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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 22042 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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    Thermo Fisher gene exp tnf mm00443258 m1
    Analysis of proinflammatory (M1) mφ markers in Hoxa3 mCh - or mCherry-treated BMDMs from non-db (ndb) and db mice. ( A ) Production of NO (as measured by nitrate as the final product in the Griess assay) in NA or CA mφs from ndb or db mice and treated with mCherry (□) or with Hoxa3 mCh (▪). ( B ) IL-12 release from NA or CA mφs from ndb or db mice, treated with mCherry (□) or Hoxa3 mCh (▪). ( C and D ) <t>TNF</t> release from NA or CA mφs from ndb and db mice, respectively, treated with mCherry (□) or with Hoxa3 mCh (▪). ( E – H ) Relative expression (RE) of Nos2 , Tnf , Cd86 , and Ccl2 to Hsp90 (reference gene, reference, n = 5; * p
    Gene Exp Tnf Mm00443258 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3964 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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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    95
    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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    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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    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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    99
    Thermo Fisher gene exp tnf hs00174128 m1
    <t>TNF-α-mediated</t> reactivation is correlated with activation of NF-κB and ATM signaling. Latently infected cells (14 dpi) were treated for 3 days with or without TNF-α at 5 ng/ml. (A to D) Flow cytometry analysis was performed to assess the activation of NF-κB (p65) and DDR (ATM, KAP-1, and γ-H2AX). The activation of p65, ATM, and KAP-1 was expressed as the ratio of phosphoprotein to total protein in comparison with untreated cells (p-p65-S536/p65, pATM-S1981/ATM, and p-Kap-S824/Kap1). γ-H2AX is expressed as the ratio of mean fluorescence intensity compared to untreated cells. The error bars represent the standard errors of the means calculated from 3 independent experiments (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).
    Gene Exp Tnf Hs00174128 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1230 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 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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    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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    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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    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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    Thermo Fisher tnf
    RIPK3 activates caspase-1 independent of MLKL unless caspase-8 is inhibited. ( a – c ) WT, Mlkl −/− and Ripk3 −/− BMDM were primed with LPS (20 ng ml −1 ) for 3 h and cultured with Q-VD-OPh (20 μM), where indicated, which was added in the last 20 min of priming. Cells were then stimulated with Cp.A (500 nM) or alum (300 μg ml −1 ) for a further 6 h. Supernatants were analyzed for ( a , c ) IL-1β and ( b ) <t>TNF</t> by <t>ELISA.</t> n =3 mice per genotype. Data are represented as mean+s.e.m. and are representative of one of three independent experiments. ( d ) WT, Mlkl −/− and Ripk3 −/− BMDM were primed with LPS for 2.5 h. In the last 20 min of priming, cells were incubated with Q-VD-OPh (20 μM) and then cultured with Cp.A (1 μM) for 5 h. Cell supernatants and lysates were analyzed by immunoblot. Representative of one of three experiments. Full-size immunoblots are presented in Supplementary Fig. 11 . ( e ) Schematic depicting how RIPK3 signals IL-1β activation based on the data presented in Figs 1 , 2 , 3 . ( f ) Lysates from WT ( Casp8 fl/fl ) littermate and caspase-8-deficient ( Casp8 LysMcre ) BMDM ( n =2 mice) were subjected to immunoblot to assess efficiency of caspase-8 deletion. Full-size immunoblots are presented in Supplementary Fig. 11 . ( g ) WT littermate and Caspase-8 LysMcre BMDM were primed for 3 h with Pam 3 Cys (2 μg ml −1 ), and as indicated treated with Nec-1 (50 μM) in the last 20 min of priming. Cells were then exposed to Cp.A (500 nM), as specified, for a further 24 h, after which IL-1β release was measured by ELISA. n =3 mice per genotype, mean+s.e.m. Representative of one of three experiments. ( h ) WT littermate and Caspase-8 LysMcre BMDM were pre-incubated with glyburide for 20 min, as indicated, and cultured with Pam 3 Cys (2 μg ml −1 ) or LPS (100 ng ml −1 ) for 24 h. Cell supernatants were assayed for IL-1β by ELISA. n =4 mice per genotype, mean+s.e.m. Representative of one of two experiments.
    Tnf, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1363 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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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    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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    Thermo Fisher gene exp tnf mm00443260 g1
    Kidney <t>TNF-α</t> expression does not change in diabetic CD11b Cre / Arg1 fl/fl mice. TNF-α mRNA levels in mouse kidney were evaluated by qRT-PCR. Levels of TNF-α mRNA were normalized to GAPDH mRNA and expressed relative to the average value for normal Arg1 fl/fl mice (arbitrarily set to 100). White bar, normal group; black bar, diabetic group. Results are means ± SE for n = 6–9 animals in each group. * P
    Gene Exp Tnf Mm00443260 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 746 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems tnf alpha
    Enhanced expression of <t>TNF-alpha</t> in PKR-deficient mice and macrophages. (A) Levels of TNF-alpha in lung homogenates. TNF-alpha in lung homogenates from Mtb-infected mice at indicated time was measured by ELISA. Means ± SD for 4 mice per strain from one experiment representative of four. *, p
    Tnf Alpha, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 587 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson tnf
    Monocytes from people with the FcγRIIA disease associated gene variant have lower IVIg-mediated anti-inflammatory responses to LPS. Monocytes from healthy control participants were stimulated with LPS (100 ng/ml) or [IVIg (5 mg/ml) + LPS (100 ng/ml)] for 24 h. Participants were genotyped for the FcγRIIA H131R polymorphism (rs1801274); CC = does not have the disease associated gene variant (low affinity), CT = heterozygous for the disease associated gene variant, and TT = homozygous for the disease associated gene variant (high affinity). Clarified cell supernatants were assayed for (A) IL-10, (B) <t>IL-12/23p40,</t> (C) IL-6, and (D) <t>TNF</t> and responses were stratified to genotype. Data are mean ± SEM from n = 11 CC participants, n = 13 CT participants, and n = 20 TT participants performed as independent experiments, assayed in duplicate. * p
    Tnf, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 1475 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp tnf hs01113624 g1
    The recombinant JEV with NS5-M19A mutation is less able to block LCFA β-oxidation and induces less cytokine expression. (A and B) A549 cells infected with JEV-WT or JEV-NS5-M19A (MOI = 10) for 5 h were changed to serum-free medium for 1 h, then incubated with PA-BSA or BSA control. (A) Real-time OCR was measured from 6 to 24 h post-infection. The OCR before PA-BSA or BSA treatment was set to 100%. (B) The AUC OCR with PA-BSA and BSA (n = 3). (C and D) A549 cells infected with the indicated JEV (MOI = 10) for 5 h were incubated with serum-free medium for 1 h before treatment with PA-BSA or BSA for 18 h. RT-qPCR analysis of relative mRNA levels of IL-6 (C) and <t>TNF-α</t> (D) (n = 3). (E-G) A549 cells were infected with JEV-WT or JEV-NS5-M19A (MOI = 10) for 24 h in serum (10% FBS)-containing medium. RT-qPCR analysis of relative mRNA levels of JEV RNA (E), IL-6 (F) and TNF-α (G) (n = 3). Data are mean±SD.*P
    Gene Exp Tnf Hs01113624 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 631 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti tnf alpha antibody
    Level of inflammatory cytokines in graft extracts after transplantation. A)IL-1.B) IL-6.C) <t>TNF-alpha.</t> Note that the expression of IL-1, IL-6 and TNF-alpha in the pancreas graft was dropped after Cis. The results were expressed as ratio of gray value of Target band and the beat-Actin bend. N=6 mice per group.
    Anti Tnf Alpha Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 366 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

    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

    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

    Analysis of proinflammatory (M1) mφ markers in Hoxa3 mCh - or mCherry-treated BMDMs from non-db (ndb) and db mice. ( A ) Production of NO (as measured by nitrate as the final product in the Griess assay) in NA or CA mφs from ndb or db mice and treated with mCherry (□) or with Hoxa3 mCh (▪). ( B ) IL-12 release from NA or CA mφs from ndb or db mice, treated with mCherry (□) or Hoxa3 mCh (▪). ( C and D ) TNF release from NA or CA mφs from ndb and db mice, respectively, treated with mCherry (□) or with Hoxa3 mCh (▪). ( E – H ) Relative expression (RE) of Nos2 , Tnf , Cd86 , and Ccl2 to Hsp90 (reference gene, reference, n = 5; * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Enforced Expression of Hoxa3 Inhibits Classical and Promotes Alternative Activation of Macrophages In Vitro and In Vivo

    doi: 10.4049/jimmunol.1501944

    Figure Lengend Snippet: Analysis of proinflammatory (M1) mφ markers in Hoxa3 mCh - or mCherry-treated BMDMs from non-db (ndb) and db mice. ( A ) Production of NO (as measured by nitrate as the final product in the Griess assay) in NA or CA mφs from ndb or db mice and treated with mCherry (□) or with Hoxa3 mCh (▪). ( B ) IL-12 release from NA or CA mφs from ndb or db mice, treated with mCherry (□) or Hoxa3 mCh (▪). ( C and D ) TNF release from NA or CA mφs from ndb and db mice, respectively, treated with mCherry (□) or with Hoxa3 mCh (▪). ( E – H ) Relative expression (RE) of Nos2 , Tnf , Cd86 , and Ccl2 to Hsp90 (reference gene, reference, n = 5; * p

    Article Snippet: Gene expression analysis qRT-PCR analysis was performed using the following TaqMan Gene Expression Assays: Itgam (Mm00434455_m1), Emr1 (Mm00802529_m1), Csf1r (Mm01266652_m1), Ym1 /Chi313 (Mm00675889_m1), Arg1 (Mm00475988_m1), Tgfβ (Mm01178820_m1), Mrc1 (Mm01329362_m1), Nos2 (Mm01309897_m1), Tnf (Mm00443258-m1), Cd86 (Mm00444543-m1), Ccl2 (Mm00441242_m1), Spi1 (Mm00488142_m1), Stat6 (Mm01160477_m1), and Socs1 (Mm00782550-s1).

    Techniques: Mouse Assay, Griess Assay, Expressing

    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

    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

    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

    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

    TNF-α-mediated reactivation is correlated with activation of NF-κB and ATM signaling. Latently infected cells (14 dpi) were treated for 3 days with or without TNF-α at 5 ng/ml. (A to D) Flow cytometry analysis was performed to assess the activation of NF-κB (p65) and DDR (ATM, KAP-1, and γ-H2AX). The activation of p65, ATM, and KAP-1 was expressed as the ratio of phosphoprotein to total protein in comparison with untreated cells (p-p65-S536/p65, pATM-S1981/ATM, and p-Kap-S824/Kap1). γ-H2AX is expressed as the ratio of mean fluorescence intensity compared to untreated cells. The error bars represent the standard errors of the means calculated from 3 independent experiments (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

    Journal: mBio

    Article Title: Tumor Necrosis Factor Alpha Induces Reactivation of Human Cytomegalovirus Independently of Myeloid Cell Differentiation following Posttranscriptional Establishment of Latency

    doi: 10.1128/mBio.01560-18

    Figure Lengend Snippet: TNF-α-mediated reactivation is correlated with activation of NF-κB and ATM signaling. Latently infected cells (14 dpi) were treated for 3 days with or without TNF-α at 5 ng/ml. (A to D) Flow cytometry analysis was performed to assess the activation of NF-κB (p65) and DDR (ATM, KAP-1, and γ-H2AX). The activation of p65, ATM, and KAP-1 was expressed as the ratio of phosphoprotein to total protein in comparison with untreated cells (p-p65-S536/p65, pATM-S1981/ATM, and p-Kap-S824/Kap1). γ-H2AX is expressed as the ratio of mean fluorescence intensity compared to untreated cells. The error bars represent the standard errors of the means calculated from 3 independent experiments (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

    Article Snippet: TaqMan gene expression assays purchased from Life Technologies were used for analysis of cellular RNAs (Hs00174128_m1 for TNF-α and Hs99999905_m1 for GAPDH).

    Techniques: Activation Assay, Infection, Flow Cytometry, Cytometry, Fluorescence

    HCMV latency is established after activation of transcription at 14 days postinfection. (A) Schematic outlining the infection model used for studies of latency and reactivation in Kasumi-3 cells. GFP + infected cells were purified by flow cytometry at 1 dpi. On day 14, latently infected cells were treated with TNF-α for 3 days to induce reactivation. (B) Representative FACS analysis of GFP expression in Kasumi-3 infected cells at 1 dpi compared to uninfected cells. FITC, fluorescein isothiocyanate; SSC, side scatter. (C) Release of viral particles into the medium was measured by a TCID 50 assay on MRC-5 cells after 2 weeks. (D) UL122 mRNA expression and DNA amount were analyzed at the indicated times postinfection and expressed relative to day 1 after normalization to GAPDH or RNase P. (E) RNA/DNA ratios of UL123, UL54, and UL32 over the course of infection. For panels C to E, statistical significance was calculated by a one-way analysis of variance with Dunnett’s multiple-comparison test ( n = 4). The error bars represent standard errors of the means, and the asterisks indicate P values (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001) calculated by the comparison to the peak (day 4 for RNA, day 8 for DNA and virus, and day 1 for the RNA/DNA ratio).

    Journal: mBio

    Article Title: Tumor Necrosis Factor Alpha Induces Reactivation of Human Cytomegalovirus Independently of Myeloid Cell Differentiation following Posttranscriptional Establishment of Latency

    doi: 10.1128/mBio.01560-18

    Figure Lengend Snippet: HCMV latency is established after activation of transcription at 14 days postinfection. (A) Schematic outlining the infection model used for studies of latency and reactivation in Kasumi-3 cells. GFP + infected cells were purified by flow cytometry at 1 dpi. On day 14, latently infected cells were treated with TNF-α for 3 days to induce reactivation. (B) Representative FACS analysis of GFP expression in Kasumi-3 infected cells at 1 dpi compared to uninfected cells. FITC, fluorescein isothiocyanate; SSC, side scatter. (C) Release of viral particles into the medium was measured by a TCID 50 assay on MRC-5 cells after 2 weeks. (D) UL122 mRNA expression and DNA amount were analyzed at the indicated times postinfection and expressed relative to day 1 after normalization to GAPDH or RNase P. (E) RNA/DNA ratios of UL123, UL54, and UL32 over the course of infection. For panels C to E, statistical significance was calculated by a one-way analysis of variance with Dunnett’s multiple-comparison test ( n = 4). The error bars represent standard errors of the means, and the asterisks indicate P values (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001) calculated by the comparison to the peak (day 4 for RNA, day 8 for DNA and virus, and day 1 for the RNA/DNA ratio).

    Article Snippet: TaqMan gene expression assays purchased from Life Technologies were used for analysis of cellular RNAs (Hs00174128_m1 for TNF-α and Hs99999905_m1 for GAPDH).

    Techniques: Activation Assay, Infection, Purification, Flow Cytometry, Cytometry, FACS, Expressing

    TNF-α induces reactivation independently of differentiation. (A) Latently infected cells (14 dpi) were treated or not with TNF-α for 3 days. On day 17, DNA and RNA from infected cells were analyzed for DNA copy number and expression of viral genes as decribed in Materials and Methods. The release of viral particles in the supernatant was measured by a TCID 50 assay. The statistical significance was calculated by unpaired t test ( n = 4). The error bars represent the standard errors of the means, and the asterisks indicate P values (*, P

    Journal: mBio

    Article Title: Tumor Necrosis Factor Alpha Induces Reactivation of Human Cytomegalovirus Independently of Myeloid Cell Differentiation following Posttranscriptional Establishment of Latency

    doi: 10.1128/mBio.01560-18

    Figure Lengend Snippet: TNF-α induces reactivation independently of differentiation. (A) Latently infected cells (14 dpi) were treated or not with TNF-α for 3 days. On day 17, DNA and RNA from infected cells were analyzed for DNA copy number and expression of viral genes as decribed in Materials and Methods. The release of viral particles in the supernatant was measured by a TCID 50 assay. The statistical significance was calculated by unpaired t test ( n = 4). The error bars represent the standard errors of the means, and the asterisks indicate P values (*, P

    Article Snippet: TaqMan gene expression assays purchased from Life Technologies were used for analysis of cellular RNAs (Hs00174128_m1 for TNF-α and Hs99999905_m1 for GAPDH).

    Techniques: Infection, Expressing

    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:

    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

    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

    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

    RIPK3 activates caspase-1 independent of MLKL unless caspase-8 is inhibited. ( a – c ) WT, Mlkl −/− and Ripk3 −/− BMDM were primed with LPS (20 ng ml −1 ) for 3 h and cultured with Q-VD-OPh (20 μM), where indicated, which was added in the last 20 min of priming. Cells were then stimulated with Cp.A (500 nM) or alum (300 μg ml −1 ) for a further 6 h. Supernatants were analyzed for ( a , c ) IL-1β and ( b ) TNF by ELISA. n =3 mice per genotype. Data are represented as mean+s.e.m. and are representative of one of three independent experiments. ( d ) WT, Mlkl −/− and Ripk3 −/− BMDM were primed with LPS for 2.5 h. In the last 20 min of priming, cells were incubated with Q-VD-OPh (20 μM) and then cultured with Cp.A (1 μM) for 5 h. Cell supernatants and lysates were analyzed by immunoblot. Representative of one of three experiments. Full-size immunoblots are presented in Supplementary Fig. 11 . ( e ) Schematic depicting how RIPK3 signals IL-1β activation based on the data presented in Figs 1 , 2 , 3 . ( f ) Lysates from WT ( Casp8 fl/fl ) littermate and caspase-8-deficient ( Casp8 LysMcre ) BMDM ( n =2 mice) were subjected to immunoblot to assess efficiency of caspase-8 deletion. Full-size immunoblots are presented in Supplementary Fig. 11 . ( g ) WT littermate and Caspase-8 LysMcre BMDM were primed for 3 h with Pam 3 Cys (2 μg ml −1 ), and as indicated treated with Nec-1 (50 μM) in the last 20 min of priming. Cells were then exposed to Cp.A (500 nM), as specified, for a further 24 h, after which IL-1β release was measured by ELISA. n =3 mice per genotype, mean+s.e.m. Representative of one of three experiments. ( h ) WT littermate and Caspase-8 LysMcre BMDM were pre-incubated with glyburide for 20 min, as indicated, and cultured with Pam 3 Cys (2 μg ml −1 ) or LPS (100 ng ml −1 ) for 24 h. Cell supernatants were assayed for IL-1β by ELISA. n =4 mice per genotype, mean+s.e.m. Representative of one of two experiments.

    Journal: Nature Communications

    Article Title: RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL

    doi: 10.1038/ncomms7282

    Figure Lengend Snippet: RIPK3 activates caspase-1 independent of MLKL unless caspase-8 is inhibited. ( a – c ) WT, Mlkl −/− and Ripk3 −/− BMDM were primed with LPS (20 ng ml −1 ) for 3 h and cultured with Q-VD-OPh (20 μM), where indicated, which was added in the last 20 min of priming. Cells were then stimulated with Cp.A (500 nM) or alum (300 μg ml −1 ) for a further 6 h. Supernatants were analyzed for ( a , c ) IL-1β and ( b ) TNF by ELISA. n =3 mice per genotype. Data are represented as mean+s.e.m. and are representative of one of three independent experiments. ( d ) WT, Mlkl −/− and Ripk3 −/− BMDM were primed with LPS for 2.5 h. In the last 20 min of priming, cells were incubated with Q-VD-OPh (20 μM) and then cultured with Cp.A (1 μM) for 5 h. Cell supernatants and lysates were analyzed by immunoblot. Representative of one of three experiments. Full-size immunoblots are presented in Supplementary Fig. 11 . ( e ) Schematic depicting how RIPK3 signals IL-1β activation based on the data presented in Figs 1 , 2 , 3 . ( f ) Lysates from WT ( Casp8 fl/fl ) littermate and caspase-8-deficient ( Casp8 LysMcre ) BMDM ( n =2 mice) were subjected to immunoblot to assess efficiency of caspase-8 deletion. Full-size immunoblots are presented in Supplementary Fig. 11 . ( g ) WT littermate and Caspase-8 LysMcre BMDM were primed for 3 h with Pam 3 Cys (2 μg ml −1 ), and as indicated treated with Nec-1 (50 μM) in the last 20 min of priming. Cells were then exposed to Cp.A (500 nM), as specified, for a further 24 h, after which IL-1β release was measured by ELISA. n =3 mice per genotype, mean+s.e.m. Representative of one of three experiments. ( h ) WT littermate and Caspase-8 LysMcre BMDM were pre-incubated with glyburide for 20 min, as indicated, and cultured with Pam 3 Cys (2 μg ml −1 ) or LPS (100 ng ml −1 ) for 24 h. Cell supernatants were assayed for IL-1β by ELISA. n =4 mice per genotype, mean+s.e.m. Representative of one of two experiments.

    Article Snippet: Cytokine ELISA IL-1β and IL-18 (R & D Systems, Ebioscience), and TNF and IL-6 (Ebioscience, R & D Systems) ELISA kits and paired G-CSF antibodies and standard (R & D Systems, Peprotech) were used to perform ELISAs on serum, joint fluid and supernatants according to the manufacturer’s instructions.

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Mouse Assay, Incubation, Western Blot, Activation Assay

    RIPK3 kinase activity is not required for MLKL-independent activation of NLRP3. ( a ) WT littermate and Caspase-8 LysMcre BMDM were primed for 3 h with Pam 3 Cys (2 μg ml −1 ), and as indicated RIPK3 inhibitor (R3 inhib GSK872; 1 μM) was added in the last 20 min of priming. Cells were then exposed to Cp.A (500 nM), as specified, for a further 24 h. Levels of IL-1β secretion were measured by ELISA. n =4 mice per genotype, mean+s.e.m. ( b , c ) WT, Ripk3 −/− and Mlkl −/− BMDM were primed for 3 h with LPS (20 ng ml −1 ) in the absence or presence of RIPK3 inhibitor (R3 inhib; 1 μM), prior to addition of Cp.A for a further 6 h. Supernatants were assayed for ( b ) IL-1β and ( c ) TNF by ELISA or death assessed by lactate dehydrogenase (LDH) activity (see Supplementary Fig. 2f ). n =3 mice per genotype, mean+s.e.m. ( d , e ) Cell supernatants ( d ) and lysates ( e ) from WT, Mlkl −/− and Ripk3 −/− BMDM primed with LPS (3 h) and treated with Q-VD-OPh (20 μM) and R3 inhibitor (1 μM, last 20 min of priming), as indicated, and subsequently treated with Cp.A (1 μM, 5 h) were analyzed by immunoblot as indicated. One of three experiments. Full-size immunoblots are presented in Supplementary Fig. 12 . ( f ) WT and caspase-1 −/− BMDM were primed for 3 h with LPS, and as indicated treated with Q-VD-OPh (20 μM) and R3 inhib (1 μM) for the last 20 min of priming. BMDM were then cultured with Cp.A (1 μM) or Alum (300 μg ml −1 ) for a further 6 h. Culture supernatants were assayed for IL-1β levels by ELISA. n =3 mice, mean+s.e.m., one of two experiments. ( g – i ) WT, Ripk3 −/− and Mlkl −/− BMDM were primed for 3 h with LPS (20 ng ml −1 ) in the presence of Q-VD-OPh (20 μM), and where indicated 1 μM RIPK3 inhibitor (R3 inhib), prior to addition of Cp.A for a further 6 h. Supernatants were assayed for ( g ) IL-1β and ( i ) TNF by ELISA, and ( h ) cell death was measured via an LDH assay. n =3 mice per genotype, mean+s.e.m. *NS, non-specific band.

    Journal: Nature Communications

    Article Title: RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL

    doi: 10.1038/ncomms7282

    Figure Lengend Snippet: RIPK3 kinase activity is not required for MLKL-independent activation of NLRP3. ( a ) WT littermate and Caspase-8 LysMcre BMDM were primed for 3 h with Pam 3 Cys (2 μg ml −1 ), and as indicated RIPK3 inhibitor (R3 inhib GSK872; 1 μM) was added in the last 20 min of priming. Cells were then exposed to Cp.A (500 nM), as specified, for a further 24 h. Levels of IL-1β secretion were measured by ELISA. n =4 mice per genotype, mean+s.e.m. ( b , c ) WT, Ripk3 −/− and Mlkl −/− BMDM were primed for 3 h with LPS (20 ng ml −1 ) in the absence or presence of RIPK3 inhibitor (R3 inhib; 1 μM), prior to addition of Cp.A for a further 6 h. Supernatants were assayed for ( b ) IL-1β and ( c ) TNF by ELISA or death assessed by lactate dehydrogenase (LDH) activity (see Supplementary Fig. 2f ). n =3 mice per genotype, mean+s.e.m. ( d , e ) Cell supernatants ( d ) and lysates ( e ) from WT, Mlkl −/− and Ripk3 −/− BMDM primed with LPS (3 h) and treated with Q-VD-OPh (20 μM) and R3 inhibitor (1 μM, last 20 min of priming), as indicated, and subsequently treated with Cp.A (1 μM, 5 h) were analyzed by immunoblot as indicated. One of three experiments. Full-size immunoblots are presented in Supplementary Fig. 12 . ( f ) WT and caspase-1 −/− BMDM were primed for 3 h with LPS, and as indicated treated with Q-VD-OPh (20 μM) and R3 inhib (1 μM) for the last 20 min of priming. BMDM were then cultured with Cp.A (1 μM) or Alum (300 μg ml −1 ) for a further 6 h. Culture supernatants were assayed for IL-1β levels by ELISA. n =3 mice, mean+s.e.m., one of two experiments. ( g – i ) WT, Ripk3 −/− and Mlkl −/− BMDM were primed for 3 h with LPS (20 ng ml −1 ) in the presence of Q-VD-OPh (20 μM), and where indicated 1 μM RIPK3 inhibitor (R3 inhib), prior to addition of Cp.A for a further 6 h. Supernatants were assayed for ( g ) IL-1β and ( i ) TNF by ELISA, and ( h ) cell death was measured via an LDH assay. n =3 mice per genotype, mean+s.e.m. *NS, non-specific band.

    Article Snippet: Cytokine ELISA IL-1β and IL-18 (R & D Systems, Ebioscience), and TNF and IL-6 (Ebioscience, R & D Systems) ELISA kits and paired G-CSF antibodies and standard (R & D Systems, Peprotech) were used to perform ELISAs on serum, joint fluid and supernatants according to the manufacturer’s instructions.

    Techniques: Activity Assay, Activation Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Mouse Assay, Western Blot, Cell Culture, Lactate Dehydrogenase Assay

    RIPK1 represses LPS-induced RIPK3 activity. ( a ) WT BMDM were either pre-treated for 30 min with Nec-1 (50 μM), or cultured with Nec-1 in the final 30 min of priming with LPS (20 ng ml −1 ) for 3 h. Cp.A (500 nM) was added, as indicated, and cells were cultured for 6 h. Supernatants were assayed for IL-1β. n =3 mice per group, mean+s.e.m. One of two experiments. ( b ) WT and Ripk1 −/− FLM, and WT and Ripk3 −/− BMDM were primed with LPS for 3 h and stimulated with Cp.A (500 nM) or Alum (300 μg ml −1 ) for a further 6 h, and supernatants and lysates were assayed by immunoblot. Full-size immunoblots are presented in Supplementary Fig. 14 . ( c – e ) WT, Ripk1 +/− and Ripk1 −/− FLDM were treated with glyburide for 20 min and then stimulated with LPS (20 ng ml −1 ) for 6–8 h. Data shows three to five embryos of each genotype. ( c , d ) Supernatants were assayed for ( c ) IL-1β and ( d ) TNF production. Data symbols represent individual mice from three experiments. ( e ) Cell lysates and supernatants were blotted for caspase-1 cleavage. n =2 individual mice (numbered). Full-size immunoblots are presented in Supplementary Fig. 14 . ( f ) WT Ripk1 +/+ , Ripk1 −/− , Ripk3 −/− and Ripk1 −/− Ripk3 −/− FLDM were labelled with cell tracker green (green) and cultured with LPS (20 ng ml −1 ) for 1 h, prior to stimulation with Cp.A, as indicated, and PI addition (red). Cells were imaged from 2h post-LPS addition every 30 min fo r 14 h. Supplementary Figure 4g shows additional treatments (LPS/Cp.A/QVD) used in this experiment. Representative images of one of three experiments. *NS, non-specific band.

    Journal: Nature Communications

    Article Title: RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL

    doi: 10.1038/ncomms7282

    Figure Lengend Snippet: RIPK1 represses LPS-induced RIPK3 activity. ( a ) WT BMDM were either pre-treated for 30 min with Nec-1 (50 μM), or cultured with Nec-1 in the final 30 min of priming with LPS (20 ng ml −1 ) for 3 h. Cp.A (500 nM) was added, as indicated, and cells were cultured for 6 h. Supernatants were assayed for IL-1β. n =3 mice per group, mean+s.e.m. One of two experiments. ( b ) WT and Ripk1 −/− FLM, and WT and Ripk3 −/− BMDM were primed with LPS for 3 h and stimulated with Cp.A (500 nM) or Alum (300 μg ml −1 ) for a further 6 h, and supernatants and lysates were assayed by immunoblot. Full-size immunoblots are presented in Supplementary Fig. 14 . ( c – e ) WT, Ripk1 +/− and Ripk1 −/− FLDM were treated with glyburide for 20 min and then stimulated with LPS (20 ng ml −1 ) for 6–8 h. Data shows three to five embryos of each genotype. ( c , d ) Supernatants were assayed for ( c ) IL-1β and ( d ) TNF production. Data symbols represent individual mice from three experiments. ( e ) Cell lysates and supernatants were blotted for caspase-1 cleavage. n =2 individual mice (numbered). Full-size immunoblots are presented in Supplementary Fig. 14 . ( f ) WT Ripk1 +/+ , Ripk1 −/− , Ripk3 −/− and Ripk1 −/− Ripk3 −/− FLDM were labelled with cell tracker green (green) and cultured with LPS (20 ng ml −1 ) for 1 h, prior to stimulation with Cp.A, as indicated, and PI addition (red). Cells were imaged from 2h post-LPS addition every 30 min fo r 14 h. Supplementary Figure 4g shows additional treatments (LPS/Cp.A/QVD) used in this experiment. Representative images of one of three experiments. *NS, non-specific band.

    Article Snippet: Cytokine ELISA IL-1β and IL-18 (R & D Systems, Ebioscience), and TNF and IL-6 (Ebioscience, R & D Systems) ELISA kits and paired G-CSF antibodies and standard (R & D Systems, Peprotech) were used to perform ELISAs on serum, joint fluid and supernatants according to the manufacturer’s instructions.

    Techniques: Activity Assay, Cell Culture, Mouse Assay, Western Blot

    XIAP is required to repress LPS- and TNF-induced IL-1β secretion. ( a – f ) WT and Xiap -deficient ( x −/− ) macrophages were pre-incubated with or without ( a – c ) LPS (20 ng ml −1 ) or ( d – f ) human Fc-TNF (100 ng ml −1 ) for 2–3 h and cultured with or without IAP antagonists of differing IAP specificities (see g ). After 24 h, cell supernatants were assayed for ( a , d ) IL-1β, ( b , e ) TNF and ( c , f ) IL-6 levels by ELISA. n =3 mice; Data are represented as mean+s.e.m., from one of three experiments. ( g ) Efficiency of functional XIAP antagonism by IAP antagonist compounds (+, high; −,low). ( h ) WT, cIAP1 −/− ( c1 −/− ), cIAP2 −/− ( c2 −/− ) and Xiap −/− ( x −/− ) BMDM were primed with LPS (20 ng ml −1 ) for 3 h and cultured with the IAP antagonist LBW242 (20 μM) or alum (320 μg ml −1 ) for a further 6 h. Secreted IL-1β was measured in supernatants by ELISA. n =3 mice; mean+s.e.m. ( i ) Yield of macrophages from WT and IAP mutant bone marrow after 6 days of culture with L929 cell conditioned media. n =3–6 mice per genotype, mean+s.e.m. ( j – l ) WT and IAP mutant macrophages were stimulated with LPS (20 ng ml −1 ) for up to 24 h, and ( j ) IL-1β, ( k ) TNF and ( l ) IL-6 levels were assayed in supernatants by ELISA. n =3–4 mice, data are represented as mean+s.e.m., one of three experiments. ( m ) Yield of WT, c1 lox/lox x −/− c2 −/− , c1 LysMcre x −/− c2 −/− and c1 ERcre x −/− c2 −/− bone marrow macrophages after 6 days of culture with L929 cell conditioned media. n =3–6 mice per genotype, mean+s.e.m. ( n , o ) WT, c1 lox/lox x −/− c2 −/− , c1 LysMcre x −/− c2 −/− and c1 ERcre x −/− c2 −/− macrophages were pulsed for 16 h with 4′-hydroxy-tamoxifen (4HT 1000, nM) and then rested for 10 h prior to stimulation with or without LPS (50 ng ml −1 ) for a further 8 h. ( n ) Secreted IL-1β was measured in supernatants by ELISA, n =3 mice per group; c1 LysMcre x −/− c2 −/− ( n =2), mean+s.d., one of three experiments, and ( o ) IL-1β and caspase-1 activation assayed by immunoblot of supernatants and lysates. Representative blot from the analysis of 4 c1 ERcre x −/− c2 −/− mice. Full-size immunoblots are presented in Supplementary Fig. 9 .

    Journal: Nature Communications

    Article Title: RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL

    doi: 10.1038/ncomms7282

    Figure Lengend Snippet: XIAP is required to repress LPS- and TNF-induced IL-1β secretion. ( a – f ) WT and Xiap -deficient ( x −/− ) macrophages were pre-incubated with or without ( a – c ) LPS (20 ng ml −1 ) or ( d – f ) human Fc-TNF (100 ng ml −1 ) for 2–3 h and cultured with or without IAP antagonists of differing IAP specificities (see g ). After 24 h, cell supernatants were assayed for ( a , d ) IL-1β, ( b , e ) TNF and ( c , f ) IL-6 levels by ELISA. n =3 mice; Data are represented as mean+s.e.m., from one of three experiments. ( g ) Efficiency of functional XIAP antagonism by IAP antagonist compounds (+, high; −,low). ( h ) WT, cIAP1 −/− ( c1 −/− ), cIAP2 −/− ( c2 −/− ) and Xiap −/− ( x −/− ) BMDM were primed with LPS (20 ng ml −1 ) for 3 h and cultured with the IAP antagonist LBW242 (20 μM) or alum (320 μg ml −1 ) for a further 6 h. Secreted IL-1β was measured in supernatants by ELISA. n =3 mice; mean+s.e.m. ( i ) Yield of macrophages from WT and IAP mutant bone marrow after 6 days of culture with L929 cell conditioned media. n =3–6 mice per genotype, mean+s.e.m. ( j – l ) WT and IAP mutant macrophages were stimulated with LPS (20 ng ml −1 ) for up to 24 h, and ( j ) IL-1β, ( k ) TNF and ( l ) IL-6 levels were assayed in supernatants by ELISA. n =3–4 mice, data are represented as mean+s.e.m., one of three experiments. ( m ) Yield of WT, c1 lox/lox x −/− c2 −/− , c1 LysMcre x −/− c2 −/− and c1 ERcre x −/− c2 −/− bone marrow macrophages after 6 days of culture with L929 cell conditioned media. n =3–6 mice per genotype, mean+s.e.m. ( n , o ) WT, c1 lox/lox x −/− c2 −/− , c1 LysMcre x −/− c2 −/− and c1 ERcre x −/− c2 −/− macrophages were pulsed for 16 h with 4′-hydroxy-tamoxifen (4HT 1000, nM) and then rested for 10 h prior to stimulation with or without LPS (50 ng ml −1 ) for a further 8 h. ( n ) Secreted IL-1β was measured in supernatants by ELISA, n =3 mice per group; c1 LysMcre x −/− c2 −/− ( n =2), mean+s.d., one of three experiments, and ( o ) IL-1β and caspase-1 activation assayed by immunoblot of supernatants and lysates. Representative blot from the analysis of 4 c1 ERcre x −/− c2 −/− mice. Full-size immunoblots are presented in Supplementary Fig. 9 .

    Article Snippet: Cytokine ELISA IL-1β and IL-18 (R & D Systems, Ebioscience), and TNF and IL-6 (Ebioscience, R & D Systems) ELISA kits and paired G-CSF antibodies and standard (R & D Systems, Peprotech) were used to perform ELISAs on serum, joint fluid and supernatants according to the manufacturer’s instructions.

    Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Mouse Assay, Functional Assay, Mutagenesis, Activation Assay, Western Blot

    Model for how XIAP and cIAPs repress inflammatory cytokine production, apoptosis and necroptosis. ( a ) When IAPs are present, TNFR1 or TLR–TRIF signalling results in IAP-mediated ubiquitylation of RIPK1 or RIPK3, respectively, to propagate pro-survival signals and gene induction. ( b ) If IAPs are inactivated but caspase-8 is present (left panel), LPS stimulation induces the ripoptosome platform to activate caspase-8 (ubiquitylated). Caspase-8 can (i) trigger apoptosis, (ii) cleave pro-IL-1β directly into its mature form or (iii) promote NLRP3-associated caspase-1 activation, by a mechanism yet to be defined. Alternatively, if both IAPs and caspase-8 are inactive (right panel), LPS induces the formation of the RIPK3–MLKL necrosome that, in addition to causing necroptotic cell death, activates the NLRP3 inflammasome. This necroptotic pathway is associated with RIPK3 and MLKL ubiquitylation, which may control RIPK3/MLKL signalling and/or stability. ( c ) Genetic loss or inhibition of cIAPs alone or XIAP and cIAPs in myeloid cells cause differential effects on cell death, cytokine production and haematopoiesis leading to spontaneous arthritis. Loss of all three IAPs leads to spontaneous systemic inflammatory disease, featuring mild joint inflammation. Disease is associated with increased cytokine release, including IL-1β and TNF, as well as apoptotic and necroptotic cell death and the accumulation of innate inflammatory cells. In contrast, loss of cIAP1/2 causes severe arthritis that is associated specifically with enhanced TNF levels and only modest effects on haematopoiesis.

    Journal: Nature Communications

    Article Title: RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL

    doi: 10.1038/ncomms7282

    Figure Lengend Snippet: Model for how XIAP and cIAPs repress inflammatory cytokine production, apoptosis and necroptosis. ( a ) When IAPs are present, TNFR1 or TLR–TRIF signalling results in IAP-mediated ubiquitylation of RIPK1 or RIPK3, respectively, to propagate pro-survival signals and gene induction. ( b ) If IAPs are inactivated but caspase-8 is present (left panel), LPS stimulation induces the ripoptosome platform to activate caspase-8 (ubiquitylated). Caspase-8 can (i) trigger apoptosis, (ii) cleave pro-IL-1β directly into its mature form or (iii) promote NLRP3-associated caspase-1 activation, by a mechanism yet to be defined. Alternatively, if both IAPs and caspase-8 are inactive (right panel), LPS induces the formation of the RIPK3–MLKL necrosome that, in addition to causing necroptotic cell death, activates the NLRP3 inflammasome. This necroptotic pathway is associated with RIPK3 and MLKL ubiquitylation, which may control RIPK3/MLKL signalling and/or stability. ( c ) Genetic loss or inhibition of cIAPs alone or XIAP and cIAPs in myeloid cells cause differential effects on cell death, cytokine production and haematopoiesis leading to spontaneous arthritis. Loss of all three IAPs leads to spontaneous systemic inflammatory disease, featuring mild joint inflammation. Disease is associated with increased cytokine release, including IL-1β and TNF, as well as apoptotic and necroptotic cell death and the accumulation of innate inflammatory cells. In contrast, loss of cIAP1/2 causes severe arthritis that is associated specifically with enhanced TNF levels and only modest effects on haematopoiesis.

    Article Snippet: Cytokine ELISA IL-1β and IL-18 (R & D Systems, Ebioscience), and TNF and IL-6 (Ebioscience, R & D Systems) ELISA kits and paired G-CSF antibodies and standard (R & D Systems, Peprotech) were used to perform ELISAs on serum, joint fluid and supernatants according to the manufacturer’s instructions.

    Techniques: Activation Assay, Inhibition

    TRIF and IAPs regulate LPS-induced ubiquitylation of RIPK3 and MLKL. ( a ) WT, Myd88 −/− and Trif −/− BMDM were cultured with neutralizing antibodies to TNF (anti-TNF [XT-22] 20 μg ml −1 ), isotype control antibodies (IC [GL113] 20 μg ml −1 ) or Nec-1 (50 μM), and primed for 2 h with LPS, as indicated. Cells were then cultured with Cp.A (500 nM), and IL-1β levels were assayed in supernatants by ELISA at 6 h. n =3 mice per group, mean+s.e.m., representative of one of three experiments. ( b ) WT and Tnf −/− BMDM were primed for 3 h with LPS, and treated with Nec-1 (50 μM) as indicated. Cells were then cultured with Cp.A (500 nM), and after 6 h IL-1β secretion was assayed by ELISA. n =3 mice per genotype, mean+s.e.m., one of three experiments. ( c , d ) WT, Myd88 −/− and Trif −/− BMDM were cultured with neutralizing antibodies to TNF (anti-TNF [XT-22] 20 μg ml −1 ), isotype control antibodies (IC [GL113] 20 μg ml −1 ) or Nec-1 (50 μM), and primed for 2 h with LPS, as indicated. Cells were then cultured with Cp.A (500 nM) for 24 h, and ( c ) IL-1β levels assayed in supernatants by ELISA and ( d ) cell death (% Dead cells) assessed by PI and FACS. n =3 mice per group, mean+s.e.m., representative of one of three experiments. ( e ) WT BMDM were treated with 50 ng ml −1 LPS (30 and 60 min) or 100ng ml −1 TNF (20 min) and ubiquitylated proteins were isolated by TUBE and analyzed by immunoblot. One of two experiments. ( f ) WT, Myd88 −/− , and Trif −/− BMDM were treated with LPS (100 ng ml −1 ) for 60 min and endogenous ubiquitylated proteins isolated by TUBE and analyzed by immunoblot. One of three experiments. ( g ) WT and Trif −/− BMDM were pre-incubated with Q-VD-OPh (20 μM) for 40 min, cultured with Cp.A 500 nM and subsequently treated with LPS (50 ng ml −1 ) for 60 or 190 min as indicated. Endogenous ubiquitylated proteins were isolated by TUBE and analyzed by immunoblot. One of three experiments.

    Journal: Nature Communications

    Article Title: RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL

    doi: 10.1038/ncomms7282

    Figure Lengend Snippet: TRIF and IAPs regulate LPS-induced ubiquitylation of RIPK3 and MLKL. ( a ) WT, Myd88 −/− and Trif −/− BMDM were cultured with neutralizing antibodies to TNF (anti-TNF [XT-22] 20 μg ml −1 ), isotype control antibodies (IC [GL113] 20 μg ml −1 ) or Nec-1 (50 μM), and primed for 2 h with LPS, as indicated. Cells were then cultured with Cp.A (500 nM), and IL-1β levels were assayed in supernatants by ELISA at 6 h. n =3 mice per group, mean+s.e.m., representative of one of three experiments. ( b ) WT and Tnf −/− BMDM were primed for 3 h with LPS, and treated with Nec-1 (50 μM) as indicated. Cells were then cultured with Cp.A (500 nM), and after 6 h IL-1β secretion was assayed by ELISA. n =3 mice per genotype, mean+s.e.m., one of three experiments. ( c , d ) WT, Myd88 −/− and Trif −/− BMDM were cultured with neutralizing antibodies to TNF (anti-TNF [XT-22] 20 μg ml −1 ), isotype control antibodies (IC [GL113] 20 μg ml −1 ) or Nec-1 (50 μM), and primed for 2 h with LPS, as indicated. Cells were then cultured with Cp.A (500 nM) for 24 h, and ( c ) IL-1β levels assayed in supernatants by ELISA and ( d ) cell death (% Dead cells) assessed by PI and FACS. n =3 mice per group, mean+s.e.m., representative of one of three experiments. ( e ) WT BMDM were treated with 50 ng ml −1 LPS (30 and 60 min) or 100ng ml −1 TNF (20 min) and ubiquitylated proteins were isolated by TUBE and analyzed by immunoblot. One of two experiments. ( f ) WT, Myd88 −/− , and Trif −/− BMDM were treated with LPS (100 ng ml −1 ) for 60 min and endogenous ubiquitylated proteins isolated by TUBE and analyzed by immunoblot. One of three experiments. ( g ) WT and Trif −/− BMDM were pre-incubated with Q-VD-OPh (20 μM) for 40 min, cultured with Cp.A 500 nM and subsequently treated with LPS (50 ng ml −1 ) for 60 or 190 min as indicated. Endogenous ubiquitylated proteins were isolated by TUBE and analyzed by immunoblot. One of three experiments.

    Article Snippet: Cytokine ELISA IL-1β and IL-18 (R & D Systems, Ebioscience), and TNF and IL-6 (Ebioscience, R & D Systems) ELISA kits and paired G-CSF antibodies and standard (R & D Systems, Peprotech) were used to perform ELISAs on serum, joint fluid and supernatants according to the manufacturer’s instructions.

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Mouse Assay, FACS, Isolation, Incubation

    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

    Kidney TNF-α expression does not change in diabetic CD11b Cre / Arg1 fl/fl mice. TNF-α mRNA levels in mouse kidney were evaluated by qRT-PCR. Levels of TNF-α mRNA were normalized to GAPDH mRNA and expressed relative to the average value for normal Arg1 fl/fl mice (arbitrarily set to 100). White bar, normal group; black bar, diabetic group. Results are means ± SE for n = 6–9 animals in each group. * P

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Distinct roles of arginases 1 and 2 in diabetic nephropathy

    doi: 10.1152/ajprenal.00158.2017

    Figure Lengend Snippet: Kidney TNF-α expression does not change in diabetic CD11b Cre / Arg1 fl/fl mice. TNF-α mRNA levels in mouse kidney were evaluated by qRT-PCR. Levels of TNF-α mRNA were normalized to GAPDH mRNA and expressed relative to the average value for normal Arg1 fl/fl mice (arbitrarily set to 100). White bar, normal group; black bar, diabetic group. Results are means ± SE for n = 6–9 animals in each group. * P

    Article Snippet: Quantitative PCR was performed in a CFX96 Real-Time System (Bio-Rad) using Taqman gene expression assays [fibronectin ( Fn1 ): Mm01256744_m1, TNF-α ( Tnf ): Mm00443260_g1, and GAPDH ( Gapdh ): Mm99999915_g1; Life Technologies, Grand Island, NY].

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR

    Arginase inhibition reduces kidney TNF-α expression in diabetic Arg2 −/− mice. TNF-α mRNA levels in mouse kidney were evaluated by qRT-PCR. Levels of mRNAs were normalized to GAPDH mRNA and expressed relative to the average value for normal wild-type mice (arbitrarily set to 100). White bar, normal group; black bar, vehicle-treated diabetic group; gray bar, BEC-treated diabetic group. Results are means ± SE for n = 7–12 animals in each group. * P

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Distinct roles of arginases 1 and 2 in diabetic nephropathy

    doi: 10.1152/ajprenal.00158.2017

    Figure Lengend Snippet: Arginase inhibition reduces kidney TNF-α expression in diabetic Arg2 −/− mice. TNF-α mRNA levels in mouse kidney were evaluated by qRT-PCR. Levels of mRNAs were normalized to GAPDH mRNA and expressed relative to the average value for normal wild-type mice (arbitrarily set to 100). White bar, normal group; black bar, vehicle-treated diabetic group; gray bar, BEC-treated diabetic group. Results are means ± SE for n = 7–12 animals in each group. * P

    Article Snippet: Quantitative PCR was performed in a CFX96 Real-Time System (Bio-Rad) using Taqman gene expression assays [fibronectin ( Fn1 ): Mm01256744_m1, TNF-α ( Tnf ): Mm00443260_g1, and GAPDH ( Gapdh ): Mm99999915_g1; Life Technologies, Grand Island, NY].

    Techniques: Inhibition, Expressing, Mouse Assay, Quantitative RT-PCR

    Enhanced expression of TNF-alpha in PKR-deficient mice and macrophages. (A) Levels of TNF-alpha in lung homogenates. TNF-alpha in lung homogenates from Mtb-infected mice at indicated time was measured by ELISA. Means ± SD for 4 mice per strain from one experiment representative of four. *, p

    Journal: PLoS ONE

    Article Title: Improved Control of Tuberculosis and Activation of Macrophages in Mice Lacking Protein Kinase R

    doi: 10.1371/journal.pone.0030512

    Figure Lengend Snippet: Enhanced expression of TNF-alpha in PKR-deficient mice and macrophages. (A) Levels of TNF-alpha in lung homogenates. TNF-alpha in lung homogenates from Mtb-infected mice at indicated time was measured by ELISA. Means ± SD for 4 mice per strain from one experiment representative of four. *, p

    Article Snippet: Purified recombinant mouse IFN-gamma, TNF-alpha and IL10 were from R & D Systems.

    Techniques: Expressing, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay

    Induction of IL-10 by Mtb and IFN-gamma and its impact on iNOS and TNF-alpha. (A) Secretion of IL-10. Macrophages (5×10 5 ) were treated with Mtb (MOI 3), IFN-gamma (10 ng/mL), or pre-treated with IFN-gamma (10 ng/mL) overnight followed by infection with Mtb (MOI 3) for 24 h. Supernatant was collected for IL-10 production by ELISA. Results were similar in additional experiments with MOIs of 0.3, 1 and 3 and IFN-gamma concentrations of 1, 10 and 100 ng/mL. (B) Induction of mature and nascent IL-10 transcripts. Macrophages (5×10 5 ) were treated with IFN-gamma (10 ng/mL) for 3 h. qRT-PCR signals for IL-10 were normalized to GAPDH. Results were similar with IFN-gamma concentrations of 1, 10 and 100 ng/mL. (C) Effect of IL-10 on nascent iNOS transcripts and secretion of nitrite. Left panel, macrophages (5×10 5 ) were treated with IL-10 for 3 h after the addition of IFN-gamma 0 ng/mL. qRT-PCR signals for iNOS were normalized to GAPDH. Right panel, macrophages (5×10 5 ) were treated with IL-10 for 48 h after the addition of IFN-gamma 0 ng/mL. Nitrite in the supernatant was measured by the Griess reaction. (D) Effect of IL10 on nascent TNF-alpha transcripts and TNF-alpha. Left panel, macrophages (5×10 5 ) were treated with IL-10 for 3 h after the addition of IFN-gamma 0 ng/mL. qRT-PCR signals for TNF-alpha were normalized to GAPDH. Right panel, macrophages (5×10 5 ) were treated with IL-10 for 48 h after the addition of IFN-gamma 0 ng/mL. TNF-alpha in the supernatant was measured by ELISA. (E) Effect of neutralizing anti-IL-10 antibody (10 µg/mL) on release of nitrite 48 h after addition of IFN-gamma (10 ng/mL). Results in (A) to (F) are means ± SD from individual experiments each with 3 replicates that are representative of at least 2 independent experiments.

    Journal: PLoS ONE

    Article Title: Improved Control of Tuberculosis and Activation of Macrophages in Mice Lacking Protein Kinase R

    doi: 10.1371/journal.pone.0030512

    Figure Lengend Snippet: Induction of IL-10 by Mtb and IFN-gamma and its impact on iNOS and TNF-alpha. (A) Secretion of IL-10. Macrophages (5×10 5 ) were treated with Mtb (MOI 3), IFN-gamma (10 ng/mL), or pre-treated with IFN-gamma (10 ng/mL) overnight followed by infection with Mtb (MOI 3) for 24 h. Supernatant was collected for IL-10 production by ELISA. Results were similar in additional experiments with MOIs of 0.3, 1 and 3 and IFN-gamma concentrations of 1, 10 and 100 ng/mL. (B) Induction of mature and nascent IL-10 transcripts. Macrophages (5×10 5 ) were treated with IFN-gamma (10 ng/mL) for 3 h. qRT-PCR signals for IL-10 were normalized to GAPDH. Results were similar with IFN-gamma concentrations of 1, 10 and 100 ng/mL. (C) Effect of IL-10 on nascent iNOS transcripts and secretion of nitrite. Left panel, macrophages (5×10 5 ) were treated with IL-10 for 3 h after the addition of IFN-gamma 0 ng/mL. qRT-PCR signals for iNOS were normalized to GAPDH. Right panel, macrophages (5×10 5 ) were treated with IL-10 for 48 h after the addition of IFN-gamma 0 ng/mL. Nitrite in the supernatant was measured by the Griess reaction. (D) Effect of IL10 on nascent TNF-alpha transcripts and TNF-alpha. Left panel, macrophages (5×10 5 ) were treated with IL-10 for 3 h after the addition of IFN-gamma 0 ng/mL. qRT-PCR signals for TNF-alpha were normalized to GAPDH. Right panel, macrophages (5×10 5 ) were treated with IL-10 for 48 h after the addition of IFN-gamma 0 ng/mL. TNF-alpha in the supernatant was measured by ELISA. (E) Effect of neutralizing anti-IL-10 antibody (10 µg/mL) on release of nitrite 48 h after addition of IFN-gamma (10 ng/mL). Results in (A) to (F) are means ± SD from individual experiments each with 3 replicates that are representative of at least 2 independent experiments.

    Article Snippet: Purified recombinant mouse IFN-gamma, TNF-alpha and IL10 were from R & D Systems.

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

    Monocytes from people with the FcγRIIA disease associated gene variant have lower IVIg-mediated anti-inflammatory responses to LPS. Monocytes from healthy control participants were stimulated with LPS (100 ng/ml) or [IVIg (5 mg/ml) + LPS (100 ng/ml)] for 24 h. Participants were genotyped for the FcγRIIA H131R polymorphism (rs1801274); CC = does not have the disease associated gene variant (low affinity), CT = heterozygous for the disease associated gene variant, and TT = homozygous for the disease associated gene variant (high affinity). Clarified cell supernatants were assayed for (A) IL-10, (B) IL-12/23p40, (C) IL-6, and (D) TNF and responses were stratified to genotype. Data are mean ± SEM from n = 11 CC participants, n = 13 CT participants, and n = 20 TT participants performed as independent experiments, assayed in duplicate. * p

    Journal: Frontiers in Immunology

    Article Title: IVIg and LPS Co-stimulation Induces IL-10 Production by Human Monocytes, Which Is Compromised by an FcγRIIA Disease-Associated Gene Variant

    doi: 10.3389/fimmu.2018.02676

    Figure Lengend Snippet: Monocytes from people with the FcγRIIA disease associated gene variant have lower IVIg-mediated anti-inflammatory responses to LPS. Monocytes from healthy control participants were stimulated with LPS (100 ng/ml) or [IVIg (5 mg/ml) + LPS (100 ng/ml)] for 24 h. Participants were genotyped for the FcγRIIA H131R polymorphism (rs1801274); CC = does not have the disease associated gene variant (low affinity), CT = heterozygous for the disease associated gene variant, and TT = homozygous for the disease associated gene variant (high affinity). Clarified cell supernatants were assayed for (A) IL-10, (B) IL-12/23p40, (C) IL-6, and (D) TNF and responses were stratified to genotype. Data are mean ± SEM from n = 11 CC participants, n = 13 CT participants, and n = 20 TT participants performed as independent experiments, assayed in duplicate. * p

    Article Snippet: ELISA kits for human IL-10, IL-12/23p40, IL-6, and TNF were from BD Biosciences (Mississauga, ON, Canada).

    Techniques: Variant Assay

    IL-10 signaling contributes to reduced LPS-induced pro-inflammatory cytokine production by IVIg-activated monocytes. In (A) monocytes from healthy control participants were left untreated (C) or stimulated with LPS (100 ng/ml), recombinant human IL-10 (rhIL-10; 400 pg/ml) or [rhIL-10 (400 pg/ml) + LPS (100 ng/ml)] for 24 h. Clarified cell supernatants were assayed for (A) IL-12/23p40, IL-6, and TNF. In (B,C) monocytes from healthy control participants were untreated (–) or pre-treated for 1 h with an IgG isotype control (IgG; 5 μg/ml) or an IL-10 receptor (IL-10R) blocking antibody (5 μg/ml). Cells were then stimulated with LPS (100 ng/ml) or [IVIg (5 mg/ml) + LPS (100 ng/ml)] for 24 h. Clarified cell supernatants were assayed for (B) IL-10, (C) IL-12/23p40, IL-6, and TNF. Data are mean ± SEM from n = 12 (A) or 8 (B,C) participants performed as independent experiments, assayed in duplicate. * p

    Journal: Frontiers in Immunology

    Article Title: IVIg and LPS Co-stimulation Induces IL-10 Production by Human Monocytes, Which Is Compromised by an FcγRIIA Disease-Associated Gene Variant

    doi: 10.3389/fimmu.2018.02676

    Figure Lengend Snippet: IL-10 signaling contributes to reduced LPS-induced pro-inflammatory cytokine production by IVIg-activated monocytes. In (A) monocytes from healthy control participants were left untreated (C) or stimulated with LPS (100 ng/ml), recombinant human IL-10 (rhIL-10; 400 pg/ml) or [rhIL-10 (400 pg/ml) + LPS (100 ng/ml)] for 24 h. Clarified cell supernatants were assayed for (A) IL-12/23p40, IL-6, and TNF. In (B,C) monocytes from healthy control participants were untreated (–) or pre-treated for 1 h with an IgG isotype control (IgG; 5 μg/ml) or an IL-10 receptor (IL-10R) blocking antibody (5 μg/ml). Cells were then stimulated with LPS (100 ng/ml) or [IVIg (5 mg/ml) + LPS (100 ng/ml)] for 24 h. Clarified cell supernatants were assayed for (B) IL-10, (C) IL-12/23p40, IL-6, and TNF. Data are mean ± SEM from n = 12 (A) or 8 (B,C) participants performed as independent experiments, assayed in duplicate. * p

    Article Snippet: ELISA kits for human IL-10, IL-12/23p40, IL-6, and TNF were from BD Biosciences (Mississauga, ON, Canada).

    Techniques: Recombinant, Blocking Assay

    FcγRIIA prevents IVIg-induced IL-10 production in monocytes from people with the disease-associated gene variant. Monocytes from healthy control participants of the non-risk genotype (CC) and risk genotype (TT) were untreated (UnRx) or pre-treated for 48 h with a non-silencing siRNA (ns) or 2 different siRNAs to the FcγRIIA (si1 or si2). (A) Cell lysates (2.5 × 10 5 cells / treatment) were prepared, separated by SDS-PAGE, analyzed by western blotting with antibodies for FcγRIIA and β-actin, as a loading control. Results are representative of n = 6 experiments for the non-risk genotype (CC) and n = 10 experiments for the risk genotype (TT); Monocytes were derived from 1 participant for each independent experiment. Densitometry for FcγRIIA normalized to β-actin and relative to the control (UnRx) are averaged and shown below each band. (B,C) Monocytes pre-treated with the ns siRNA control were unstimulated [control (C)] or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or both, for 24 h, and monocytes pre-treated with FcγRIIA si1 and si2 were stimulated with IVIg (5 mg/ml) + LPS (100 ng/ml). Clarified cell supernatants were assayed for (B) IL-10, (C) IL-12/23p40, IL-6, and TNF. Data are mean ± SEM and are representative of n = 6 experiments for the non-risk genotype (CC) and n = 10 experiments for the risk genotype (TT). Monocytes were derived from 1 participant for each independent experiment and assayed in duplicate. * p

    Journal: Frontiers in Immunology

    Article Title: IVIg and LPS Co-stimulation Induces IL-10 Production by Human Monocytes, Which Is Compromised by an FcγRIIA Disease-Associated Gene Variant

    doi: 10.3389/fimmu.2018.02676

    Figure Lengend Snippet: FcγRIIA prevents IVIg-induced IL-10 production in monocytes from people with the disease-associated gene variant. Monocytes from healthy control participants of the non-risk genotype (CC) and risk genotype (TT) were untreated (UnRx) or pre-treated for 48 h with a non-silencing siRNA (ns) or 2 different siRNAs to the FcγRIIA (si1 or si2). (A) Cell lysates (2.5 × 10 5 cells / treatment) were prepared, separated by SDS-PAGE, analyzed by western blotting with antibodies for FcγRIIA and β-actin, as a loading control. Results are representative of n = 6 experiments for the non-risk genotype (CC) and n = 10 experiments for the risk genotype (TT); Monocytes were derived from 1 participant for each independent experiment. Densitometry for FcγRIIA normalized to β-actin and relative to the control (UnRx) are averaged and shown below each band. (B,C) Monocytes pre-treated with the ns siRNA control were unstimulated [control (C)] or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or both, for 24 h, and monocytes pre-treated with FcγRIIA si1 and si2 were stimulated with IVIg (5 mg/ml) + LPS (100 ng/ml). Clarified cell supernatants were assayed for (B) IL-10, (C) IL-12/23p40, IL-6, and TNF. Data are mean ± SEM and are representative of n = 6 experiments for the non-risk genotype (CC) and n = 10 experiments for the risk genotype (TT). Monocytes were derived from 1 participant for each independent experiment and assayed in duplicate. * p

    Article Snippet: ELISA kits for human IL-10, IL-12/23p40, IL-6, and TNF were from BD Biosciences (Mississauga, ON, Canada).

    Techniques: Variant Assay, SDS Page, Western Blot, Derivative Assay

    IVIg increases IL-10 production and reduces pro-inflammatory cytokine production in LPS-stimulated human monocytes. Monocytes from healthy control participants were unstimulated [Control (C)] or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or both, for 24 h. Clarified cell supernatants were assayed for (A) IL-10, (B) IL-12/23p40, IL-6, or TNF by ELISA. Data are mean ± SEM with n = 16 participants performed as independent experiments, and assayed in duplicate. * p

    Journal: Frontiers in Immunology

    Article Title: IVIg and LPS Co-stimulation Induces IL-10 Production by Human Monocytes, Which Is Compromised by an FcγRIIA Disease-Associated Gene Variant

    doi: 10.3389/fimmu.2018.02676

    Figure Lengend Snippet: IVIg increases IL-10 production and reduces pro-inflammatory cytokine production in LPS-stimulated human monocytes. Monocytes from healthy control participants were unstimulated [Control (C)] or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or both, for 24 h. Clarified cell supernatants were assayed for (A) IL-10, (B) IL-12/23p40, IL-6, or TNF by ELISA. Data are mean ± SEM with n = 16 participants performed as independent experiments, and assayed in duplicate. * p

    Article Snippet: ELISA kits for human IL-10, IL-12/23p40, IL-6, and TNF were from BD Biosciences (Mississauga, ON, Canada).

    Techniques: Enzyme-linked Immunosorbent Assay

    The recombinant JEV with NS5-M19A mutation is less able to block LCFA β-oxidation and induces less cytokine expression. (A and B) A549 cells infected with JEV-WT or JEV-NS5-M19A (MOI = 10) for 5 h were changed to serum-free medium for 1 h, then incubated with PA-BSA or BSA control. (A) Real-time OCR was measured from 6 to 24 h post-infection. The OCR before PA-BSA or BSA treatment was set to 100%. (B) The AUC OCR with PA-BSA and BSA (n = 3). (C and D) A549 cells infected with the indicated JEV (MOI = 10) for 5 h were incubated with serum-free medium for 1 h before treatment with PA-BSA or BSA for 18 h. RT-qPCR analysis of relative mRNA levels of IL-6 (C) and TNF-α (D) (n = 3). (E-G) A549 cells were infected with JEV-WT or JEV-NS5-M19A (MOI = 10) for 24 h in serum (10% FBS)-containing medium. RT-qPCR analysis of relative mRNA levels of JEV RNA (E), IL-6 (F) and TNF-α (G) (n = 3). Data are mean±SD.*P

    Journal: PLoS Pathogens

    Article Title: Japanese Encephalitis Virus Nonstructural Protein NS5 Interacts with Mitochondrial Trifunctional Protein and Impairs Fatty Acid β-Oxidation

    doi: 10.1371/journal.ppat.1004750

    Figure Lengend Snippet: The recombinant JEV with NS5-M19A mutation is less able to block LCFA β-oxidation and induces less cytokine expression. (A and B) A549 cells infected with JEV-WT or JEV-NS5-M19A (MOI = 10) for 5 h were changed to serum-free medium for 1 h, then incubated with PA-BSA or BSA control. (A) Real-time OCR was measured from 6 to 24 h post-infection. The OCR before PA-BSA or BSA treatment was set to 100%. (B) The AUC OCR with PA-BSA and BSA (n = 3). (C and D) A549 cells infected with the indicated JEV (MOI = 10) for 5 h were incubated with serum-free medium for 1 h before treatment with PA-BSA or BSA for 18 h. RT-qPCR analysis of relative mRNA levels of IL-6 (C) and TNF-α (D) (n = 3). (E-G) A549 cells were infected with JEV-WT or JEV-NS5-M19A (MOI = 10) for 24 h in serum (10% FBS)-containing medium. RT-qPCR analysis of relative mRNA levels of JEV RNA (E), IL-6 (F) and TNF-α (G) (n = 3). Data are mean±SD.*P

    Article Snippet: Quantitative RT-PCR (RT-qPCR) Total cellular RNA was prepared with use of an RNeasy Mini Kit (Qiagen, Hilden, Germany) and cDNA was reverse-transcribed from 1 μg total RNA by use of the SuperScript III First-Strand Synthesis System (Invitrogen). qPCR involved use of TaqMan Universal PCR Master Mix (Invitrogen) with commercial probes for IL-6 (Hs00985639 and Mm00446190), TNF-α (Hs01113624 and Mm00443260), IL-10 (Hs00961622), IL-4 (Hs00174122), IL-13 (Hs00174379), IFN-β (Hs01077958) and GAPDH (Hs02758991 and Mm99999915) (Applied Biosystems, Foster City, CA, USA) as well as primers for JEV viral RNA (5΄-AGAACGGAAGATAACCATGACTAAA-3΄and 5΄-CCGCGTTTCAGCATATTGAT-3΄).

    Techniques: Recombinant, Mutagenesis, Blocking Assay, Expressing, Infection, Incubation, Quantitative RT-PCR

    Reduced neuroinvasiveness of NS5-M19A—mutated JEV in challenged mice. (A) Survival in C57BL/6 mice infected with 10 3 or 10 5 PFU JEV-WT or JEV-NS5-M19A by an intraperitoneal (i.p.) plus i.c. route. The animal number ( n ) and survival rate for each group are shown. (B) Plaque-forming assay of virus titers in brain tissues of mice inoculated with JEV-WT or JEV-NS5-M19A (10 5 PFU) (n = 3). (C-E) RT-qPCR of relative JEV RNA (C), IL-6 (D), and TNF-α (E) mRNA levels in brain tissues (n = 3). (F) ELISA of IL-6 protein levels in the sera samples (n = 3). Data are mean±SD.*P

    Journal: PLoS Pathogens

    Article Title: Japanese Encephalitis Virus Nonstructural Protein NS5 Interacts with Mitochondrial Trifunctional Protein and Impairs Fatty Acid β-Oxidation

    doi: 10.1371/journal.ppat.1004750

    Figure Lengend Snippet: Reduced neuroinvasiveness of NS5-M19A—mutated JEV in challenged mice. (A) Survival in C57BL/6 mice infected with 10 3 or 10 5 PFU JEV-WT or JEV-NS5-M19A by an intraperitoneal (i.p.) plus i.c. route. The animal number ( n ) and survival rate for each group are shown. (B) Plaque-forming assay of virus titers in brain tissues of mice inoculated with JEV-WT or JEV-NS5-M19A (10 5 PFU) (n = 3). (C-E) RT-qPCR of relative JEV RNA (C), IL-6 (D), and TNF-α (E) mRNA levels in brain tissues (n = 3). (F) ELISA of IL-6 protein levels in the sera samples (n = 3). Data are mean±SD.*P

    Article Snippet: Quantitative RT-PCR (RT-qPCR) Total cellular RNA was prepared with use of an RNeasy Mini Kit (Qiagen, Hilden, Germany) and cDNA was reverse-transcribed from 1 μg total RNA by use of the SuperScript III First-Strand Synthesis System (Invitrogen). qPCR involved use of TaqMan Universal PCR Master Mix (Invitrogen) with commercial probes for IL-6 (Hs00985639 and Mm00446190), TNF-α (Hs01113624 and Mm00443260), IL-10 (Hs00961622), IL-4 (Hs00174122), IL-13 (Hs00174379), IFN-β (Hs01077958) and GAPDH (Hs02758991 and Mm99999915) (Applied Biosystems, Foster City, CA, USA) as well as primers for JEV viral RNA (5΄-AGAACGGAAGATAACCATGACTAAA-3΄and 5΄-CCGCGTTTCAGCATATTGAT-3΄).

    Techniques: Mouse Assay, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Reduced neurovirulence of NS5-M19A—mutated JEV in challenged mice. (A) Survival in C57BL/6 mice infected with 0.2, 2 or 20 plaque-forming units (PFU) of JEV-WT or JEV-NS5-M19A by an intracerebral (i.c.) injection. The animal number ( n ) and survival rate for each group are shown. (B-D) RT-qPCR of relative JEV RNA (B), IL-6 (C), and TNF-α (D) mRNA levels in brain tissues of mice inoculated with JEV-WT or JEV-NS5-M19A (20 PFU) (n = 3). Data are mean±SD.*P

    Journal: PLoS Pathogens

    Article Title: Japanese Encephalitis Virus Nonstructural Protein NS5 Interacts with Mitochondrial Trifunctional Protein and Impairs Fatty Acid β-Oxidation

    doi: 10.1371/journal.ppat.1004750

    Figure Lengend Snippet: Reduced neurovirulence of NS5-M19A—mutated JEV in challenged mice. (A) Survival in C57BL/6 mice infected with 0.2, 2 or 20 plaque-forming units (PFU) of JEV-WT or JEV-NS5-M19A by an intracerebral (i.c.) injection. The animal number ( n ) and survival rate for each group are shown. (B-D) RT-qPCR of relative JEV RNA (B), IL-6 (C), and TNF-α (D) mRNA levels in brain tissues of mice inoculated with JEV-WT or JEV-NS5-M19A (20 PFU) (n = 3). Data are mean±SD.*P

    Article Snippet: Quantitative RT-PCR (RT-qPCR) Total cellular RNA was prepared with use of an RNeasy Mini Kit (Qiagen, Hilden, Germany) and cDNA was reverse-transcribed from 1 μg total RNA by use of the SuperScript III First-Strand Synthesis System (Invitrogen). qPCR involved use of TaqMan Universal PCR Master Mix (Invitrogen) with commercial probes for IL-6 (Hs00985639 and Mm00446190), TNF-α (Hs01113624 and Mm00443260), IL-10 (Hs00961622), IL-4 (Hs00174122), IL-13 (Hs00174379), IFN-β (Hs01077958) and GAPDH (Hs02758991 and Mm99999915) (Applied Biosystems, Foster City, CA, USA) as well as primers for JEV viral RNA (5΄-AGAACGGAAGATAACCATGACTAAA-3΄and 5΄-CCGCGTTTCAGCATATTGAT-3΄).

    Techniques: Mouse Assay, Infection, Injection, Quantitative RT-PCR

    Impaired LCFA β-oxidation and cytokine induction in NS5-overexpressing cells. (A) AUC OCR for NS5-overexpressing and vector control-A549 cells incubated with serum-free medium for 1 h, then treated with PA-BSA or BSA control for 18 h (n = 2). (B and C) NS5-overexpressing and vector control-A549 cells were cultured with serum-free medium for 1 h, then incubated with PA-BSA or BSA for 24 h. RT-qPCR analysis of the relative mRNA levels of IL-6 (B) and TNF-α (C) (n = 3). Data are mean±SD. **P

    Journal: PLoS Pathogens

    Article Title: Japanese Encephalitis Virus Nonstructural Protein NS5 Interacts with Mitochondrial Trifunctional Protein and Impairs Fatty Acid β-Oxidation

    doi: 10.1371/journal.ppat.1004750

    Figure Lengend Snippet: Impaired LCFA β-oxidation and cytokine induction in NS5-overexpressing cells. (A) AUC OCR for NS5-overexpressing and vector control-A549 cells incubated with serum-free medium for 1 h, then treated with PA-BSA or BSA control for 18 h (n = 2). (B and C) NS5-overexpressing and vector control-A549 cells were cultured with serum-free medium for 1 h, then incubated with PA-BSA or BSA for 24 h. RT-qPCR analysis of the relative mRNA levels of IL-6 (B) and TNF-α (C) (n = 3). Data are mean±SD. **P

    Article Snippet: Quantitative RT-PCR (RT-qPCR) Total cellular RNA was prepared with use of an RNeasy Mini Kit (Qiagen, Hilden, Germany) and cDNA was reverse-transcribed from 1 μg total RNA by use of the SuperScript III First-Strand Synthesis System (Invitrogen). qPCR involved use of TaqMan Universal PCR Master Mix (Invitrogen) with commercial probes for IL-6 (Hs00985639 and Mm00446190), TNF-α (Hs01113624 and Mm00443260), IL-10 (Hs00961622), IL-4 (Hs00174122), IL-13 (Hs00174379), IFN-β (Hs01077958) and GAPDH (Hs02758991 and Mm99999915) (Applied Biosystems, Foster City, CA, USA) as well as primers for JEV viral RNA (5΄-AGAACGGAAGATAACCATGACTAAA-3΄and 5΄-CCGCGTTTCAGCATATTGAT-3΄).

    Techniques: Plasmid Preparation, Incubation, Cell Culture, Quantitative RT-PCR

    Level of inflammatory cytokines in graft extracts after transplantation. A)IL-1.B) IL-6.C) TNF-alpha. Note that the expression of IL-1, IL-6 and TNF-alpha in the pancreas graft was dropped after Cis. The results were expressed as ratio of gray value of Target band and the beat-Actin bend. N=6 mice per group.

    Journal: International Journal of Biological Sciences

    Article Title: Pretreatment of Cisplatin in Recipients Attenuates Post-Transplantation Pancreatitis in Murine Model

    doi: 10.7150/ijbs.3656

    Figure Lengend Snippet: Level of inflammatory cytokines in graft extracts after transplantation. A)IL-1.B) IL-6.C) TNF-alpha. Note that the expression of IL-1, IL-6 and TNF-alpha in the pancreas graft was dropped after Cis. The results were expressed as ratio of gray value of Target band and the beat-Actin bend. N=6 mice per group.

    Article Snippet: Agents and Antibodies Cis was purchased from Sigma Inc., All the antibodies used in this study are listed below: Apop-Tag Peroxidase In Situ Apoptosis Detection Kit (cat#S7100; Chemicon International Inc., Billerica, MA, USA), Anti-IL-1-beta antibody (cat#ab9722,abcam,UK), Anti-IL6 antibody (cat#ab6672, abcam,UK), Anti-TNF alpha antibody (cat#ab6671, abcam,UK), Mouse Myeloperoxidase /MPO Antibody(cat# 392105, R & D Systems, Inc).

    Techniques: Transplantation Assay, Expressing, Mouse Assay