and TNFα (5 ng/ml; PeproTech, Inc., Rocky Hill, NJ, USA) or 7.5 µ M DTX (Actavis Generics, Dublin, Ireland).

    Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation

    DTX induces senescence in the B16 cell line. (A) Senescence-associated β-galactosidase activity in B16 cells treated with DTX or IFNγ + TNFα for 4 days. (B) The size and granularity of control or IFNγ + TNFα-treated, senescent B16 cells was determined by forward and side scatter flow cytometry analysis. (C) Autofluorescence of the B16 control cells is presented in light grey, DTX-treated in black and IFNγ + TNFα-treated in grey. (D) Reverse transcription-quantitative polymerase chain reaction quantification of p21 in control, DTX- and IFNγ + TNFα-treated B16 cells. (E) Immunoblotting detection of mouse p21 in control, DTX- and IFNγ + TNFα-treated B16 cells harvested on day 4. GAPDH was used as a loading control. Representative results from at least three independent experiments are presented. Data are presented as the mean ± standard deviation. ** P

    Journal: International Journal of Oncology

    Article Title: Distinct phenotypes and ‘bystander’ effects of senescent tumour cells induced by docetaxel or immunomodulatory cytokines

    doi: 10.3892/ijo.2018.4553

    Figure Lengend Snippet: DTX induces senescence in the B16 cell line. (A) Senescence-associated β-galactosidase activity in B16 cells treated with DTX or IFNγ + TNFα for 4 days. (B) The size and granularity of control or IFNγ + TNFα-treated, senescent B16 cells was determined by forward and side scatter flow cytometry analysis. (C) Autofluorescence of the B16 control cells is presented in light grey, DTX-treated in black and IFNγ + TNFα-treated in grey. (D) Reverse transcription-quantitative polymerase chain reaction quantification of p21 in control, DTX- and IFNγ + TNFα-treated B16 cells. (E) Immunoblotting detection of mouse p21 in control, DTX- and IFNγ + TNFα-treated B16 cells harvested on day 4. GAPDH was used as a loading control. Representative results from at least three independent experiments are presented. Data are presented as the mean ± standard deviation. ** P

    Article Snippet: Induction of ‘primary’ premature senescence TC-1 and B16 cells were cultured in fresh RPMI-1640 medium for 24 h, following which the medium was removed and replaced with medium containing either recombinant IFNγ (50 U/ml; R & D Systems, Inc., Minneapolis, MN, USA) and TNFα (5 ng/ml; PeproTech, Inc., Rocky Hill, NJ, USA) or 7.5 µ M DTX (Actavis Generics, Dublin, Ireland).

    Techniques: Activity Assay, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Standard Deviation

    Analysis of TC-1 and B16 cell proliferation during ‘primary’ induction. (A) TC-1 and (B) B16 cells were treated with DTX and IFNγ + TNFα. Cell proliferation was determined by counting the cell number on days 4 (TC-1 and B16) and 7 (B16 only). Control cells were passaged on day 4. Data are presented as the mean ± standard deviation. *** P

    Journal: International Journal of Oncology

    Article Title: Distinct phenotypes and ‘bystander’ effects of senescent tumour cells induced by docetaxel or immunomodulatory cytokines

    doi: 10.3892/ijo.2018.4553

    Figure Lengend Snippet: Analysis of TC-1 and B16 cell proliferation during ‘primary’ induction. (A) TC-1 and (B) B16 cells were treated with DTX and IFNγ + TNFα. Cell proliferation was determined by counting the cell number on days 4 (TC-1 and B16) and 7 (B16 only). Control cells were passaged on day 4. Data are presented as the mean ± standard deviation. *** P

    Article Snippet: Induction of ‘primary’ premature senescence TC-1 and B16 cells were cultured in fresh RPMI-1640 medium for 24 h, following which the medium was removed and replaced with medium containing either recombinant IFNγ (50 U/ml; R & D Systems, Inc., Minneapolis, MN, USA) and TNFα (5 ng/ml; PeproTech, Inc., Rocky Hill, NJ, USA) or 7.5 µ M DTX (Actavis Generics, Dublin, Ireland).

    Techniques: Standard Deviation

    Effects of Banhasasim-tang (BHSST) on production of chemokines in HaCaT cells. Production of RANTES (a), TARC (b), MDC (c), and IL-8 (d) were measured using the culture supernatant. Cells were cotreated with BHSST (125, 250, or 500 μ g/mL), and TNF- α and IFN- γ (TI, each 10 ng/mL) for 24 h. Silymarin (6.25, 12.5, or 25 μ g/mL) was used as a positive control. Values are expressed as mean ± SEM of three independent experiments. ## P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Traditional Herbal Formula Banhasasim-tang Exerts Anti-Inflammatory Effects in RAW 264.7 Macrophages and HaCaT Keratinocytes

    doi: 10.1155/2015/728380

    Figure Lengend Snippet: Effects of Banhasasim-tang (BHSST) on production of chemokines in HaCaT cells. Production of RANTES (a), TARC (b), MDC (c), and IL-8 (d) were measured using the culture supernatant. Cells were cotreated with BHSST (125, 250, or 500 μ g/mL), and TNF- α and IFN- γ (TI, each 10 ng/mL) for 24 h. Silymarin (6.25, 12.5, or 25 μ g/mL) was used as a positive control. Values are expressed as mean ± SEM of three independent experiments. ## P

    Article Snippet: Mouse TNF-α and IL-6 ELISA kits and Trizol reagent were obtained from Invitrogen (Camarillo, CA, USA).

    Techniques: Positive Control

    Effect of Banhasasim-tang (BHSST) on TNF- α and IFN- γ -induced STAT1 activation in HaCaT cells. (a) Expression of total and phosphorylated STAT1 was determined by Western blotting. Cells were cotreated with BHSST (125, 250, or 500 μ g/mL), and TNF- α and IFN- γ (TI, each 10 ng/mL) for 30 min. Silymarin (12.5 or 25 μ g/mL) was used as a positive control. Results are representative of at least three independent experiments. (b) Cellular localization of STAT1 was examined by immunofluorescence staining. Cells were cotreated with BHSST (500 μ g/mL), and TI for 30 min. The cells were fixed with 4% (v/v) methanol-free formaldehyde solution (pH 7.4), stained with anti-STAT1 (red). The stained cells were mounted with medium containing DAPI (blue) and visualized under an Olympus FLUOVIEW FV 10i confocal microscope.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Traditional Herbal Formula Banhasasim-tang Exerts Anti-Inflammatory Effects in RAW 264.7 Macrophages and HaCaT Keratinocytes

    doi: 10.1155/2015/728380

    Figure Lengend Snippet: Effect of Banhasasim-tang (BHSST) on TNF- α and IFN- γ -induced STAT1 activation in HaCaT cells. (a) Expression of total and phosphorylated STAT1 was determined by Western blotting. Cells were cotreated with BHSST (125, 250, or 500 μ g/mL), and TNF- α and IFN- γ (TI, each 10 ng/mL) for 30 min. Silymarin (12.5 or 25 μ g/mL) was used as a positive control. Results are representative of at least three independent experiments. (b) Cellular localization of STAT1 was examined by immunofluorescence staining. Cells were cotreated with BHSST (500 μ g/mL), and TI for 30 min. The cells were fixed with 4% (v/v) methanol-free formaldehyde solution (pH 7.4), stained with anti-STAT1 (red). The stained cells were mounted with medium containing DAPI (blue) and visualized under an Olympus FLUOVIEW FV 10i confocal microscope.

    Article Snippet: Mouse TNF-α and IL-6 ELISA kits and Trizol reagent were obtained from Invitrogen (Camarillo, CA, USA).

    Techniques: Activation Assay, Expressing, Western Blot, Positive Control, Immunofluorescence, Staining, Microscopy

    Cytotoxic effects of Banhasasim-tang (BHSST) in RAW 264.7 cells and HaCaT cells. RAW 264.7 cells (a) and HaCaT cells (c) were seeded in to 96-well plates and treated with various concentrations of BHSST for 24 h. (b) RAW 264.7 cells were cotreated with various concentrations of BHSST, and LPS (1 μ g/mL) for 24 h. (d) HaCaT cells were cotreated with various concentrations of BHSST, and TNF- α and IFN- γ (each 10 ng/mL, TI) for 24 h. Cell viability was assessed using a CCK-8 kit. The values are expressed as mean ± SEM of three independent experiments.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Traditional Herbal Formula Banhasasim-tang Exerts Anti-Inflammatory Effects in RAW 264.7 Macrophages and HaCaT Keratinocytes

    doi: 10.1155/2015/728380

    Figure Lengend Snippet: Cytotoxic effects of Banhasasim-tang (BHSST) in RAW 264.7 cells and HaCaT cells. RAW 264.7 cells (a) and HaCaT cells (c) were seeded in to 96-well plates and treated with various concentrations of BHSST for 24 h. (b) RAW 264.7 cells were cotreated with various concentrations of BHSST, and LPS (1 μ g/mL) for 24 h. (d) HaCaT cells were cotreated with various concentrations of BHSST, and TNF- α and IFN- γ (each 10 ng/mL, TI) for 24 h. Cell viability was assessed using a CCK-8 kit. The values are expressed as mean ± SEM of three independent experiments.

    Article Snippet: Mouse TNF-α and IL-6 ELISA kits and Trizol reagent were obtained from Invitrogen (Camarillo, CA, USA).

    Techniques: CCK-8 Assay

    Influence of TNF-α on the activity of glutamate transporters from wild-type or hSOD1 G93A cortical astrocytes. Glutamate transporter activity was evaluated by measuring velocity of d -[ 3 H]-aspartate uptake (50 nmol/L) in wild-type and hSOD1 G93A astrocytes treated or not with TNF-α (20 ng/mL) for 72 h. (A) shows total uptake while (B) and (C) illustrate GLT-1- and GLAST-dependent uptake, respectively evaluated in presence of the selective inhibitors WAY-213613 (100 µmol/L) and UCPH-101 (10 µmol/L). Shown are mean with SEM from five independent experiments realized in quintuplicate. # p

    Journal: PLoS ONE

    Article Title: Differential Regulation of Glutamate Transporter Subtypes by Pro-Inflammatory Cytokine TNF-? in Cortical Astrocytes from a Rat Model of Amyotrophic Lateral Sclerosis

    doi: 10.1371/journal.pone.0097649

    Figure Lengend Snippet: Influence of TNF-α on the activity of glutamate transporters from wild-type or hSOD1 G93A cortical astrocytes. Glutamate transporter activity was evaluated by measuring velocity of d -[ 3 H]-aspartate uptake (50 nmol/L) in wild-type and hSOD1 G93A astrocytes treated or not with TNF-α (20 ng/mL) for 72 h. (A) shows total uptake while (B) and (C) illustrate GLT-1- and GLAST-dependent uptake, respectively evaluated in presence of the selective inhibitors WAY-213613 (100 µmol/L) and UCPH-101 (10 µmol/L). Shown are mean with SEM from five independent experiments realized in quintuplicate. # p

    Article Snippet: Then, astrocyte treatment with TNF-α (20 ng/mL, AbD Serotec, Oxford, England) in the same medium was commenced and lasted 72 h; alternatively, astrocytes were incubated for 48 h with cycloheximide (10 µg/mL, Sigma Aldrich), supplemented or not with 20 ng/mL TNF-α.

    Techniques: Activity Assay

    Influence of TNF-α on GLAST, GLT-1a and GLT-1b mRNAs and proteins in cortical astrocytes from wild-type or hSOD1 G93A rats. Expression of GLAST (A), GLT-1a (C) and GLT-1b (E) mRNAs as number of copies per microgram of RNA was estimated by RT-qPCR in control conditions or after 72 h exposure to TNF-α (20 ng/mL) using the corresponding cloned cDNA sequences as standards. Data shown are means with SEM conducted from six independent experiments performed in duplicate. Expression of GLAST (B), GLT-1a (D) and GLT-1b (F) proteins was examined in cells maintained in control conditions or treated with TNF-α (20 ng/mL) for 72 h. Immunoblots shown are representative of six independent experiments. Data indicate the levels of the protein of interest normalized to GAPDH and represents means with SEM. * p

    Journal: PLoS ONE

    Article Title: Differential Regulation of Glutamate Transporter Subtypes by Pro-Inflammatory Cytokine TNF-? in Cortical Astrocytes from a Rat Model of Amyotrophic Lateral Sclerosis

    doi: 10.1371/journal.pone.0097649

    Figure Lengend Snippet: Influence of TNF-α on GLAST, GLT-1a and GLT-1b mRNAs and proteins in cortical astrocytes from wild-type or hSOD1 G93A rats. Expression of GLAST (A), GLT-1a (C) and GLT-1b (E) mRNAs as number of copies per microgram of RNA was estimated by RT-qPCR in control conditions or after 72 h exposure to TNF-α (20 ng/mL) using the corresponding cloned cDNA sequences as standards. Data shown are means with SEM conducted from six independent experiments performed in duplicate. Expression of GLAST (B), GLT-1a (D) and GLT-1b (F) proteins was examined in cells maintained in control conditions or treated with TNF-α (20 ng/mL) for 72 h. Immunoblots shown are representative of six independent experiments. Data indicate the levels of the protein of interest normalized to GAPDH and represents means with SEM. * p

    Article Snippet: Then, astrocyte treatment with TNF-α (20 ng/mL, AbD Serotec, Oxford, England) in the same medium was commenced and lasted 72 h; alternatively, astrocytes were incubated for 48 h with cycloheximide (10 µg/mL, Sigma Aldrich), supplemented or not with 20 ng/mL TNF-α.

    Techniques: Expressing, Quantitative RT-PCR, Clone Assay, Western Blot

    Effect of protein synthesis inhibition on the protein expression of GLAST, GLT-1a and GLT-1b on wild-type or hSOD1 G93A astrocytes. (A) Protein expression of GLAST, GLT-1a and GLT-1b was examined by immunoblotting in astrocytes from wild-type and hSOD1 G93A maintained in culture either in control conditions or treated with TNF-α (20 ng/mL) and/or using the inhibitor of protein synthesis cycloheximide (10 µg/mL) for 48 h. Immunoblots shown are representative of four independent experiments. Data obtained after densitometric analyses of GLAST (B), GLT-1a (C) and GLT-1b (D) proteins are means with SEM normalized to GAPDH and expressed in percent of the signal obtained for wild-type astrocytes cultured in control conditions. * p

    Journal: PLoS ONE

    Article Title: Differential Regulation of Glutamate Transporter Subtypes by Pro-Inflammatory Cytokine TNF-? in Cortical Astrocytes from a Rat Model of Amyotrophic Lateral Sclerosis

    doi: 10.1371/journal.pone.0097649

    Figure Lengend Snippet: Effect of protein synthesis inhibition on the protein expression of GLAST, GLT-1a and GLT-1b on wild-type or hSOD1 G93A astrocytes. (A) Protein expression of GLAST, GLT-1a and GLT-1b was examined by immunoblotting in astrocytes from wild-type and hSOD1 G93A maintained in culture either in control conditions or treated with TNF-α (20 ng/mL) and/or using the inhibitor of protein synthesis cycloheximide (10 µg/mL) for 48 h. Immunoblots shown are representative of four independent experiments. Data obtained after densitometric analyses of GLAST (B), GLT-1a (C) and GLT-1b (D) proteins are means with SEM normalized to GAPDH and expressed in percent of the signal obtained for wild-type astrocytes cultured in control conditions. * p

    Article Snippet: Then, astrocyte treatment with TNF-α (20 ng/mL, AbD Serotec, Oxford, England) in the same medium was commenced and lasted 72 h; alternatively, astrocytes were incubated for 48 h with cycloheximide (10 µg/mL, Sigma Aldrich), supplemented or not with 20 ng/mL TNF-α.

    Techniques: Inhibition, Expressing, Western Blot, Cell Culture

    TM5441 pre-treatment and siPAI-1 improved TNF-α-induced mitochondrial dysfunction in HepG2 cells HepG2 cells were pretreated with 20 μM TM5441 before the exposure of 50 ng/ml TNF-α for 24 hours. mRNA expressions of ( A ) PAI-1 and ( B) mitochondria biogenesis-related genes, such as PGC-1α, mtDNA, TFAM, NRF1, and NRF2 were measured by RT-qPCR. Then, PAI-1 is downregulated with siRNA in which confirmed by immunoblot analysis. ( C ) 20 nM siPAI-1 was transfected 24 hours before the exposure of 50 ng/ml TNF-α for 24 hour. The mRNA gene expression of ( D ) PAI-1 and ( E ) the mitochondrial function-related genes were measured by RT-qPCR. Data are presented as mean±SE of 4-5 experiments. * p

    Journal: Oncotarget

    Article Title: TM5441, a plasminogen activator inhibitor-1 inhibitor, protects against high fat diet-induced non-alcoholic fatty liver disease

    doi: 10.18632/oncotarget.21120

    Figure Lengend Snippet: TM5441 pre-treatment and siPAI-1 improved TNF-α-induced mitochondrial dysfunction in HepG2 cells HepG2 cells were pretreated with 20 μM TM5441 before the exposure of 50 ng/ml TNF-α for 24 hours. mRNA expressions of ( A ) PAI-1 and ( B) mitochondria biogenesis-related genes, such as PGC-1α, mtDNA, TFAM, NRF1, and NRF2 were measured by RT-qPCR. Then, PAI-1 is downregulated with siRNA in which confirmed by immunoblot analysis. ( C ) 20 nM siPAI-1 was transfected 24 hours before the exposure of 50 ng/ml TNF-α for 24 hour. The mRNA gene expression of ( D ) PAI-1 and ( E ) the mitochondrial function-related genes were measured by RT-qPCR. Data are presented as mean±SE of 4-5 experiments. * p

    Article Snippet: Growth arrested and synchronized cells were exposed with 50 ng/mL tumor necrosis factor-α/TNF-α (Sigma-Aldrich, St. Louis, MO, USA) or 25 and 50 nM mouse recombinant PAI-1 (EMD Millipore, Billerica, MA, USA) in the presence and absence of 20 μM TM5441.

    Techniques: Pyrolysis Gas Chromatography, Quantitative RT-PCR, Transfection, Expressing

    Early TM5441 treatment suppressed hepatic inflammation in HFD mice ( A , B ) Anti-inflammatory effect of TM5441 was analyzed through quantifying positive F4/80 IHC staining area in the liver section. Magnification, 200×; scale bar, 50 μm. Pro-inflammatory mRNA expressions in the liver, such as ( C ) F4/80, ( D ) MCP-1 ( E ) TNF-α, ( F ) NLRP3, were measured by RT-qPCR. Data are shown as mean ± SE of 7–8 mice. * p

    Journal: Oncotarget

    Article Title: TM5441, a plasminogen activator inhibitor-1 inhibitor, protects against high fat diet-induced non-alcoholic fatty liver disease

    doi: 10.18632/oncotarget.21120

    Figure Lengend Snippet: Early TM5441 treatment suppressed hepatic inflammation in HFD mice ( A , B ) Anti-inflammatory effect of TM5441 was analyzed through quantifying positive F4/80 IHC staining area in the liver section. Magnification, 200×; scale bar, 50 μm. Pro-inflammatory mRNA expressions in the liver, such as ( C ) F4/80, ( D ) MCP-1 ( E ) TNF-α, ( F ) NLRP3, were measured by RT-qPCR. Data are shown as mean ± SE of 7–8 mice. * p

    Article Snippet: Growth arrested and synchronized cells were exposed with 50 ng/mL tumor necrosis factor-α/TNF-α (Sigma-Aldrich, St. Louis, MO, USA) or 25 and 50 nM mouse recombinant PAI-1 (EMD Millipore, Billerica, MA, USA) in the presence and absence of 20 μM TM5441.

    Techniques: Mouse Assay, Immunohistochemistry, Staining, Quantitative RT-PCR