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  • 94
    Thermo Fisher tnf α
    Tnf α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 21729 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tnf α
    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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    R&D Systems tnf α
    Recombinant HNE impairs <t>TNF-α</t> neutralizing ability of the anti-TNF-α biologics. ( A ) Luciferase activity from a TNFR cell line expressed as a percentage (%) referred to the highest luminescence induced by TNF-α positive stimulus. TNFR-reporter HeLa cells were stimulated for 6 hours with TNF-α, which had been previously incubated (1h at 37ºC) with HNE-treated infliximab, adalimumab, or etanercept, with or without elafin. Cells incubated with TNF-α for 1 hour served as a positive control. Mean ± standard deviation; three independent experiments performed in triplicates. *p
    Tnf α, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 26790 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam tnf α
    p-ERK1/2 inhibitor protects the retina from light-induced inflammation. The release of proinflammatory cytokine in the retina was detected with Western blotting for <t>TNF-α</t> and IL-1β. (a) Light injury significantly increased the expression of TNF-α compared with normal group. p-ERK1/2 inhibitor significantly decreased the overexpression of TNF-α compared with light injury group. (b) Light injury significantly increased the expression of IL-1β compared with normal group. p-ERK1/2 inhibitor significantly decreased the overexpression of IL-1β compared with light injury group. * P
    Tnf α, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 4059 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson tnf α
    MiR-145 directly targets MKK4. ( a ) A schematic illustrating the design of luciferase reporters with the WT MKK4 3′-UTR or the site-directed mutant MKK4 3′-UTR. ( b and c ) Expression level of MKK4 in chondrocytes transfected with miR-145 mimics, inhibitor or their negative controls. ( d ) Immunoblotting of MKK4 and p-MKK4 in chondrocytes transfected at different doses (10, 20, and 40 nM) and then stimulated with <t>TNF-</t> α . ( e and f ) Expression level of miR-145 and MKK4 in chondrocytes transfected with miR-145 overexpression plasmid, inhibition plasmid or empty plasmids (GV268, GV249). ( g ) The mRNA level of MKK4 in chondrocytes stimulated with TNF- α for different time points. ( h ) Immunoblotting of MKK4 in chondrocytes transfected with miR-145 mimics at different doses (10, 20, and 40 nM) and then stimulated with TNF- α . ( i ) Effect of miR-145 mimics or inhibitor on the luciferase activity of WT MKK4 3′-UTR (UTR) or MUT MKK4 3′-UTR (MT) reporter in SW1353 cells. ( j ) MKK4 expression level in different tissues from rat. ( k ) Immunoblotting of MKK4 and its downstream molecules in cartilage samples from OA patients ( n =3) or normal controls ( n =2). ( l and m ) Immunostaining of MKK4 and p-MKK4 in human ( n =6) and DMM-operated rat OA cartilage ( n =6). Scale bar: × 100, 50 μ m; × 200, 20 μ m. Data represent the mean±S.E.M. of at least n =4 independent experiments. * P
    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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    Thermo Fisher tumor necrosis factor alpha tnf α
    VLP effectively stimulates BMDCs in vitro to secrete cytokines. BMDCs were stimulated with M2e5x, M1 VLP, or protein (0.3, 1, 5 μg/mL) for 24 h. ( a – c ) The levels of cytokines ( a ) <t>TNF-α,</t> ( b ) IL-6, ( c ) IFN-γ were determined in the culture supernatants by ELISA assay. Un: Medium only, P: proteins, V: VLP. The statistical significance was confirmed by one-way ANOVA and Dunnett’s multiple comparison test. Error bars indicate the means ± SEM of concentrations from individual animals. *; p
    Tumor Necrosis Factor Alpha Tnf α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 757 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc tnf α
    mCD40L-induced <t>TNF</t> -α does not contribute to CD40L-induced cell death. ( a ) EJ cells were infected with 100 MOI of either RAdMock (AdM) or RAdnCD40L (AdnL) or left uninfected as a negative control for 24 h, RNA was extracted utilising the EZ-RNA total isolation kit and the cDNA was prepared by reverse transcription. The expression of TNF -α was examined by qRT-PCR technique. Results are mean of triplicate samples ±S.D. ( b ) EJ cells were infected with 100 MOI of either RAdMock (AdM) or RAdnCD40L (AdnL) or left uninfected as a negative control and plated at a density of 6000/100 μ l/well in 96-well microplate. AdnL-infected cells were either treated with 1, 3 or 5 μ g/ml of TNF -α monoclonal neutralising antibody or left untreated as a control for 28 h. Cell viability was then assessed using the WST-1 assay. Results are mean of triplicate samples ±S.D.
    Tnf α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1810 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeproTech tnf α
    <t>TNF-α</t> induces Bcl6 gene expression in the hepatocytes and Bcl6 overexpression upregulates the TNF-α levels in vivo . (A) The RNA levels of Tnf- α, Il-6, Il-12, Cxcl9, and Cxcl10 were examined by RT-qPCR in B6.2- and B6.2S-injected mouse liver isolated during the HBV clearance stage. (B) AML12 cells were treated with or without TNF-α (100 ng/ml) for the indicated time periods and Bcl6 RNA was analyzed by RT-qPCR. The induction fold means the ratio of the Bcl6 RNA levels in the presence of TNF-α over those without TNF-α. (C) AML12 and Huh-7 cells were transfected with 1 μg of vector or the Bcl6 -expressing plasmid. Two days later, the TNF-α RNA levels were analyzed by RT-qPCR analysis. (D) The liver RNA from the B6.2 or the (B6.2 + Bcl6 ) mice at 2 w.p.i. were analyzed for Tnf- α expression by RT-qPCR. In (A–D) , * P
    Tnf α, supplied by PeproTech, used in various techniques. Bioz Stars score: 99/100, based on 5243 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher tnfα
    <t>TNF-α</t> induces Bcl6 gene expression in the hepatocytes and Bcl6 overexpression upregulates the TNF-α levels in vivo . (A) The RNA levels of Tnf- α, Il-6, Il-12, Cxcl9, and Cxcl10 were examined by RT-qPCR in B6.2- and B6.2S-injected mouse liver isolated during the HBV clearance stage. (B) AML12 cells were treated with or without TNF-α (100 ng/ml) for the indicated time periods and Bcl6 RNA was analyzed by RT-qPCR. The induction fold means the ratio of the Bcl6 RNA levels in the presence of TNF-α over those without TNF-α. (C) AML12 and Huh-7 cells were transfected with 1 μg of vector or the Bcl6 -expressing plasmid. Two days later, the TNF-α RNA levels were analyzed by RT-qPCR analysis. (D) The liver RNA from the B6.2 or the (B6.2 + Bcl6 ) mice at 2 w.p.i. were analyzed for Tnf- α expression by RT-qPCR. In (A–D) , * P
    Tnfα, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 5782 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tumor necrosis factor tnf α
    Regulatory effects of curculigoside on the expression levels of NF-κB p65 (C) and IκB in <t>TNF-α-stimulated</t> MH7A cells. Cells were treated with TNF-α (10 ng/ml) for 12 h, exposed to curculigoside (4 and 16 µg/ml) for a further 24 h and then subjected to western blotting assays to determine the expression levels of NF-κB and IκB (n=4). **P
    Tumor Necrosis Factor Tnf α, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, 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
    CD4+ T cell responses to S . Typhi proteins presented by targets exposed to one of the four recombinant S . Typhi proteins. Ex vivo PBMC from a volunteer collected 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets exposed to 0.5ug/ml with one of the four recombinant S . Typhi proteins: SifA, OmpC, FliC, and GroEL. Untreated B-LCL targets (media) were used as controls. After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and <t>TNF-α)</t> and/or CD107a/b molecules was evaluated by flow cytometry. Shown are the CD4 + T cell responses from a representative volunteer. Numbers represent the percentage of positive cells.
    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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    Thermo Fisher tumor necrosis factor α tnf α
    CD4+ T cell responses to S . Typhi proteins presented by targets exposed to one of the four recombinant S . Typhi proteins. Ex vivo PBMC from a volunteer collected 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets exposed to 0.5ug/ml with one of the four recombinant S . Typhi proteins: SifA, OmpC, FliC, and GroEL. Untreated B-LCL targets (media) were used as controls. After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and <t>TNF-α)</t> and/or CD107a/b molecules was evaluated by flow cytometry. Shown are the CD4 + T cell responses from a representative volunteer. Numbers represent the percentage of positive cells.
    Tumor Necrosis Factor α Tnf α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 945 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeproTech recombinant human tnf α
    CeCyld inhibits TNF-induced IL8 expression. HeLa cells (10 5 cells per well in 12-well plates) were transfected with 0.1μg, 1.4μg and 0.4μg of plasmids expressing HsCYLD, CeCYLD and CeCYLDC774S respectively. After 24h, each transfected cell population was split equally in two new wells and 24 h later, half of each transfected cell population was left untreated or treated with <t>TNFα</t> (40ng/ml, 1h). The cells were then harvested and from each cell population RNA and total protein was extracted. The overexpressed FLAG-tagged proteins were immuno-precipitated and the products of immunoprecipitation were used to assess the expression levels of each protein by immuoblotting. (A) Values are shown as the mean +/- standard error of relative IL8 expression induction from four independent experiments of RT-PCR. The values that were compared statistically are indicated by horizontal lines. (B) Immunoblot showing the expression levels of immuno-precipitated proteins with anti-FLAG antibody (IP:αFLAG) along with the immunoglobulin heavy chains (Ig-HC) and β-actin in the whole cell lysate (WCL). *p
    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
    <t>TNF-α-mediated</t> G1 arrest in Panc1 cells. One hundred thousand ( A ) Panc1 cells or ( B ) PancTu-I cells were cultured in 6-well plates overnight. After 24 h, cells were left untreated (medium) or were treated with 10 ng/mL IFN-γ, 10 ng/mL TNF-α or 10 ng/mL IFN-γ together with TNF-α as indicated for 24 h. Cell cycle distribution of living cells obtained by gating in the forward/sideward scatter was determined using PI staining and flow cytometry. Numbers in the figures represent the percentage of the different cell cycle phases. One representative result is shown as a histogram (upper panel), and three independent experiments are represented as pie diagrams (lower panels). SD is
    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
    Relationships of RIP1 with <t>TNF-α</t> and LVD in human GBC patients. Notes: ( A ) TNF-α and RIP1 protein expression and lymphatic vessel expression in GBC tissues were analyzed using immunohistochemical staining using TNF-α, RIP1, and D2-40 antibodies, respectively (100×; 200×). ( B , D ) Relationships of the average optical density of RIP1 with optical density of TNF-α and LVD in the GBC samples. ( C ) Lymphatic vessels based on D2-40 immunostaining were flattened and invaded by GBC cells. ** p
    Tnf α, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 2573 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems mouse tnf α
    Relationships of RIP1 with <t>TNF-α</t> and LVD in human GBC patients. Notes: ( A ) TNF-α and RIP1 protein expression and lymphatic vessel expression in GBC tissues were analyzed using immunohistochemical staining using TNF-α, RIP1, and D2-40 antibodies, respectively (100×; 200×). ( B , D ) Relationships of the average optical density of RIP1 with optical density of TNF-α and LVD in the GBC samples. ( C ) Lymphatic vessels based on D2-40 immunostaining were flattened and invaded by GBC cells. ** p
    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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    Thermo Fisher mouse tnf α
    Relationships of RIP1 with <t>TNF-α</t> and LVD in human GBC patients. Notes: ( A ) TNF-α and RIP1 protein expression and lymphatic vessel expression in GBC tissues were analyzed using immunohistochemical staining using TNF-α, RIP1, and D2-40 antibodies, respectively (100×; 200×). ( B , D ) Relationships of the average optical density of RIP1 with optical density of TNF-α and LVD in the GBC samples. ( C ) Lymphatic vessels based on D2-40 immunostaining were flattened and invaded by GBC cells. ** p
    Mouse Tnf α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 482 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tumor necrosis factor α tnf α
    Relationships of RIP1 with <t>TNF-α</t> and LVD in human GBC patients. Notes: ( A ) TNF-α and RIP1 protein expression and lymphatic vessel expression in GBC tissues were analyzed using immunohistochemical staining using TNF-α, RIP1, and D2-40 antibodies, respectively (100×; 200×). ( B , D ) Relationships of the average optical density of RIP1 with optical density of TNF-α and LVD in the GBC samples. ( C ) Lymphatic vessels based on D2-40 immunostaining were flattened and invaded by GBC cells. ** p
    Tumor Necrosis Factor α Tnf α, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 538 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti tnf α
    <t>TNF-α</t> is sufficient to induce HF TAT and is crucial for WIHN. ( a , b ) Intracutaneous injection of TNF-α can induce the HF telogen–anagen transition at the injection site in 7-week (W)-old mice (refractory phase) and 9-week-old mice (competent phase) ( a ), and the number of TNF-α-induced anagen hair follicles in the two different group was quantified ( b ). Seven-week-old mice, n =6 for each group; 9-week-old mice, n =8 for each group. ( c , d ) Wounding to the skin induced more anagen HFs in 9-week-old mice than in 7-week-old mice ( c ), and the number of anagen HFs in the two different groups was quantified ( d ). n =7 for each age group. ( e , f ) WIH-A analysis in TNFR1 −/− mice, TNFR2 −/− mice and WT mice ( e ), and the number of anagen HFs in different groups was quantified ( f ). Wild-type (C57BL/6) mice, n =7; TNFR1 −/− mice, n =6; TNFR2 −/− mice, n =9. ( g , h ) Wound-induced anagen HFs in mice constitutively expressing TNF-α (Tg-TNF-α) were significantly increased compared with WT mice, and the number of anagen HFs in the two different groups was quantified ( h ). Control mice (WT), n =6; Tg-TNF-α mice, n =5. ( i ) Bioluminescent imaging of Tnf-Luc-eGFP mice at different days (D) after wounding showed changes in the TNF-α level in the wound. Areas with high TNF-α levels (green/red) shifted from the wound (W) periphery to the wound centre with the progression of wound healing. n =15 for Tnf-Luc-eGFP mice, and n =10 for wild-type (WT) mice. ( j ) IF analysis of sections from the PWD-14 wound showed the presence of F4/80 + macrophages in the wound, which largely co-localized with TNF-α. Scale bar, 50 μm. ( k , l ) WIHN analysis in WT, TNFA −/− and Tg-TNF-α mice at PWD-30 ( k ), and the number of neogenic HFs in wounds was quantified ( l ). n =6 for both wild-type and TNFA −/− mice; n =12 for Tg-TNF-α mice. Scale bars, 2 mm. Data are expressed as the mean±s.e.m. * P
    Anti Tnf α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 715 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant tnf α
    PP242 alleviated the change of autophagy flux caused by <t>TNF-α.</t> ( A , B ) Western blot analysis of P62 and LC3B-II after TNF-α (10 ng/mL, 48 h) and/or PP242 (1 μm, 24 h) treatment. Data were shown as mean ± SD and replicated three times. ** p
    Recombinant Tnf α, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 505 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Recombinant HNE impairs TNF-α neutralizing ability of the anti-TNF-α biologics. ( A ) Luciferase activity from a TNFR cell line expressed as a percentage (%) referred to the highest luminescence induced by TNF-α positive stimulus. TNFR-reporter HeLa cells were stimulated for 6 hours with TNF-α, which had been previously incubated (1h at 37ºC) with HNE-treated infliximab, adalimumab, or etanercept, with or without elafin. Cells incubated with TNF-α for 1 hour served as a positive control. Mean ± standard deviation; three independent experiments performed in triplicates. *p

    Journal: Journal of Inflammation Research

    Article Title: Human Neutrophil Elastase Proteolytic Activity in Ulcerative Colitis Favors the Loss of Function of Therapeutic Monoclonal Antibodies

    doi: 10.2147/JIR.S234710

    Figure Lengend Snippet: Recombinant HNE impairs TNF-α neutralizing ability of the anti-TNF-α biologics. ( A ) Luciferase activity from a TNFR cell line expressed as a percentage (%) referred to the highest luminescence induced by TNF-α positive stimulus. TNFR-reporter HeLa cells were stimulated for 6 hours with TNF-α, which had been previously incubated (1h at 37ºC) with HNE-treated infliximab, adalimumab, or etanercept, with or without elafin. Cells incubated with TNF-α for 1 hour served as a positive control. Mean ± standard deviation; three independent experiments performed in triplicates. *p

    Article Snippet: Untreated anti-TNF agents, HNE and DMEM were also pre-incubated with human recombinant TNF-α and included as controls.

    Techniques: Recombinant, Luciferase, Activity Assay, Incubation, Positive Control, Standard Deviation

    p-ERK1/2 inhibitor protects the retina from light-induced inflammation. The release of proinflammatory cytokine in the retina was detected with Western blotting for TNF-α and IL-1β. (a) Light injury significantly increased the expression of TNF-α compared with normal group. p-ERK1/2 inhibitor significantly decreased the overexpression of TNF-α compared with light injury group. (b) Light injury significantly increased the expression of IL-1β compared with normal group. p-ERK1/2 inhibitor significantly decreased the overexpression of IL-1β compared with light injury group. * P

    Journal: Chinese Medical Journal

    Article Title: Effect of Phosphorylated-Extracellular Regulated Kinase 1/2 Inhibitor on Retina from Light-induced Photoreceptor Degeneration

    doi: 10.4103/0366-6999.246064

    Figure Lengend Snippet: p-ERK1/2 inhibitor protects the retina from light-induced inflammation. The release of proinflammatory cytokine in the retina was detected with Western blotting for TNF-α and IL-1β. (a) Light injury significantly increased the expression of TNF-α compared with normal group. p-ERK1/2 inhibitor significantly decreased the overexpression of TNF-α compared with light injury group. (b) Light injury significantly increased the expression of IL-1β compared with normal group. p-ERK1/2 inhibitor significantly decreased the overexpression of IL-1β compared with light injury group. * P

    Article Snippet: The membranes were blocked with 5% nonfat milk in Tris-buffered saline containing Tween-20 for 1 h and then incubated with antibodies directed against the following proteins: p-ERK1/2 (1:100, Cat #4370, CST, Boston, MA, USA), TNF-α (1:1000, Cat #ab6671, Abcam, Cambridge, MA, USA), IL-1β (1:1000, Cat #ab9722, Abcam, Cambridge, MA, USA), caspase 3 (1:1000, Cat #ab13847, Abcam, Cambridge, MA, USA), and activated caspase 3 (1:1000, Cat #ab2302, Abcam, Cambridge, MA, USA).

    Techniques: Western Blot, Expressing, Over Expression

    Liver inflammation triggered by intrahepatic deposited lupus immunoglobulin G (IgG) depends on macrophages and TNF-α. (A) Immunohistochemistry detected Kupffer cells (F4/80+), dendritic cells (CD11c+), T cells (CD3+), and B cells (CD20+) in liver sections from lupus-prone mice. The results were from three independent experiments. Original magnification, 200× (B) Severity of liver inflammation and serum alanine aminotransferase (ALT) level 48 h after intrahepatic injection of systemic lupus erythematosus (SLE)-IgG (200 µg) in RAG-1 –/– and wild-type mice (the result is representative of three independent experiments, n = 5 mice per group/experiment). (C) Severity of liver inflammation and serum ALT level 48 h after intrahepatic injection of SLE-IgG (200 µg) in macrophage-depleted mice with clodronate liposomes ( Clod .)-pretreated and control mice. ** p

    Journal: Frontiers in Immunology

    Article Title: Role of Hepatic Deposited Immunoglobulin G in the Pathogenesis of Liver Damage in Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2018.01457

    Figure Lengend Snippet: Liver inflammation triggered by intrahepatic deposited lupus immunoglobulin G (IgG) depends on macrophages and TNF-α. (A) Immunohistochemistry detected Kupffer cells (F4/80+), dendritic cells (CD11c+), T cells (CD3+), and B cells (CD20+) in liver sections from lupus-prone mice. The results were from three independent experiments. Original magnification, 200× (B) Severity of liver inflammation and serum alanine aminotransferase (ALT) level 48 h after intrahepatic injection of systemic lupus erythematosus (SLE)-IgG (200 µg) in RAG-1 –/– and wild-type mice (the result is representative of three independent experiments, n = 5 mice per group/experiment). (C) Severity of liver inflammation and serum ALT level 48 h after intrahepatic injection of SLE-IgG (200 µg) in macrophage-depleted mice with clodronate liposomes ( Clod .)-pretreated and control mice. ** p

    Article Snippet: TNF-α and IFN-γ were detected using primary antibodies (Abs): anti-TNF-α (Abcam, ab6671) and anti-IFN-γ (Bioworld technology, BS3486) at 1/1,000 dilution.

    Techniques: Immunohistochemistry, Mouse Assay, Injection

    Synergistic effect of macrophage and natural killer (NK) cells on hepatic apoptosis induced by lupus immunoglobulin G (IgG). (A) Western blot showing the levels of TNF-α and IFN-γ in the liver from C57BL/6 mice with intrahepatic injection of the same dose of systemic lupus erythematosus (SLE) and normal serum, SLE, and normal IgG. (B) Flow cytometry analysis of NK cell activation in the liver from B6 lpr (25 w) and age-matched control mice using anti-NK1.1, anti-CD3e and anti CD69 antibodies. The result is representative of three independent experiments, n = 6 mice per group/experiment. (C) Flow cytometry analysis of NK cell activation in the liver from C57BL/6 mice with intrahepatic injection of SLE-serum (treated for 72 h), SLE-IgG (treated for 48 h), or phosphate-buffered saline (treated 48 h) using anti-NK1.1, anti-CD3e and anti CD69 antibodies (the result is representative of three independent experiments, n = 5 mice per group/experiment). (D) Severity of liver inflammation 48 h after intrahepatic injection of SLE-IgG in C57BL/6 mice with or without NK depletion using anti-ASGM1 antibody treatment. * p

    Journal: Frontiers in Immunology

    Article Title: Role of Hepatic Deposited Immunoglobulin G in the Pathogenesis of Liver Damage in Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2018.01457

    Figure Lengend Snippet: Synergistic effect of macrophage and natural killer (NK) cells on hepatic apoptosis induced by lupus immunoglobulin G (IgG). (A) Western blot showing the levels of TNF-α and IFN-γ in the liver from C57BL/6 mice with intrahepatic injection of the same dose of systemic lupus erythematosus (SLE) and normal serum, SLE, and normal IgG. (B) Flow cytometry analysis of NK cell activation in the liver from B6 lpr (25 w) and age-matched control mice using anti-NK1.1, anti-CD3e and anti CD69 antibodies. The result is representative of three independent experiments, n = 6 mice per group/experiment. (C) Flow cytometry analysis of NK cell activation in the liver from C57BL/6 mice with intrahepatic injection of SLE-serum (treated for 72 h), SLE-IgG (treated for 48 h), or phosphate-buffered saline (treated 48 h) using anti-NK1.1, anti-CD3e and anti CD69 antibodies (the result is representative of three independent experiments, n = 5 mice per group/experiment). (D) Severity of liver inflammation 48 h after intrahepatic injection of SLE-IgG in C57BL/6 mice with or without NK depletion using anti-ASGM1 antibody treatment. * p

    Article Snippet: TNF-α and IFN-γ were detected using primary antibodies (Abs): anti-TNF-α (Abcam, ab6671) and anti-IFN-γ (Bioworld technology, BS3486) at 1/1,000 dilution.

    Techniques: Western Blot, Mouse Assay, Injection, Flow Cytometry, Cytometry, Activation Assay

    MiR-145 directly targets MKK4. ( a ) A schematic illustrating the design of luciferase reporters with the WT MKK4 3′-UTR or the site-directed mutant MKK4 3′-UTR. ( b and c ) Expression level of MKK4 in chondrocytes transfected with miR-145 mimics, inhibitor or their negative controls. ( d ) Immunoblotting of MKK4 and p-MKK4 in chondrocytes transfected at different doses (10, 20, and 40 nM) and then stimulated with TNF- α . ( e and f ) Expression level of miR-145 and MKK4 in chondrocytes transfected with miR-145 overexpression plasmid, inhibition plasmid or empty plasmids (GV268, GV249). ( g ) The mRNA level of MKK4 in chondrocytes stimulated with TNF- α for different time points. ( h ) Immunoblotting of MKK4 in chondrocytes transfected with miR-145 mimics at different doses (10, 20, and 40 nM) and then stimulated with TNF- α . ( i ) Effect of miR-145 mimics or inhibitor on the luciferase activity of WT MKK4 3′-UTR (UTR) or MUT MKK4 3′-UTR (MT) reporter in SW1353 cells. ( j ) MKK4 expression level in different tissues from rat. ( k ) Immunoblotting of MKK4 and its downstream molecules in cartilage samples from OA patients ( n =3) or normal controls ( n =2). ( l and m ) Immunostaining of MKK4 and p-MKK4 in human ( n =6) and DMM-operated rat OA cartilage ( n =6). Scale bar: × 100, 50 μ m; × 200, 20 μ m. Data represent the mean±S.E.M. of at least n =4 independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: MicroRNA-145 attenuates TNF-α-driven cartilage matrix degradation in osteoarthritis via direct suppression of MKK4

    doi: 10.1038/cddis.2017.522

    Figure Lengend Snippet: MiR-145 directly targets MKK4. ( a ) A schematic illustrating the design of luciferase reporters with the WT MKK4 3′-UTR or the site-directed mutant MKK4 3′-UTR. ( b and c ) Expression level of MKK4 in chondrocytes transfected with miR-145 mimics, inhibitor or their negative controls. ( d ) Immunoblotting of MKK4 and p-MKK4 in chondrocytes transfected at different doses (10, 20, and 40 nM) and then stimulated with TNF- α . ( e and f ) Expression level of miR-145 and MKK4 in chondrocytes transfected with miR-145 overexpression plasmid, inhibition plasmid or empty plasmids (GV268, GV249). ( g ) The mRNA level of MKK4 in chondrocytes stimulated with TNF- α for different time points. ( h ) Immunoblotting of MKK4 in chondrocytes transfected with miR-145 mimics at different doses (10, 20, and 40 nM) and then stimulated with TNF- α . ( i ) Effect of miR-145 mimics or inhibitor on the luciferase activity of WT MKK4 3′-UTR (UTR) or MUT MKK4 3′-UTR (MT) reporter in SW1353 cells. ( j ) MKK4 expression level in different tissues from rat. ( k ) Immunoblotting of MKK4 and its downstream molecules in cartilage samples from OA patients ( n =3) or normal controls ( n =2). ( l and m ) Immunostaining of MKK4 and p-MKK4 in human ( n =6) and DMM-operated rat OA cartilage ( n =6). Scale bar: × 100, 50 μ m; × 200, 20 μ m. Data represent the mean±S.E.M. of at least n =4 independent experiments. * P

    Article Snippet: Enzyme-linked immunosorbent assay The cytokine and the degradation products of collagen II (C2C) production from the samples of human OA cartilage and surgery-induced OA in rats were assessed with TNF-α (560479; BD Biosciences, San Jose, CA, USA) or C2C (YM8649; Yuan Mu Bioscience, Shanghai, China) ELISA Kits (enzyme-linked immunosorbent assay).

    Techniques: Luciferase, Mutagenesis, Expressing, Transfection, Over Expression, Plasmid Preparation, Inhibition, Activity Assay, Immunostaining

    MiR-145 suppresses TNF- α -induced activation of JNK and p38 pathways. ( a and b ) Chondrocytes were transfected with miR-145 mimics, inhibitor, or their negative controls and then stimulated with TNF- α for different time periods as described. Protein levels of the main molecules involved in the NF- κ B and MAPK signaling pathways were analyzed by immunoblotting. ( c ) The nuclear import of p-c-Jun and p-ATF2 induced by TNF- α in chondrocytes transfected with miR-145 mimics, inhibitor, or their negative controls. Scale bar: 10 μ m. ( d ) The mRNA levels of MMP-3, MMP-13, and Adamts-5 in chondrocytes transfected with miR-145 inhibitor alone or in combination with SB203508 and SP600125 and then treated with TNF- α . ( e ) ChIP assays for binding of c-Jun or ATF2 to the promoter region of MMP-3 , MMP-13 , or Adamts-5 in TNF- α -treated chondrocytes. Normal rabbit IgG was used as the negative control. ( f ) Safranin-O staining and OARSI grade in sham- and DMM-operated rat IA injected with SP600125, SB203508 or vehicle ( n =8). Scale bar: × 50, 100 μ m; × 100, 50 μ m. Data represent the mean±S.E.M. of at least n =4 independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: MicroRNA-145 attenuates TNF-α-driven cartilage matrix degradation in osteoarthritis via direct suppression of MKK4

    doi: 10.1038/cddis.2017.522

    Figure Lengend Snippet: MiR-145 suppresses TNF- α -induced activation of JNK and p38 pathways. ( a and b ) Chondrocytes were transfected with miR-145 mimics, inhibitor, or their negative controls and then stimulated with TNF- α for different time periods as described. Protein levels of the main molecules involved in the NF- κ B and MAPK signaling pathways were analyzed by immunoblotting. ( c ) The nuclear import of p-c-Jun and p-ATF2 induced by TNF- α in chondrocytes transfected with miR-145 mimics, inhibitor, or their negative controls. Scale bar: 10 μ m. ( d ) The mRNA levels of MMP-3, MMP-13, and Adamts-5 in chondrocytes transfected with miR-145 inhibitor alone or in combination with SB203508 and SP600125 and then treated with TNF- α . ( e ) ChIP assays for binding of c-Jun or ATF2 to the promoter region of MMP-3 , MMP-13 , or Adamts-5 in TNF- α -treated chondrocytes. Normal rabbit IgG was used as the negative control. ( f ) Safranin-O staining and OARSI grade in sham- and DMM-operated rat IA injected with SP600125, SB203508 or vehicle ( n =8). Scale bar: × 50, 100 μ m; × 100, 50 μ m. Data represent the mean±S.E.M. of at least n =4 independent experiments. * P

    Article Snippet: Enzyme-linked immunosorbent assay The cytokine and the degradation products of collagen II (C2C) production from the samples of human OA cartilage and surgery-induced OA in rats were assessed with TNF-α (560479; BD Biosciences, San Jose, CA, USA) or C2C (YM8649; Yuan Mu Bioscience, Shanghai, China) ELISA Kits (enzyme-linked immunosorbent assay).

    Techniques: Activation Assay, Transfection, Chromatin Immunoprecipitation, Binding Assay, Negative Control, Staining, IA, Injection

    Requirement for NF- κ B-binding site in the regulation of miR-145. ( a ) Expression level of miR-145 in chondrocytes pretreated with MAPK inhibitors (SB203508, SP600125, or PD98059, 10 μ M) or NF- κ B inhibitor (BAY11-7082, 5 μ M) for 2 h and then cultured with or without TNF- α for 12 h. ( b ) The mRNA levels of p65, traf2, and tradd in chondrocytes transfected with p65 siRNA, traf2 siRNA, tradd siRNA, or negative control (scramble siRNA, Scr-siR). ( c ) The protein level of p65 in chondrocytes transfected with p65 siRNA or Scr-siR. ( d ) Immunoblotting of p65 and p-p65 in chondrocytes transfected with siRNAs for p65 , traf2 , or tradd . ( e ) Expression level of miR-145 in chondrocytes transfected as described above and then cultured with or without TNF- α . ( f ) Human, mouse, and rat sequences of putative NF- κ B-binding sites (red) and their flanking regions in miR-145 promoter. ( g ) Binding of NF- κ B subunit p65 to miR-145 promoter was determined by the ChIP assay; normal rabbit IgG was used as the negative control. ( h ) Luciferase activity in lysates of SW1353 cells transfected with luciferase reporter plasmids of empty vector, miR-145 promoter, or miR-145 promoter with mutation of the p65-binding site, and then left unstimulated or stimulated with TNF- α for 12 h. Results were presented relative to Renilla luciferase activity. Data represent the mean±S.E.M. of at least n =4 independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: MicroRNA-145 attenuates TNF-α-driven cartilage matrix degradation in osteoarthritis via direct suppression of MKK4

    doi: 10.1038/cddis.2017.522

    Figure Lengend Snippet: Requirement for NF- κ B-binding site in the regulation of miR-145. ( a ) Expression level of miR-145 in chondrocytes pretreated with MAPK inhibitors (SB203508, SP600125, or PD98059, 10 μ M) or NF- κ B inhibitor (BAY11-7082, 5 μ M) for 2 h and then cultured with or without TNF- α for 12 h. ( b ) The mRNA levels of p65, traf2, and tradd in chondrocytes transfected with p65 siRNA, traf2 siRNA, tradd siRNA, or negative control (scramble siRNA, Scr-siR). ( c ) The protein level of p65 in chondrocytes transfected with p65 siRNA or Scr-siR. ( d ) Immunoblotting of p65 and p-p65 in chondrocytes transfected with siRNAs for p65 , traf2 , or tradd . ( e ) Expression level of miR-145 in chondrocytes transfected as described above and then cultured with or without TNF- α . ( f ) Human, mouse, and rat sequences of putative NF- κ B-binding sites (red) and their flanking regions in miR-145 promoter. ( g ) Binding of NF- κ B subunit p65 to miR-145 promoter was determined by the ChIP assay; normal rabbit IgG was used as the negative control. ( h ) Luciferase activity in lysates of SW1353 cells transfected with luciferase reporter plasmids of empty vector, miR-145 promoter, or miR-145 promoter with mutation of the p65-binding site, and then left unstimulated or stimulated with TNF- α for 12 h. Results were presented relative to Renilla luciferase activity. Data represent the mean±S.E.M. of at least n =4 independent experiments. * P

    Article Snippet: Enzyme-linked immunosorbent assay The cytokine and the degradation products of collagen II (C2C) production from the samples of human OA cartilage and surgery-induced OA in rats were assessed with TNF-α (560479; BD Biosciences, San Jose, CA, USA) or C2C (YM8649; Yuan Mu Bioscience, Shanghai, China) ELISA Kits (enzyme-linked immunosorbent assay).

    Techniques: Binding Assay, Expressing, Cell Culture, Transfection, Negative Control, Chromatin Immunoprecipitation, Luciferase, Activity Assay, Plasmid Preparation, Mutagenesis

    MiR-145 counteracts cartilage matrix degradation in surgery-induced OA. ( a ) The diagrammatic sketch of agomir/antagomir-145 with specific chemical modifications. ( b ) Schematic of the animal experiments (for each group, n =6, rat). ( c and d ) Transfection efficiency of agomir labeled by cy5 in articular cartilage or chondrocytes compared with the saline group ( n =6). Scale bar: 50 μ m. ( e ) Safranin-O staining and OARSI grade in sham-operated and DMM-operated rat IA injected with agomir-145, antagomir-145, or PBS ( n =8). Scale bar: × 50, 100 μ m; × 100, 50 μ m. ( f ) Immunostaining of MMP-3, MMP-13, p-MKK4, p-c-Jun, and p-ATF2 in cartilage tissue of sham- and DMM-operated rat IA injected with agomir-145, antagomir-145, or PBS ( n =8). Scale bar: 20 μ m. ( g ) Diagram depicting the signaling pathways for miR-145 in the regulation of TNF- α -induced expression of matrix-degrading enzymes in chondrocytes. P, phosphorylation. Data represent the mean±S.E.M. of at least n =4 independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: MicroRNA-145 attenuates TNF-α-driven cartilage matrix degradation in osteoarthritis via direct suppression of MKK4

    doi: 10.1038/cddis.2017.522

    Figure Lengend Snippet: MiR-145 counteracts cartilage matrix degradation in surgery-induced OA. ( a ) The diagrammatic sketch of agomir/antagomir-145 with specific chemical modifications. ( b ) Schematic of the animal experiments (for each group, n =6, rat). ( c and d ) Transfection efficiency of agomir labeled by cy5 in articular cartilage or chondrocytes compared with the saline group ( n =6). Scale bar: 50 μ m. ( e ) Safranin-O staining and OARSI grade in sham-operated and DMM-operated rat IA injected with agomir-145, antagomir-145, or PBS ( n =8). Scale bar: × 50, 100 μ m; × 100, 50 μ m. ( f ) Immunostaining of MMP-3, MMP-13, p-MKK4, p-c-Jun, and p-ATF2 in cartilage tissue of sham- and DMM-operated rat IA injected with agomir-145, antagomir-145, or PBS ( n =8). Scale bar: 20 μ m. ( g ) Diagram depicting the signaling pathways for miR-145 in the regulation of TNF- α -induced expression of matrix-degrading enzymes in chondrocytes. P, phosphorylation. Data represent the mean±S.E.M. of at least n =4 independent experiments. * P

    Article Snippet: Enzyme-linked immunosorbent assay The cytokine and the degradation products of collagen II (C2C) production from the samples of human OA cartilage and surgery-induced OA in rats were assessed with TNF-α (560479; BD Biosciences, San Jose, CA, USA) or C2C (YM8649; Yuan Mu Bioscience, Shanghai, China) ELISA Kits (enzyme-linked immunosorbent assay).

    Techniques: Transfection, Labeling, Staining, IA, Injection, Immunostaining, Expressing

    MiR-145 represses TNF- α -induced expression of matrix-degrading enzymes in chondrocytes. ( a–d ) Chondrocytes were transfected with miR-145 mimics, inhibitor, or their negative controls at a final concentration of 40 nM. At 24 h after transfection, ( a ) the expression level of miR-145 was measured by qRT-PCR and normalized to U6. Cell apoptosis was detected by ( b ) Annexin V-FITC/propidium iodide double staining with FACS analysis and ( d ) TUNEL staining. Scale bar: 10 μ m. ( c ) Quantitative results of the FACS analysis. ( e ) The mRNA and ( f ) protein levels of MMP-3, MMP-13, and Adamts-5 in chondrocytes transfected with miR-145 mimics, inhibitor, or their negative controls, and then cultured with or without TNF- α . ( g ) Immunofluorescence of MMP-3, MMP-13, and Adamts-5. Scale bar: 50 μ m. ( h ) The mRNA levels of MMP-3, MMP-13, and Adamts-5 in chondrocytes transfected as described above and then stimulated with TNF- α for different time periods as indicated. Data represent the mean±S.E.M. of at least n =4 independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: MicroRNA-145 attenuates TNF-α-driven cartilage matrix degradation in osteoarthritis via direct suppression of MKK4

    doi: 10.1038/cddis.2017.522

    Figure Lengend Snippet: MiR-145 represses TNF- α -induced expression of matrix-degrading enzymes in chondrocytes. ( a–d ) Chondrocytes were transfected with miR-145 mimics, inhibitor, or their negative controls at a final concentration of 40 nM. At 24 h after transfection, ( a ) the expression level of miR-145 was measured by qRT-PCR and normalized to U6. Cell apoptosis was detected by ( b ) Annexin V-FITC/propidium iodide double staining with FACS analysis and ( d ) TUNEL staining. Scale bar: 10 μ m. ( c ) Quantitative results of the FACS analysis. ( e ) The mRNA and ( f ) protein levels of MMP-3, MMP-13, and Adamts-5 in chondrocytes transfected with miR-145 mimics, inhibitor, or their negative controls, and then cultured with or without TNF- α . ( g ) Immunofluorescence of MMP-3, MMP-13, and Adamts-5. Scale bar: 50 μ m. ( h ) The mRNA levels of MMP-3, MMP-13, and Adamts-5 in chondrocytes transfected as described above and then stimulated with TNF- α for different time periods as indicated. Data represent the mean±S.E.M. of at least n =4 independent experiments. * P

    Article Snippet: Enzyme-linked immunosorbent assay The cytokine and the degradation products of collagen II (C2C) production from the samples of human OA cartilage and surgery-induced OA in rats were assessed with TNF-α (560479; BD Biosciences, San Jose, CA, USA) or C2C (YM8649; Yuan Mu Bioscience, Shanghai, China) ELISA Kits (enzyme-linked immunosorbent assay).

    Techniques: Expressing, Transfection, Concentration Assay, Quantitative RT-PCR, Double Staining, FACS, TUNEL Assay, Staining, Cell Culture, Immunofluorescence

    Expression level of miR-145 in TNF- α -stimulated chondrocytes and OA cartilage. ( a ) The mRNA levels of proinflammatory cytokines in rat cartilage obtained from the sham group ( n =6) and DMM groups ( n =6) 15, 30, 45, and 60 days after the surgery. ( b ) Production of TNF- α and C2C in rat serum obtained from the sham group ( n =9), DMM groups ( n =6–9), DMSO group ( n =7–9), and CC-5013 group ( n =7-10) were measured by ELISA. ( c ) Cartilage destruction and OARSI grade in sham- and DMM-operated (60 days) rat IA injected with CC-5013 or vehicle ( n =8). ( d ) Heatmaps of miRNAs differentially expressed in TNF- α -stimulated chondrocytes. ( e ) Expression levels of the indicated miRNAs in TNF- α -stimulated chondrocytes. ( f ) Quantitative real-time PCR (qRT-PCR) validation of miR-92a, miR-23b, and miR-145 expression in chondrocytes stimulated with TNF- α at different doses for 24 h. ( g ) Expression of the five miRNAs in chondrocytes stimulated with TNF- α for different time periods were analyzed by qRT-PCR. ( h and i ) Immunoblotting of catabolic and anabolic factors, expression of miR-145, ELISA results of TNF- α , and hematoxylin and eosin (HE) staining in cartilage from OA patients ( n =3) or normal ( n =2). Scale bar: 20 μ m. ( j ) Correlation between miR-145 and TNF- α in the cartilage of OA patients and DMM-operated rat. ( k ) Expression level of miR-145 in rat cartilage obtained from the sham group ( n =6), DMM groups ( n =6), DMSO group ( n =6), and CC-5013 group ( n =6). Data represent the mean±S.E.M. of at least n =4 independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: MicroRNA-145 attenuates TNF-α-driven cartilage matrix degradation in osteoarthritis via direct suppression of MKK4

    doi: 10.1038/cddis.2017.522

    Figure Lengend Snippet: Expression level of miR-145 in TNF- α -stimulated chondrocytes and OA cartilage. ( a ) The mRNA levels of proinflammatory cytokines in rat cartilage obtained from the sham group ( n =6) and DMM groups ( n =6) 15, 30, 45, and 60 days after the surgery. ( b ) Production of TNF- α and C2C in rat serum obtained from the sham group ( n =9), DMM groups ( n =6–9), DMSO group ( n =7–9), and CC-5013 group ( n =7-10) were measured by ELISA. ( c ) Cartilage destruction and OARSI grade in sham- and DMM-operated (60 days) rat IA injected with CC-5013 or vehicle ( n =8). ( d ) Heatmaps of miRNAs differentially expressed in TNF- α -stimulated chondrocytes. ( e ) Expression levels of the indicated miRNAs in TNF- α -stimulated chondrocytes. ( f ) Quantitative real-time PCR (qRT-PCR) validation of miR-92a, miR-23b, and miR-145 expression in chondrocytes stimulated with TNF- α at different doses for 24 h. ( g ) Expression of the five miRNAs in chondrocytes stimulated with TNF- α for different time periods were analyzed by qRT-PCR. ( h and i ) Immunoblotting of catabolic and anabolic factors, expression of miR-145, ELISA results of TNF- α , and hematoxylin and eosin (HE) staining in cartilage from OA patients ( n =3) or normal ( n =2). Scale bar: 20 μ m. ( j ) Correlation between miR-145 and TNF- α in the cartilage of OA patients and DMM-operated rat. ( k ) Expression level of miR-145 in rat cartilage obtained from the sham group ( n =6), DMM groups ( n =6), DMSO group ( n =6), and CC-5013 group ( n =6). Data represent the mean±S.E.M. of at least n =4 independent experiments. * P

    Article Snippet: Enzyme-linked immunosorbent assay The cytokine and the degradation products of collagen II (C2C) production from the samples of human OA cartilage and surgery-induced OA in rats were assessed with TNF-α (560479; BD Biosciences, San Jose, CA, USA) or C2C (YM8649; Yuan Mu Bioscience, Shanghai, China) ELISA Kits (enzyme-linked immunosorbent assay).

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, IA, Injection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Staining

    MKK4 regulates TNF- α -triggered matrix-degrading enzymes and cartilage degradation. ( a and b ) The mRNA and protein levels of MKK4 in chondrocytes transfected with Scr-siR or MKK4 siRNA (#1). ( c and d ) The mRNA level of MKK4 and protein levels of MKK4, p-MKK4, and p-c-Jun in chondrocytes infected with Len-C (lentivirus containing empty vector), Len-sh- MKK4 (lentivirus containing sh- MKK4 ), or Len- MKK4 (lentivirus containing the entire CDS sequence of MKK4 ) at the indicated multiplicity of infection (MOI=50 or 100). ( e – g ) Chondrocytes were infected with Len-C, Len-sh- MKK4 , or Len- MKK4 (100 MOI). At 48 h after infection, the cells were stimulated with TNF- α . The mRNA levels of MMP-3, MMP-13, and Adamts-5 were evaluated by qRT-PCR. The protein levels of catabolic factors, anabolic factors, MKK4, and its downstream molecules were measured by immunoblotting. ( h ) Safranin-O staining and OARSI grade in sham- and DMM-operated rat IA injected with Len-C, Len-sh- MKK4 , or Len- MKK4 ( n =8). Scale bar: 50 μ m. ( i ) Immunostaining of p-MKK4, MMP-3, and MMP-13 in cartilage tissue of sham-operated and DMM-operated rat IA injected with Len-C, Len-sh- MKK4 , or Len- MKK4 ( n =8). Scale bar: 20 μ m. Data represent the mean±S.E.M. of at least n =4 independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: MicroRNA-145 attenuates TNF-α-driven cartilage matrix degradation in osteoarthritis via direct suppression of MKK4

    doi: 10.1038/cddis.2017.522

    Figure Lengend Snippet: MKK4 regulates TNF- α -triggered matrix-degrading enzymes and cartilage degradation. ( a and b ) The mRNA and protein levels of MKK4 in chondrocytes transfected with Scr-siR or MKK4 siRNA (#1). ( c and d ) The mRNA level of MKK4 and protein levels of MKK4, p-MKK4, and p-c-Jun in chondrocytes infected with Len-C (lentivirus containing empty vector), Len-sh- MKK4 (lentivirus containing sh- MKK4 ), or Len- MKK4 (lentivirus containing the entire CDS sequence of MKK4 ) at the indicated multiplicity of infection (MOI=50 or 100). ( e – g ) Chondrocytes were infected with Len-C, Len-sh- MKK4 , or Len- MKK4 (100 MOI). At 48 h after infection, the cells were stimulated with TNF- α . The mRNA levels of MMP-3, MMP-13, and Adamts-5 were evaluated by qRT-PCR. The protein levels of catabolic factors, anabolic factors, MKK4, and its downstream molecules were measured by immunoblotting. ( h ) Safranin-O staining and OARSI grade in sham- and DMM-operated rat IA injected with Len-C, Len-sh- MKK4 , or Len- MKK4 ( n =8). Scale bar: 50 μ m. ( i ) Immunostaining of p-MKK4, MMP-3, and MMP-13 in cartilage tissue of sham-operated and DMM-operated rat IA injected with Len-C, Len-sh- MKK4 , or Len- MKK4 ( n =8). Scale bar: 20 μ m. Data represent the mean±S.E.M. of at least n =4 independent experiments. * P

    Article Snippet: Enzyme-linked immunosorbent assay The cytokine and the degradation products of collagen II (C2C) production from the samples of human OA cartilage and surgery-induced OA in rats were assessed with TNF-α (560479; BD Biosciences, San Jose, CA, USA) or C2C (YM8649; Yuan Mu Bioscience, Shanghai, China) ELISA Kits (enzyme-linked immunosorbent assay).

    Techniques: Transfection, Infection, Plasmid Preparation, Sequencing, Quantitative RT-PCR, Staining, IA, Injection, Immunostaining

    PTPN1 negatively regulates the JNK pathway via JNK dephosphorylation. ( A–C) Phosphorylation levels of each kinase were examined by immunoblot analysis. PTPN1 was incubated with phosphorylated form of (A) HA-MLK3 and (B) GST-MKK7. (C) Phosphorylated GST-JNK was incubated with PTPN1 (30 min, 37 °C), and then GST-cJun and ATP were added to perform a JNK kinase assay. (D) Flag-PTPN1 was expressed in 293 T cells. Control cells were transfected with an empty vector. After TNFα-stimulation, samples were prepared at the indicated time-points. JNK phosphorylation levels, with or without PTPN1 expression, were examined by immunoblot analysis using an anti-dual phospho-JNK antibody. (E) The density of immunoblotted bands detected using the anti-dual phospho-JNK antibody is plotted. (* p

    Journal: Scientific Reports

    Article Title: Identification of PTPN1 as a novel negative regulator of the JNK MAPK pathway using a synthetic screening for pathway-specific phosphatases

    doi: 10.1038/s41598-017-13494-x

    Figure Lengend Snippet: PTPN1 negatively regulates the JNK pathway via JNK dephosphorylation. ( A–C) Phosphorylation levels of each kinase were examined by immunoblot analysis. PTPN1 was incubated with phosphorylated form of (A) HA-MLK3 and (B) GST-MKK7. (C) Phosphorylated GST-JNK was incubated with PTPN1 (30 min, 37 °C), and then GST-cJun and ATP were added to perform a JNK kinase assay. (D) Flag-PTPN1 was expressed in 293 T cells. Control cells were transfected with an empty vector. After TNFα-stimulation, samples were prepared at the indicated time-points. JNK phosphorylation levels, with or without PTPN1 expression, were examined by immunoblot analysis using an anti-dual phospho-JNK antibody. (E) The density of immunoblotted bands detected using the anti-dual phospho-JNK antibody is plotted. (* p

    Article Snippet: Prior to treatment with TNFα, cells were incubated in serum-free medium (2 h), and then stimulated with TNFα (BD) for the indicated time.

    Techniques: De-Phosphorylation Assay, Incubation, Kinase Assay, Transfection, Plasmid Preparation, Expressing

    PTPN1 regulates JNK-related cell death response. ( A) Flag-PTPN1 or Flag-PTPN1 D181A was expressed in 293 T cells. Control cells were transfected with an empty vector. Upon TNFα-stimulation, protein expression and JNK phosphorylation were each examined by immunoblotting. (B) JNK activity was assessed using a luciferase reporter gene assay. The data are presented as the mean ± SD (repeats were performed in triplicate). (** p

    Journal: Scientific Reports

    Article Title: Identification of PTPN1 as a novel negative regulator of the JNK MAPK pathway using a synthetic screening for pathway-specific phosphatases

    doi: 10.1038/s41598-017-13494-x

    Figure Lengend Snippet: PTPN1 regulates JNK-related cell death response. ( A) Flag-PTPN1 or Flag-PTPN1 D181A was expressed in 293 T cells. Control cells were transfected with an empty vector. Upon TNFα-stimulation, protein expression and JNK phosphorylation were each examined by immunoblotting. (B) JNK activity was assessed using a luciferase reporter gene assay. The data are presented as the mean ± SD (repeats were performed in triplicate). (** p

    Article Snippet: Prior to treatment with TNFα, cells were incubated in serum-free medium (2 h), and then stimulated with TNFα (BD) for the indicated time.

    Techniques: Transfection, Plasmid Preparation, Expressing, Activity Assay, Luciferase, Reporter Gene Assay

    PTPN1 is a novel negative regulator of the JNK pathway. ( A) Schematic illustration of the screening method. Phosphatases were expressed as JIP1-phosphatase fusion proteins and a decrease in JNK signaling was monitored. (B) The decrease of JNK phosphorylation by JIP1-phosphatase fusion was examined by immunoblot analysis using an anti-dual phospho-JNK antibody. 293 T cells were incubated for 24 h after transfection, and then treated with TNFα (15 min). (C) Negative regulation of the JNK pathway by the analyzed phosphatases was confirmed by co-expression of individual phosphatases and JIP1. All experiments were performed at least three times.

    Journal: Scientific Reports

    Article Title: Identification of PTPN1 as a novel negative regulator of the JNK MAPK pathway using a synthetic screening for pathway-specific phosphatases

    doi: 10.1038/s41598-017-13494-x

    Figure Lengend Snippet: PTPN1 is a novel negative regulator of the JNK pathway. ( A) Schematic illustration of the screening method. Phosphatases were expressed as JIP1-phosphatase fusion proteins and a decrease in JNK signaling was monitored. (B) The decrease of JNK phosphorylation by JIP1-phosphatase fusion was examined by immunoblot analysis using an anti-dual phospho-JNK antibody. 293 T cells were incubated for 24 h after transfection, and then treated with TNFα (15 min). (C) Negative regulation of the JNK pathway by the analyzed phosphatases was confirmed by co-expression of individual phosphatases and JIP1. All experiments were performed at least three times.

    Article Snippet: Prior to treatment with TNFα, cells were incubated in serum-free medium (2 h), and then stimulated with TNFα (BD) for the indicated time.

    Techniques: Incubation, Transfection, Expressing

    PTPN1 interacts with the components of the JNK pathway. PTPN1 interaction with (A) MLK3, (B) MKK7, (C) JNK, and (D) JIP1 was observed via immunoprecipitation assay. Each sample was prepared by TNFα-treatment at the indicated time-point. All experiments were performed at least three times.

    Journal: Scientific Reports

    Article Title: Identification of PTPN1 as a novel negative regulator of the JNK MAPK pathway using a synthetic screening for pathway-specific phosphatases

    doi: 10.1038/s41598-017-13494-x

    Figure Lengend Snippet: PTPN1 interacts with the components of the JNK pathway. PTPN1 interaction with (A) MLK3, (B) MKK7, (C) JNK, and (D) JIP1 was observed via immunoprecipitation assay. Each sample was prepared by TNFα-treatment at the indicated time-point. All experiments were performed at least three times.

    Article Snippet: Prior to treatment with TNFα, cells were incubated in serum-free medium (2 h), and then stimulated with TNFα (BD) for the indicated time.

    Techniques: Immunoprecipitation

    VLP effectively stimulates BMDCs in vitro to secrete cytokines. BMDCs were stimulated with M2e5x, M1 VLP, or protein (0.3, 1, 5 μg/mL) for 24 h. ( a – c ) The levels of cytokines ( a ) TNF-α, ( b ) IL-6, ( c ) IFN-γ were determined in the culture supernatants by ELISA assay. Un: Medium only, P: proteins, V: VLP. The statistical significance was confirmed by one-way ANOVA and Dunnett’s multiple comparison test. Error bars indicate the means ± SEM of concentrations from individual animals. *; p

    Journal: Vaccines

    Article Title: Virus-Like Particles Are a Superior Platform for Presenting M2e Epitopes to Prime Humoral and Cellular Immunity against Influenza Virus

    doi: 10.3390/vaccines6040066

    Figure Lengend Snippet: VLP effectively stimulates BMDCs in vitro to secrete cytokines. BMDCs were stimulated with M2e5x, M1 VLP, or protein (0.3, 1, 5 μg/mL) for 24 h. ( a – c ) The levels of cytokines ( a ) TNF-α, ( b ) IL-6, ( c ) IFN-γ were determined in the culture supernatants by ELISA assay. Un: Medium only, P: proteins, V: VLP. The statistical significance was confirmed by one-way ANOVA and Dunnett’s multiple comparison test. Error bars indicate the means ± SEM of concentrations from individual animals. *; p

    Article Snippet: Briefly, interleukin-6 (IL-6), IL-10, tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) kits (eBioscience, San Diego, CA, USA) were used to determine the level of cytokines in the BALF and culture supernatants according to the manufacturer’s procedures.

    Techniques: In Vitro, Enzyme-linked Immunosorbent Assay

    mCD40L-induced TNF -α does not contribute to CD40L-induced cell death. ( a ) EJ cells were infected with 100 MOI of either RAdMock (AdM) or RAdnCD40L (AdnL) or left uninfected as a negative control for 24 h, RNA was extracted utilising the EZ-RNA total isolation kit and the cDNA was prepared by reverse transcription. The expression of TNF -α was examined by qRT-PCR technique. Results are mean of triplicate samples ±S.D. ( b ) EJ cells were infected with 100 MOI of either RAdMock (AdM) or RAdnCD40L (AdnL) or left uninfected as a negative control and plated at a density of 6000/100 μ l/well in 96-well microplate. AdnL-infected cells were either treated with 1, 3 or 5 μ g/ml of TNF -α monoclonal neutralising antibody or left untreated as a control for 28 h. Cell viability was then assessed using the WST-1 assay. Results are mean of triplicate samples ±S.D.

    Journal: Cell Death & Disease

    Article Title: NORE1A induction by membrane-bound CD40L (mCD40L) contributes to CD40L-induced cell death and G1 growth arrest in p21-mediated mechanism

    doi: 10.1038/cddis.2016.52

    Figure Lengend Snippet: mCD40L-induced TNF -α does not contribute to CD40L-induced cell death. ( a ) EJ cells were infected with 100 MOI of either RAdMock (AdM) or RAdnCD40L (AdnL) or left uninfected as a negative control for 24 h, RNA was extracted utilising the EZ-RNA total isolation kit and the cDNA was prepared by reverse transcription. The expression of TNF -α was examined by qRT-PCR technique. Results are mean of triplicate samples ±S.D. ( b ) EJ cells were infected with 100 MOI of either RAdMock (AdM) or RAdnCD40L (AdnL) or left uninfected as a negative control and plated at a density of 6000/100 μ l/well in 96-well microplate. AdnL-infected cells were either treated with 1, 3 or 5 μ g/ml of TNF -α monoclonal neutralising antibody or left untreated as a control for 28 h. Cell viability was then assessed using the WST-1 assay. Results are mean of triplicate samples ±S.D.

    Article Snippet: To examine whether TNF-α contributes the AdnL-induced cell death, cells were treated with escalating doses of TNF-α -neutralising monoclonal antibody (1, 3 and 5 μ g/ml) (Clone D2H4 mAb#11969, Cell Signalling), followed by assessment of cell viability.

    Techniques: Infection, Negative Control, Isolation, Expressing, Quantitative RT-PCR, WST-1 Assay

    TNF-α induces Bcl6 gene expression in the hepatocytes and Bcl6 overexpression upregulates the TNF-α levels in vivo . (A) The RNA levels of Tnf- α, Il-6, Il-12, Cxcl9, and Cxcl10 were examined by RT-qPCR in B6.2- and B6.2S-injected mouse liver isolated during the HBV clearance stage. (B) AML12 cells were treated with or without TNF-α (100 ng/ml) for the indicated time periods and Bcl6 RNA was analyzed by RT-qPCR. The induction fold means the ratio of the Bcl6 RNA levels in the presence of TNF-α over those without TNF-α. (C) AML12 and Huh-7 cells were transfected with 1 μg of vector or the Bcl6 -expressing plasmid. Two days later, the TNF-α RNA levels were analyzed by RT-qPCR analysis. (D) The liver RNA from the B6.2 or the (B6.2 + Bcl6 ) mice at 2 w.p.i. were analyzed for Tnf- α expression by RT-qPCR. In (A–D) , * P

    Journal: Frontiers in Microbiology

    Article Title: B-Cell Lymphoma 6 (BCL6) Is a Host Restriction Factor That Can Suppress HBV Gene Expression and Modulate Immune Responses

    doi: 10.3389/fmicb.2018.03253

    Figure Lengend Snippet: TNF-α induces Bcl6 gene expression in the hepatocytes and Bcl6 overexpression upregulates the TNF-α levels in vivo . (A) The RNA levels of Tnf- α, Il-6, Il-12, Cxcl9, and Cxcl10 were examined by RT-qPCR in B6.2- and B6.2S-injected mouse liver isolated during the HBV clearance stage. (B) AML12 cells were treated with or without TNF-α (100 ng/ml) for the indicated time periods and Bcl6 RNA was analyzed by RT-qPCR. The induction fold means the ratio of the Bcl6 RNA levels in the presence of TNF-α over those without TNF-α. (C) AML12 and Huh-7 cells were transfected with 1 μg of vector or the Bcl6 -expressing plasmid. Two days later, the TNF-α RNA levels were analyzed by RT-qPCR analysis. (D) The liver RNA from the B6.2 or the (B6.2 + Bcl6 ) mice at 2 w.p.i. were analyzed for Tnf- α expression by RT-qPCR. In (A–D) , * P

    Article Snippet: The cytokines used for stimulating Bcl6 expression or chemokine induction included TNF-α (315-01A, PeProtech, USA) and IFN-γ (485MI, R & D, USA).

    Techniques: Expressing, Over Expression, In Vivo, Quantitative RT-PCR, Injection, Isolation, Transfection, Plasmid Preparation, Mouse Assay

    BCL6 expression synergizes TNF-α-induced Cxcl9/Cxcl10 expression and enhances immune cell infiltration into the liver. (A,B) AML12 cells, (C) Huh-7 cells, were transfected with 1 μg of vector or the Bcl6 -expressing plasmid. Two days later, the cells were treated with or without (A,C) TNF-α (100 ng/ml) or (B) IFN-γ (50 ng/ml) for 3 h. RNA was extracted and analyzed for Cxcl9/Cxcl10 expression by RT-qPCR. (D) The liver RNA from the B6.2 or the (B6.2 + Bcl6 ) mice at 2 w.p.i. were analyzed for Cxcl9/Cxcl10 expression by RT-qPCR and (E) the liver tissues were analyzed by hematoxylin eosin staining. The arrows indicate the infiltrating immune cells. The images are displayed at 400X magnification. The average number of infiltrating foci were obtained from 15 different fields for each mouse ( N = 5) at 200X magnification and presented as the mean ± SD . LPF, low power field. * P

    Journal: Frontiers in Microbiology

    Article Title: B-Cell Lymphoma 6 (BCL6) Is a Host Restriction Factor That Can Suppress HBV Gene Expression and Modulate Immune Responses

    doi: 10.3389/fmicb.2018.03253

    Figure Lengend Snippet: BCL6 expression synergizes TNF-α-induced Cxcl9/Cxcl10 expression and enhances immune cell infiltration into the liver. (A,B) AML12 cells, (C) Huh-7 cells, were transfected with 1 μg of vector or the Bcl6 -expressing plasmid. Two days later, the cells were treated with or without (A,C) TNF-α (100 ng/ml) or (B) IFN-γ (50 ng/ml) for 3 h. RNA was extracted and analyzed for Cxcl9/Cxcl10 expression by RT-qPCR. (D) The liver RNA from the B6.2 or the (B6.2 + Bcl6 ) mice at 2 w.p.i. were analyzed for Cxcl9/Cxcl10 expression by RT-qPCR and (E) the liver tissues were analyzed by hematoxylin eosin staining. The arrows indicate the infiltrating immune cells. The images are displayed at 400X magnification. The average number of infiltrating foci were obtained from 15 different fields for each mouse ( N = 5) at 200X magnification and presented as the mean ± SD . LPF, low power field. * P

    Article Snippet: The cytokines used for stimulating Bcl6 expression or chemokine induction included TNF-α (315-01A, PeProtech, USA) and IFN-γ (485MI, R & D, USA).

    Techniques: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Mouse Assay, Staining

    Working model for the induction and anti-HBV activity of BCL6 in vivo ), followed by infiltration of many other immune cells. (2) These immune cells produce diverse cytokines in the liver. (3) The inflammatory cytokines such as TNF-α can upregulate Bcl6 expression. (4) BCL6 can repress HBV promoter activity, and (5) meanwhile synergizes TNF-α signaling to produce large amounts of CXCL9/CXCL10 chemokines. (6) The chemokines recruit more immune cells infiltrating to the livers and (7) produce higher levels of cytokines and chemokines. These immune cells, cytokines together with the BCL6 functions may ultimately lead to HBV clearance.

    Journal: Frontiers in Microbiology

    Article Title: B-Cell Lymphoma 6 (BCL6) Is a Host Restriction Factor That Can Suppress HBV Gene Expression and Modulate Immune Responses

    doi: 10.3389/fmicb.2018.03253

    Figure Lengend Snippet: Working model for the induction and anti-HBV activity of BCL6 in vivo ), followed by infiltration of many other immune cells. (2) These immune cells produce diverse cytokines in the liver. (3) The inflammatory cytokines such as TNF-α can upregulate Bcl6 expression. (4) BCL6 can repress HBV promoter activity, and (5) meanwhile synergizes TNF-α signaling to produce large amounts of CXCL9/CXCL10 chemokines. (6) The chemokines recruit more immune cells infiltrating to the livers and (7) produce higher levels of cytokines and chemokines. These immune cells, cytokines together with the BCL6 functions may ultimately lead to HBV clearance.

    Article Snippet: The cytokines used for stimulating Bcl6 expression or chemokine induction included TNF-α (315-01A, PeProtech, USA) and IFN-γ (485MI, R & D, USA).

    Techniques: Activity Assay, In Vivo, Expressing

    Regulatory effects of curculigoside on the expression levels of NF-κB p65 (C) and IκB in TNF-α-stimulated MH7A cells. Cells were treated with TNF-α (10 ng/ml) for 12 h, exposed to curculigoside (4 and 16 µg/ml) for a further 24 h and then subjected to western blotting assays to determine the expression levels of NF-κB and IκB (n=4). **P

    Journal: Molecular Medicine Reports

    Article Title: Curculigoside exerts significant anti-arthritic effects in vivo and in vitro via regulation of the JAK/STAT/NF-κB signaling pathway

    doi: 10.3892/mmr.2019.9854

    Figure Lengend Snippet: Regulatory effects of curculigoside on the expression levels of NF-κB p65 (C) and IκB in TNF-α-stimulated MH7A cells. Cells were treated with TNF-α (10 ng/ml) for 12 h, exposed to curculigoside (4 and 16 µg/ml) for a further 24 h and then subjected to western blotting assays to determine the expression levels of NF-κB and IκB (n=4). **P

    Article Snippet: Tumor necrosis factor (TNF)-α (cat. no. RAB0477), dimethyl sulfoxide (DMSO) and IκB (cat. no. SAB1305978) were purchased from Sigma-Aldrich; Merck KGaA.

    Techniques: Expressing, Western Blot

    Regulatory effects of curculigoside on the expression levels of JAK1, JAK3 and STAT3 in TNF-α-stimulated MH7A cells were determined. Cells were treated with TNF-α (10 ng/ml) for 12 h, exposed to curculigoside (4 and 16 µg/ml) for a further 24 h and then subjected to western blotting assays to determine the expression levels of NF-κB and IκB (n=4). **P

    Journal: Molecular Medicine Reports

    Article Title: Curculigoside exerts significant anti-arthritic effects in vivo and in vitro via regulation of the JAK/STAT/NF-κB signaling pathway

    doi: 10.3892/mmr.2019.9854

    Figure Lengend Snippet: Regulatory effects of curculigoside on the expression levels of JAK1, JAK3 and STAT3 in TNF-α-stimulated MH7A cells were determined. Cells were treated with TNF-α (10 ng/ml) for 12 h, exposed to curculigoside (4 and 16 µg/ml) for a further 24 h and then subjected to western blotting assays to determine the expression levels of NF-κB and IκB (n=4). **P

    Article Snippet: Tumor necrosis factor (TNF)-α (cat. no. RAB0477), dimethyl sulfoxide (DMSO) and IκB (cat. no. SAB1305978) were purchased from Sigma-Aldrich; Merck KGaA.

    Techniques: Expressing, Western Blot

    Effects of curculigoside on the serum levels of TNF-α, IL-1β, IL-6, IL-10, IL-12 and IL-17A in type II collagen-induced arthritis rats. Data are presented as the mean ± standard deviation (n=10), **P

    Journal: Molecular Medicine Reports

    Article Title: Curculigoside exerts significant anti-arthritic effects in vivo and in vitro via regulation of the JAK/STAT/NF-κB signaling pathway

    doi: 10.3892/mmr.2019.9854

    Figure Lengend Snippet: Effects of curculigoside on the serum levels of TNF-α, IL-1β, IL-6, IL-10, IL-12 and IL-17A in type II collagen-induced arthritis rats. Data are presented as the mean ± standard deviation (n=10), **P

    Article Snippet: Tumor necrosis factor (TNF)-α (cat. no. RAB0477), dimethyl sulfoxide (DMSO) and IκB (cat. no. SAB1305978) were purchased from Sigma-Aldrich; Merck KGaA.

    Techniques: Standard Deviation

    CD4+ T cell responses to S . Typhi proteins presented by targets exposed to one of the four recombinant S . Typhi proteins. Ex vivo PBMC from a volunteer collected 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets exposed to 0.5ug/ml with one of the four recombinant S . Typhi proteins: SifA, OmpC, FliC, and GroEL. Untreated B-LCL targets (media) were used as controls. After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was evaluated by flow cytometry. Shown are the CD4 + T cell responses from a representative volunteer. Numbers represent the percentage of positive cells.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Use of a novel antigen expressing system to study the Salmonella enterica serovar Typhi protein recognition by T cells

    doi: 10.1371/journal.pntd.0005912

    Figure Lengend Snippet: CD4+ T cell responses to S . Typhi proteins presented by targets exposed to one of the four recombinant S . Typhi proteins. Ex vivo PBMC from a volunteer collected 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets exposed to 0.5ug/ml with one of the four recombinant S . Typhi proteins: SifA, OmpC, FliC, and GroEL. Untreated B-LCL targets (media) were used as controls. After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was evaluated by flow cytometry. Shown are the CD4 + T cell responses from a representative volunteer. Numbers represent the percentage of positive cells.

    Article Snippet: Monoclonal antibodies for surface and intracellular staining Cells were stained with monoclonal antibodies (mAbs) to CD69 (clone TPI-55-3) (Beckman-Coulter, Miami, FL), CD4 (clone RPA-T4), CD8 (clone HIT8a), CD107a and b (clones H4A3 and H4B4 respectively), interferon (IFN)-γ (clone B27), tumor necrosis factor (TNF)-α (clone MAb11) (BD Pharmingen, San Diego, CA, USA), CD14 (clone TuK4), CD19 (clone SJ25-C1), CD45 (clone H130) (Invitrogen), interleukin (IL)-17A (clone eBio64DEC17) (eBioscience, San Diego, CA), and CD3 (clone OKT3)(Biolegend, San Diego, CA).

    Techniques: Recombinant, Ex Vivo, Cell Culture, Incubation, Staining, Flow Cytometry, Cytometry

    Volunteer responses to S . Typhi proteins. Ex vivo PBMC from 7 immunized volunteers collected before (day 0) and 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets infected at an 1:30 MOI with one of the four recombinant E . coli expressing S . Typhi and Hly antigens: Hly / SifA (SifA), Hly / FliC (FliC), Hly / GroEL (GroEL) and Hly / OmpC (OmpC). After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was analyzed by flow cytometry. Two T cell subset responses (i.e., CD4 + and CD8 + T cells) were evaluated. ( A ) Heat-map of the multifunctionality of CD4 + and CD8 + T cells based on expression of cytokines and CD107a/b antigens. Percentages correspond to the net responses calculated by subtracting the T cell responses to B-LCLs infected with recombinant E . coli expressing S . Typhi/Hly proteins from the responses to the controls (B-LCL expressing Hly only). Volunteers were considered responders if the net responses of the PBMC collected 42 days after immunization were greater than 0.1 from the net responses of PBMC collected before immunization. ( B ) Cumulative frequency of responders to any functional test.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Use of a novel antigen expressing system to study the Salmonella enterica serovar Typhi protein recognition by T cells

    doi: 10.1371/journal.pntd.0005912

    Figure Lengend Snippet: Volunteer responses to S . Typhi proteins. Ex vivo PBMC from 7 immunized volunteers collected before (day 0) and 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets infected at an 1:30 MOI with one of the four recombinant E . coli expressing S . Typhi and Hly antigens: Hly / SifA (SifA), Hly / FliC (FliC), Hly / GroEL (GroEL) and Hly / OmpC (OmpC). After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was analyzed by flow cytometry. Two T cell subset responses (i.e., CD4 + and CD8 + T cells) were evaluated. ( A ) Heat-map of the multifunctionality of CD4 + and CD8 + T cells based on expression of cytokines and CD107a/b antigens. Percentages correspond to the net responses calculated by subtracting the T cell responses to B-LCLs infected with recombinant E . coli expressing S . Typhi/Hly proteins from the responses to the controls (B-LCL expressing Hly only). Volunteers were considered responders if the net responses of the PBMC collected 42 days after immunization were greater than 0.1 from the net responses of PBMC collected before immunization. ( B ) Cumulative frequency of responders to any functional test.

    Article Snippet: Monoclonal antibodies for surface and intracellular staining Cells were stained with monoclonal antibodies (mAbs) to CD69 (clone TPI-55-3) (Beckman-Coulter, Miami, FL), CD4 (clone RPA-T4), CD8 (clone HIT8a), CD107a and b (clones H4A3 and H4B4 respectively), interferon (IFN)-γ (clone B27), tumor necrosis factor (TNF)-α (clone MAb11) (BD Pharmingen, San Diego, CA, USA), CD14 (clone TuK4), CD19 (clone SJ25-C1), CD45 (clone H130) (Invitrogen), interleukin (IL)-17A (clone eBio64DEC17) (eBioscience, San Diego, CA), and CD3 (clone OKT3)(Biolegend, San Diego, CA).

    Techniques: Ex Vivo, Cell Culture, Infection, Recombinant, Expressing, Incubation, Staining, Flow Cytometry, Cytometry, Functional Assay

    CD8+ T cell responses to S . Typhi proteins presented by targets exposed to one of the four recombinant S . Typhi proteins. Ex vivo PBMC from a volunteer collected 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets exposed to 0.5ug/ml with one of the four recombinant S . Typhi proteins: SifA, OmpC, FliC, and GroEL. Untreated B-LCL targets (media) were used as controls. After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was evaluated by flow cytometry. Shown are the CD8 + T cell responses from a representative volunteer. Numbers represent the percentage of positive cells.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Use of a novel antigen expressing system to study the Salmonella enterica serovar Typhi protein recognition by T cells

    doi: 10.1371/journal.pntd.0005912

    Figure Lengend Snippet: CD8+ T cell responses to S . Typhi proteins presented by targets exposed to one of the four recombinant S . Typhi proteins. Ex vivo PBMC from a volunteer collected 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets exposed to 0.5ug/ml with one of the four recombinant S . Typhi proteins: SifA, OmpC, FliC, and GroEL. Untreated B-LCL targets (media) were used as controls. After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was evaluated by flow cytometry. Shown are the CD8 + T cell responses from a representative volunteer. Numbers represent the percentage of positive cells.

    Article Snippet: Monoclonal antibodies for surface and intracellular staining Cells were stained with monoclonal antibodies (mAbs) to CD69 (clone TPI-55-3) (Beckman-Coulter, Miami, FL), CD4 (clone RPA-T4), CD8 (clone HIT8a), CD107a and b (clones H4A3 and H4B4 respectively), interferon (IFN)-γ (clone B27), tumor necrosis factor (TNF)-α (clone MAb11) (BD Pharmingen, San Diego, CA, USA), CD14 (clone TuK4), CD19 (clone SJ25-C1), CD45 (clone H130) (Invitrogen), interleukin (IL)-17A (clone eBio64DEC17) (eBioscience, San Diego, CA), and CD3 (clone OKT3)(Biolegend, San Diego, CA).

    Techniques: Recombinant, Ex Vivo, Cell Culture, Incubation, Staining, Flow Cytometry, Cytometry

    Percentage of T cell subsets specific to any S . Typhi protein. Ex vivo PBMC from 7 volunteers collected before and 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets infected at 1:30 MOI with one of the four recombinant E . coli expressing S . Typhi and Hly genes: Hly / SifA (SifA), Hly / FliC (FliC), Hly / GroEL (GroEL) and Hly / OmpC (OmpC). After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was analyzed by flow cytometry. Two T cell subset responses (i.e., CD4 + and CD8 + T cells) were evaluated. Net responses were calculated by subtracting the T cell responses to B-LCLs infected with recombinant E . coli expressing S . Typhi/Hly antigens from the responses to the controls (B-LCL expressing Hly only). Increases over day 0 were calculated by subtracting the net responses of the PBMC collected 42 days after immunization from the net responses of PBMC collected before immunization. Bars represent mean ± SE.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Use of a novel antigen expressing system to study the Salmonella enterica serovar Typhi protein recognition by T cells

    doi: 10.1371/journal.pntd.0005912

    Figure Lengend Snippet: Percentage of T cell subsets specific to any S . Typhi protein. Ex vivo PBMC from 7 volunteers collected before and 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets infected at 1:30 MOI with one of the four recombinant E . coli expressing S . Typhi and Hly genes: Hly / SifA (SifA), Hly / FliC (FliC), Hly / GroEL (GroEL) and Hly / OmpC (OmpC). After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was analyzed by flow cytometry. Two T cell subset responses (i.e., CD4 + and CD8 + T cells) were evaluated. Net responses were calculated by subtracting the T cell responses to B-LCLs infected with recombinant E . coli expressing S . Typhi/Hly antigens from the responses to the controls (B-LCL expressing Hly only). Increases over day 0 were calculated by subtracting the net responses of the PBMC collected 42 days after immunization from the net responses of PBMC collected before immunization. Bars represent mean ± SE.

    Article Snippet: Monoclonal antibodies for surface and intracellular staining Cells were stained with monoclonal antibodies (mAbs) to CD69 (clone TPI-55-3) (Beckman-Coulter, Miami, FL), CD4 (clone RPA-T4), CD8 (clone HIT8a), CD107a and b (clones H4A3 and H4B4 respectively), interferon (IFN)-γ (clone B27), tumor necrosis factor (TNF)-α (clone MAb11) (BD Pharmingen, San Diego, CA, USA), CD14 (clone TuK4), CD19 (clone SJ25-C1), CD45 (clone H130) (Invitrogen), interleukin (IL)-17A (clone eBio64DEC17) (eBioscience, San Diego, CA), and CD3 (clone OKT3)(Biolegend, San Diego, CA).

    Techniques: Ex Vivo, Cell Culture, Infection, Recombinant, Expressing, Incubation, Staining, Flow Cytometry, Cytometry

    CD8+ T cell responses to S . Typhi proteins presented by targets infected with recombinant E . coli . Ex vivo PBMC from a volunteer collected 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets infected at 1:30 MOI with one of the four recombinant E . coli expressing S . Typhi/Hly ( Hly / SifA (SifA), Hly / FliC (FliC), Hly / GroEL (GroEL) and Hly / OmpC (OmpC)) or only Hly (control) proteins. After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was evaluated by flow cytometry. Shown are the CD8 + T cell responses from a representative volunteer. Numbers represent the percentage of positive cells.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Use of a novel antigen expressing system to study the Salmonella enterica serovar Typhi protein recognition by T cells

    doi: 10.1371/journal.pntd.0005912

    Figure Lengend Snippet: CD8+ T cell responses to S . Typhi proteins presented by targets infected with recombinant E . coli . Ex vivo PBMC from a volunteer collected 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets infected at 1:30 MOI with one of the four recombinant E . coli expressing S . Typhi/Hly ( Hly / SifA (SifA), Hly / FliC (FliC), Hly / GroEL (GroEL) and Hly / OmpC (OmpC)) or only Hly (control) proteins. After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was evaluated by flow cytometry. Shown are the CD8 + T cell responses from a representative volunteer. Numbers represent the percentage of positive cells.

    Article Snippet: Monoclonal antibodies for surface and intracellular staining Cells were stained with monoclonal antibodies (mAbs) to CD69 (clone TPI-55-3) (Beckman-Coulter, Miami, FL), CD4 (clone RPA-T4), CD8 (clone HIT8a), CD107a and b (clones H4A3 and H4B4 respectively), interferon (IFN)-γ (clone B27), tumor necrosis factor (TNF)-α (clone MAb11) (BD Pharmingen, San Diego, CA, USA), CD14 (clone TuK4), CD19 (clone SJ25-C1), CD45 (clone H130) (Invitrogen), interleukin (IL)-17A (clone eBio64DEC17) (eBioscience, San Diego, CA), and CD3 (clone OKT3)(Biolegend, San Diego, CA).

    Techniques: Infection, Recombinant, Ex Vivo, Cell Culture, Expressing, Incubation, Staining, Flow Cytometry, Cytometry

    CD4+ T cell responses to S . Typhi proteins presented by targets infected with recombinant E . coli . Ex vivo PBMC from a volunteer collected 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets infected at 1:30 MOI with one of the four recombinant E . coli expressing S . Typhi/Hly ( Hly / SifA (SifA), Hly / FliC (FliC), Hly / GroEL (GroEL) and Hly / OmpC (OmpC)) or only Hly (control) proteins. After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was evaluated by flow cytometry. Shown are the CD4+ T cell responses from a representative volunteer. Numbers represent the percentage of positive cells.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Use of a novel antigen expressing system to study the Salmonella enterica serovar Typhi protein recognition by T cells

    doi: 10.1371/journal.pntd.0005912

    Figure Lengend Snippet: CD4+ T cell responses to S . Typhi proteins presented by targets infected with recombinant E . coli . Ex vivo PBMC from a volunteer collected 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets infected at 1:30 MOI with one of the four recombinant E . coli expressing S . Typhi/Hly ( Hly / SifA (SifA), Hly / FliC (FliC), Hly / GroEL (GroEL) and Hly / OmpC (OmpC)) or only Hly (control) proteins. After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was evaluated by flow cytometry. Shown are the CD4+ T cell responses from a representative volunteer. Numbers represent the percentage of positive cells.

    Article Snippet: Monoclonal antibodies for surface and intracellular staining Cells were stained with monoclonal antibodies (mAbs) to CD69 (clone TPI-55-3) (Beckman-Coulter, Miami, FL), CD4 (clone RPA-T4), CD8 (clone HIT8a), CD107a and b (clones H4A3 and H4B4 respectively), interferon (IFN)-γ (clone B27), tumor necrosis factor (TNF)-α (clone MAb11) (BD Pharmingen, San Diego, CA, USA), CD14 (clone TuK4), CD19 (clone SJ25-C1), CD45 (clone H130) (Invitrogen), interleukin (IL)-17A (clone eBio64DEC17) (eBioscience, San Diego, CA), and CD3 (clone OKT3)(Biolegend, San Diego, CA).

    Techniques: Infection, Recombinant, Ex Vivo, Cell Culture, Expressing, Incubation, Staining, Flow Cytometry, Cytometry

    CeCyld inhibits TNF-induced IL8 expression. HeLa cells (10 5 cells per well in 12-well plates) were transfected with 0.1μg, 1.4μg and 0.4μg of plasmids expressing HsCYLD, CeCYLD and CeCYLDC774S respectively. After 24h, each transfected cell population was split equally in two new wells and 24 h later, half of each transfected cell population was left untreated or treated with TNFα (40ng/ml, 1h). The cells were then harvested and from each cell population RNA and total protein was extracted. The overexpressed FLAG-tagged proteins were immuno-precipitated and the products of immunoprecipitation were used to assess the expression levels of each protein by immuoblotting. (A) Values are shown as the mean +/- standard error of relative IL8 expression induction from four independent experiments of RT-PCR. The values that were compared statistically are indicated by horizontal lines. (B) Immunoblot showing the expression levels of immuno-precipitated proteins with anti-FLAG antibody (IP:αFLAG) along with the immunoglobulin heavy chains (Ig-HC) and β-actin in the whole cell lysate (WCL). *p

    Journal: PLoS ONE

    Article Title: Functional analysis of the C. elegans cyld-1 gene reveals extensive similarity with its human homolog

    doi: 10.1371/journal.pone.0191864

    Figure Lengend Snippet: CeCyld inhibits TNF-induced IL8 expression. HeLa cells (10 5 cells per well in 12-well plates) were transfected with 0.1μg, 1.4μg and 0.4μg of plasmids expressing HsCYLD, CeCYLD and CeCYLDC774S respectively. After 24h, each transfected cell population was split equally in two new wells and 24 h later, half of each transfected cell population was left untreated or treated with TNFα (40ng/ml, 1h). The cells were then harvested and from each cell population RNA and total protein was extracted. The overexpressed FLAG-tagged proteins were immuno-precipitated and the products of immunoprecipitation were used to assess the expression levels of each protein by immuoblotting. (A) Values are shown as the mean +/- standard error of relative IL8 expression induction from four independent experiments of RT-PCR. The values that were compared statistically are indicated by horizontal lines. (B) Immunoblot showing the expression levels of immuno-precipitated proteins with anti-FLAG antibody (IP:αFLAG) along with the immunoglobulin heavy chains (Ig-HC) and β-actin in the whole cell lysate (WCL). *p

    Article Snippet: After 48h the cells were induced using 40ng/ml TNFα (Peprotech, 300-01A) for 1h.

    Techniques: Expressing, Transfection, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    TNF-α-mediated G1 arrest in Panc1 cells. One hundred thousand ( A ) Panc1 cells or ( B ) PancTu-I cells were cultured in 6-well plates overnight. After 24 h, cells were left untreated (medium) or were treated with 10 ng/mL IFN-γ, 10 ng/mL TNF-α or 10 ng/mL IFN-γ together with TNF-α as indicated for 24 h. Cell cycle distribution of living cells obtained by gating in the forward/sideward scatter was determined using PI staining and flow cytometry. Numbers in the figures represent the percentage of the different cell cycle phases. One representative result is shown as a histogram (upper panel), and three independent experiments are represented as pie diagrams (lower panels). SD is

    Journal: Cells

    Article Title: Influence of Indoleamine-2,3-Dioxygenase and Its Metabolite Kynurenine on γδ T Cell Cytotoxicity against Ductal Pancreatic Adenocarcinoma Cells

    doi: 10.3390/cells9051140

    Figure Lengend Snippet: TNF-α-mediated G1 arrest in Panc1 cells. One hundred thousand ( A ) Panc1 cells or ( B ) PancTu-I cells were cultured in 6-well plates overnight. After 24 h, cells were left untreated (medium) or were treated with 10 ng/mL IFN-γ, 10 ng/mL TNF-α or 10 ng/mL IFN-γ together with TNF-α as indicated for 24 h. Cell cycle distribution of living cells obtained by gating in the forward/sideward scatter was determined using PI staining and flow cytometry. Numbers in the figures represent the percentage of the different cell cycle phases. One representative result is shown as a histogram (upper panel), and three independent experiments are represented as pie diagrams (lower panels). SD is

    Article Snippet: Human granzyme B was measured by a sandwich DuoSet ELISA kit (# DY1154 from R & D System), and human TNF-α and IFN-γ by human TNF-α or an IFN-γ DuoSet ELISA kit (both from R & D Systems), respectively, in duplicates, following the procedures outlined by the manufacturer.

    Techniques: Cell Culture, Staining, Flow Cytometry

    Intracellular expression of IDO in PDAC cells and release of γδ T cell mediators after coculture with PDAC cells. ( A , B ) Intracellular pan-IDO expression was analyzed by staining the indicated different PDAC cells with anti-pan-IDO-APC mAb (clone #700838) and by FACS Calibur Analyzer. ( a ) Histograms depicted are representative results of three different experiments. Thin black and grey lines represent isotype controls and thick black and grey lines represent the appropriate pan-IDO expression in the indicated PDAC cells. ( B ) Mean fluorescence intensity (MFI) of pan-IDO expression of three different experiments +/- SD is shown. ( C ) The 5–10 × 10 3 Panc1 and PancTu-I cells were cultured in complete medium for 28 h in 96-well plates. Thereafter, PDAC cells were cocultured with short-term activated Vγ9Vδ2 γδ T cells of PDAC patients in 12.5 IU/mL rIL-2 at an E/T ratio of 25:1 in medium or stimulated with 1 μg/mL tribody [(HER2) 2 ×Vγ9]. IFN-γ, TNF-α and granzyme B release was determined in the supernatant after 6 and 24 h using ELISA. Each symbol represents the data of one donor, and the lines represent the median values of different independent experiments. Significances are shown as P values; * = p

    Journal: Cells

    Article Title: Influence of Indoleamine-2,3-Dioxygenase and Its Metabolite Kynurenine on γδ T Cell Cytotoxicity against Ductal Pancreatic Adenocarcinoma Cells

    doi: 10.3390/cells9051140

    Figure Lengend Snippet: Intracellular expression of IDO in PDAC cells and release of γδ T cell mediators after coculture with PDAC cells. ( A , B ) Intracellular pan-IDO expression was analyzed by staining the indicated different PDAC cells with anti-pan-IDO-APC mAb (clone #700838) and by FACS Calibur Analyzer. ( a ) Histograms depicted are representative results of three different experiments. Thin black and grey lines represent isotype controls and thick black and grey lines represent the appropriate pan-IDO expression in the indicated PDAC cells. ( B ) Mean fluorescence intensity (MFI) of pan-IDO expression of three different experiments +/- SD is shown. ( C ) The 5–10 × 10 3 Panc1 and PancTu-I cells were cultured in complete medium for 28 h in 96-well plates. Thereafter, PDAC cells were cocultured with short-term activated Vγ9Vδ2 γδ T cells of PDAC patients in 12.5 IU/mL rIL-2 at an E/T ratio of 25:1 in medium or stimulated with 1 μg/mL tribody [(HER2) 2 ×Vγ9]. IFN-γ, TNF-α and granzyme B release was determined in the supernatant after 6 and 24 h using ELISA. Each symbol represents the data of one donor, and the lines represent the median values of different independent experiments. Significances are shown as P values; * = p

    Article Snippet: Human granzyme B was measured by a sandwich DuoSet ELISA kit (# DY1154 from R & D System), and human TNF-α and IFN-γ by human TNF-α or an IFN-γ DuoSet ELISA kit (both from R & D Systems), respectively, in duplicates, following the procedures outlined by the manufacturer.

    Techniques: Expressing, Staining, FACS, Fluorescence, Cell Culture, Enzyme-linked Immunosorbent Assay

    Relationships of RIP1 with TNF-α and LVD in human GBC patients. Notes: ( A ) TNF-α and RIP1 protein expression and lymphatic vessel expression in GBC tissues were analyzed using immunohistochemical staining using TNF-α, RIP1, and D2-40 antibodies, respectively (100×; 200×). ( B , D ) Relationships of the average optical density of RIP1 with optical density of TNF-α and LVD in the GBC samples. ( C ) Lymphatic vessels based on D2-40 immunostaining were flattened and invaded by GBC cells. ** p

    Journal: OncoTargets and therapy

    Article Title: RIP1 regulates TNF-α-mediated lymphangiogenesis and lymphatic metastasis in gallbladder cancer by modulating the NF-κB-VEGF-C pathway

    doi: 10.2147/OTT.S159026

    Figure Lengend Snippet: Relationships of RIP1 with TNF-α and LVD in human GBC patients. Notes: ( A ) TNF-α and RIP1 protein expression and lymphatic vessel expression in GBC tissues were analyzed using immunohistochemical staining using TNF-α, RIP1, and D2-40 antibodies, respectively (100×; 200×). ( B , D ) Relationships of the average optical density of RIP1 with optical density of TNF-α and LVD in the GBC samples. ( C ) Lymphatic vessels based on D2-40 immunostaining were flattened and invaded by GBC cells. ** p

    Article Snippet: Immunohistochemistry The primary antibodies used for immunohistochemistry targeted the following proteins: TNF-α (sc-52746, 1:100; Santa Cruz Biotechnology Inc.), RIP1 (ab72139, 1:500; Abcam), D2-40 (1:150; Maixin-Bio, Fuzhou, People’s Republic of China), and LYVE-1 (AF2125, 1:250; R & D Systems, Inc).

    Techniques: Expressing, Immunohistochemistry, Staining, Immunostaining

    Knockdown of RIP1 in GBC cells impaired TNF-α-mediated tube formation in HDLECs. Notes: Red fluorescent probe-labeled HDLECs were cocultured with the established NOZ or GBC-SD cell groups (control, LV-siNC, and LV-siRIP1) and analyzed using a fluorescence microscope, and the number of tubes was calculated. ( A , C ) 50 ng/mL of recombinant human TNF-α enhanced tube formation in HDLECs, and siRIP1 impaired this enhancement by TNF-α. Transfection of PcDNA3.1-RIP1 vector into LV-siRIP1 cells reversed this impairment. ( B , D ) Supplementation of recombinant human VEGF-C protein (50 ng/mL) into the LV-siRIP1 cell group reversed the impairment of TNF-α-enhanced tube formation. C and D; n=3, mean±SEM * p

    Journal: OncoTargets and therapy

    Article Title: RIP1 regulates TNF-α-mediated lymphangiogenesis and lymphatic metastasis in gallbladder cancer by modulating the NF-κB-VEGF-C pathway

    doi: 10.2147/OTT.S159026

    Figure Lengend Snippet: Knockdown of RIP1 in GBC cells impaired TNF-α-mediated tube formation in HDLECs. Notes: Red fluorescent probe-labeled HDLECs were cocultured with the established NOZ or GBC-SD cell groups (control, LV-siNC, and LV-siRIP1) and analyzed using a fluorescence microscope, and the number of tubes was calculated. ( A , C ) 50 ng/mL of recombinant human TNF-α enhanced tube formation in HDLECs, and siRIP1 impaired this enhancement by TNF-α. Transfection of PcDNA3.1-RIP1 vector into LV-siRIP1 cells reversed this impairment. ( B , D ) Supplementation of recombinant human VEGF-C protein (50 ng/mL) into the LV-siRIP1 cell group reversed the impairment of TNF-α-enhanced tube formation. C and D; n=3, mean±SEM * p

    Article Snippet: Immunohistochemistry The primary antibodies used for immunohistochemistry targeted the following proteins: TNF-α (sc-52746, 1:100; Santa Cruz Biotechnology Inc.), RIP1 (ab72139, 1:500; Abcam), D2-40 (1:150; Maixin-Bio, Fuzhou, People’s Republic of China), and LYVE-1 (AF2125, 1:250; R & D Systems, Inc).

    Techniques: Labeling, Fluorescence, Microscopy, Recombinant, Transfection, Plasmid Preparation

    RIP1 is essential for TNF-α-mediated NF-κB activation. Notes: ( A ) NF-κB-luciferase activity was examined in the siNC and siRIP1 cell groups after being stimulated with 50 ng/ml of recombinant human TNF-α or left unstimulated for 24 h. Transfection of PcDNA3.1-RIP1 vector into the siRIP1 cell groups reversed the impairment of TNF-α-mediated NF-κB activation. ( B ) Western blot analyses of RIP1, iκBα, and p-iκBα expression in protein extracts from the siNC and siRIP cell groups that were stimulated with 50 ng/mL of recombinant human TNF-α or left unstimulated for 24 h. ( C–E ) Transfection of silκBα into NOZ or GBC-SD cells effectively inhibited iκBα mRNA and protein expression. (F) NF-κB-luciferase activity assays showed that knockdown of lκBα could reverse the impairment of TNF-α-mediated NF-κB activation in the siRIP1 cell groups. (G) immunoprecipitation analysis showed that TAK1 and NEMO are associated with RIP1. B, C, D and F; n=3, mean±SEM; * p

    Journal: OncoTargets and therapy

    Article Title: RIP1 regulates TNF-α-mediated lymphangiogenesis and lymphatic metastasis in gallbladder cancer by modulating the NF-κB-VEGF-C pathway

    doi: 10.2147/OTT.S159026

    Figure Lengend Snippet: RIP1 is essential for TNF-α-mediated NF-κB activation. Notes: ( A ) NF-κB-luciferase activity was examined in the siNC and siRIP1 cell groups after being stimulated with 50 ng/ml of recombinant human TNF-α or left unstimulated for 24 h. Transfection of PcDNA3.1-RIP1 vector into the siRIP1 cell groups reversed the impairment of TNF-α-mediated NF-κB activation. ( B ) Western blot analyses of RIP1, iκBα, and p-iκBα expression in protein extracts from the siNC and siRIP cell groups that were stimulated with 50 ng/mL of recombinant human TNF-α or left unstimulated for 24 h. ( C–E ) Transfection of silκBα into NOZ or GBC-SD cells effectively inhibited iκBα mRNA and protein expression. (F) NF-κB-luciferase activity assays showed that knockdown of lκBα could reverse the impairment of TNF-α-mediated NF-κB activation in the siRIP1 cell groups. (G) immunoprecipitation analysis showed that TAK1 and NEMO are associated with RIP1. B, C, D and F; n=3, mean±SEM; * p

    Article Snippet: Immunohistochemistry The primary antibodies used for immunohistochemistry targeted the following proteins: TNF-α (sc-52746, 1:100; Santa Cruz Biotechnology Inc.), RIP1 (ab72139, 1:500; Abcam), D2-40 (1:150; Maixin-Bio, Fuzhou, People’s Republic of China), and LYVE-1 (AF2125, 1:250; R & D Systems, Inc).

    Techniques: Activation Assay, Luciferase, Activity Assay, Recombinant, Transfection, Plasmid Preparation, Western Blot, Expressing, Immunoprecipitation

    RIP1 regulates TNF-α-mediated VEGF-C expression through the NF-κB pathway. Notes: ( A , C , D ) The VEGF-C mRNA and protein in the siNC and siRIP1 cell groups were extracted and examined via qPCR, Western blotting, and ELISA afterbeing stimulated with 50 ng/mL of recombinant human TNF-α or left unstimulated for 24 h. ( B , E , F ) The qPCR, immunoblotting, and ELISA analyses indicated that Bay-11-7082 abolished the TNF-α-mediated induction of VEGF-C mRNA and protein expression in NOZ and GBC-SD cells. ( G , H ) Luciferase activity assays showed that siRIP1 and Bay-11-7082 significantly impaired TNF-α-enhanced pGL3B-332 (containing the VEGF-C promoter sequence) luciferase activity. A, B, D, F, G and H; n=3, mean±SEM; * p

    Journal: OncoTargets and therapy

    Article Title: RIP1 regulates TNF-α-mediated lymphangiogenesis and lymphatic metastasis in gallbladder cancer by modulating the NF-κB-VEGF-C pathway

    doi: 10.2147/OTT.S159026

    Figure Lengend Snippet: RIP1 regulates TNF-α-mediated VEGF-C expression through the NF-κB pathway. Notes: ( A , C , D ) The VEGF-C mRNA and protein in the siNC and siRIP1 cell groups were extracted and examined via qPCR, Western blotting, and ELISA afterbeing stimulated with 50 ng/mL of recombinant human TNF-α or left unstimulated for 24 h. ( B , E , F ) The qPCR, immunoblotting, and ELISA analyses indicated that Bay-11-7082 abolished the TNF-α-mediated induction of VEGF-C mRNA and protein expression in NOZ and GBC-SD cells. ( G , H ) Luciferase activity assays showed that siRIP1 and Bay-11-7082 significantly impaired TNF-α-enhanced pGL3B-332 (containing the VEGF-C promoter sequence) luciferase activity. A, B, D, F, G and H; n=3, mean±SEM; * p

    Article Snippet: Immunohistochemistry The primary antibodies used for immunohistochemistry targeted the following proteins: TNF-α (sc-52746, 1:100; Santa Cruz Biotechnology Inc.), RIP1 (ab72139, 1:500; Abcam), D2-40 (1:150; Maixin-Bio, Fuzhou, People’s Republic of China), and LYVE-1 (AF2125, 1:250; R & D Systems, Inc).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Luciferase, Activity Assay, Sequencing

    Knockdown of RIP1 in GBC cells impaired TNF-α-mediated lymphatic vessel formation and metastasis in vivo. Notes: ( A ) The operation performed in the murine transplantation model of GBC. (a-1) The ventral skin was opened. (a-2) The gallbladder was exposed. (a-3) The three established cell groups (NOZ-control, NOZ-LV-siNC, and NOZ-LV-siRIP1) were implanted into the gallbladder. (a-4) The nude mice presented with dyscrasia; scale bars =5 mm. ( B ) Metastatic lymph nodes (blue arrows) were primarily localized to the hepatoduodenal ligament. (b-1) (−), (b-2) (+, blue arrows); scale bars =5 mm. ( C ) Hematoxylin and eosin staining (c-1: 100×, (c-2): 200×): cancer cell invasion (red arrows) was detected via the lymphoid follicles. ( D ) The tumor lymphatic vessels (red arrows) were analyzed via immunohistochemical staining using an LYVE-1 antibody (100×). ( E ) The LVN of the orthotopic xenograft tumors was counted. ( F ) The size of the lymph nodes was counted (E and F; n=5, mean±SEM). * p

    Journal: OncoTargets and therapy

    Article Title: RIP1 regulates TNF-α-mediated lymphangiogenesis and lymphatic metastasis in gallbladder cancer by modulating the NF-κB-VEGF-C pathway

    doi: 10.2147/OTT.S159026

    Figure Lengend Snippet: Knockdown of RIP1 in GBC cells impaired TNF-α-mediated lymphatic vessel formation and metastasis in vivo. Notes: ( A ) The operation performed in the murine transplantation model of GBC. (a-1) The ventral skin was opened. (a-2) The gallbladder was exposed. (a-3) The three established cell groups (NOZ-control, NOZ-LV-siNC, and NOZ-LV-siRIP1) were implanted into the gallbladder. (a-4) The nude mice presented with dyscrasia; scale bars =5 mm. ( B ) Metastatic lymph nodes (blue arrows) were primarily localized to the hepatoduodenal ligament. (b-1) (−), (b-2) (+, blue arrows); scale bars =5 mm. ( C ) Hematoxylin and eosin staining (c-1: 100×, (c-2): 200×): cancer cell invasion (red arrows) was detected via the lymphoid follicles. ( D ) The tumor lymphatic vessels (red arrows) were analyzed via immunohistochemical staining using an LYVE-1 antibody (100×). ( E ) The LVN of the orthotopic xenograft tumors was counted. ( F ) The size of the lymph nodes was counted (E and F; n=5, mean±SEM). * p

    Article Snippet: Immunohistochemistry The primary antibodies used for immunohistochemistry targeted the following proteins: TNF-α (sc-52746, 1:100; Santa Cruz Biotechnology Inc.), RIP1 (ab72139, 1:500; Abcam), D2-40 (1:150; Maixin-Bio, Fuzhou, People’s Republic of China), and LYVE-1 (AF2125, 1:250; R & D Systems, Inc).

    Techniques: In Vivo, Transplantation Assay, Mouse Assay, Staining, Immunohistochemistry

    Knockdown of RIP1 impaired the TNF-α-enhanced association of NF-κB with the VEGF-C promoter region. Notes: Chromatin was extracted from the siNC and siRIP1 cell groups and sheared after the cells were stimulated with 50 ng/mL of recombinant human TNF-α or left unstimulated for 24 h. NF-κB immunoprecipitation reactions were performed with 5 µg of NF-κB antibody; the VEGF-C promoter fragment (−389/−278) containing the NF-κB binding sites in the immunoprecipitated and the input samples was amplified with the appropriate primers. Rabbit serum was used as a negative control. Abbreviations: TNF-α, tumor necrosis factor alpha; VEGF, vascular endothelial growth factor; IP, immunoprecipitation.

    Journal: OncoTargets and therapy

    Article Title: RIP1 regulates TNF-α-mediated lymphangiogenesis and lymphatic metastasis in gallbladder cancer by modulating the NF-κB-VEGF-C pathway

    doi: 10.2147/OTT.S159026

    Figure Lengend Snippet: Knockdown of RIP1 impaired the TNF-α-enhanced association of NF-κB with the VEGF-C promoter region. Notes: Chromatin was extracted from the siNC and siRIP1 cell groups and sheared after the cells were stimulated with 50 ng/mL of recombinant human TNF-α or left unstimulated for 24 h. NF-κB immunoprecipitation reactions were performed with 5 µg of NF-κB antibody; the VEGF-C promoter fragment (−389/−278) containing the NF-κB binding sites in the immunoprecipitated and the input samples was amplified with the appropriate primers. Rabbit serum was used as a negative control. Abbreviations: TNF-α, tumor necrosis factor alpha; VEGF, vascular endothelial growth factor; IP, immunoprecipitation.

    Article Snippet: Immunohistochemistry The primary antibodies used for immunohistochemistry targeted the following proteins: TNF-α (sc-52746, 1:100; Santa Cruz Biotechnology Inc.), RIP1 (ab72139, 1:500; Abcam), D2-40 (1:150; Maixin-Bio, Fuzhou, People’s Republic of China), and LYVE-1 (AF2125, 1:250; R & D Systems, Inc).

    Techniques: Recombinant, Immunoprecipitation, Binding Assay, Amplification, Negative Control

    TNF-α enhances RIP1 mRNA and protein expression. Notes: ( A , B ) The Jurkat, GBC-SD, and NOZ cells were stimulated with 10, 30, 50, and 100 ng/mL of recombinant human TNF-α for 24 h. qPCR indicated that TNF-α dose and time dependently enhanced RIP1 mRNA levels in the three cell lines, and the strongest effect was observed with a concentration of 50 ng/mL. ( C , E ) The GBC-SD and NOZ cells were stimulated with 10, 30, 50, and 100 ng/mL of recombinant human TNF-α for 24 h. Western blotting indicated that TNF-α dose dependently enhanced RIP1 protein levels, and the strongest effect was observed with a concentration of 50 ng/mL. ( D , F ) GBC-SD and NOZ cells stimulated with 50 ng/mL of recombinant human TNF-α for 6, 12, and 24 h. Western blotting indicated that TNF-α time dependently enhanced RIP1 protein levels. A, B, E, F; n=3, mean±SEM; * p

    Journal: OncoTargets and therapy

    Article Title: RIP1 regulates TNF-α-mediated lymphangiogenesis and lymphatic metastasis in gallbladder cancer by modulating the NF-κB-VEGF-C pathway

    doi: 10.2147/OTT.S159026

    Figure Lengend Snippet: TNF-α enhances RIP1 mRNA and protein expression. Notes: ( A , B ) The Jurkat, GBC-SD, and NOZ cells were stimulated with 10, 30, 50, and 100 ng/mL of recombinant human TNF-α for 24 h. qPCR indicated that TNF-α dose and time dependently enhanced RIP1 mRNA levels in the three cell lines, and the strongest effect was observed with a concentration of 50 ng/mL. ( C , E ) The GBC-SD and NOZ cells were stimulated with 10, 30, 50, and 100 ng/mL of recombinant human TNF-α for 24 h. Western blotting indicated that TNF-α dose dependently enhanced RIP1 protein levels, and the strongest effect was observed with a concentration of 50 ng/mL. ( D , F ) GBC-SD and NOZ cells stimulated with 50 ng/mL of recombinant human TNF-α for 6, 12, and 24 h. Western blotting indicated that TNF-α time dependently enhanced RIP1 protein levels. A, B, E, F; n=3, mean±SEM; * p

    Article Snippet: Immunohistochemistry The primary antibodies used for immunohistochemistry targeted the following proteins: TNF-α (sc-52746, 1:100; Santa Cruz Biotechnology Inc.), RIP1 (ab72139, 1:500; Abcam), D2-40 (1:150; Maixin-Bio, Fuzhou, People’s Republic of China), and LYVE-1 (AF2125, 1:250; R & D Systems, Inc).

    Techniques: Expressing, Recombinant, Real-time Polymerase Chain Reaction, Concentration Assay, Western Blot

    TNF-α is sufficient to induce HF TAT and is crucial for WIHN. ( a , b ) Intracutaneous injection of TNF-α can induce the HF telogen–anagen transition at the injection site in 7-week (W)-old mice (refractory phase) and 9-week-old mice (competent phase) ( a ), and the number of TNF-α-induced anagen hair follicles in the two different group was quantified ( b ). Seven-week-old mice, n =6 for each group; 9-week-old mice, n =8 for each group. ( c , d ) Wounding to the skin induced more anagen HFs in 9-week-old mice than in 7-week-old mice ( c ), and the number of anagen HFs in the two different groups was quantified ( d ). n =7 for each age group. ( e , f ) WIH-A analysis in TNFR1 −/− mice, TNFR2 −/− mice and WT mice ( e ), and the number of anagen HFs in different groups was quantified ( f ). Wild-type (C57BL/6) mice, n =7; TNFR1 −/− mice, n =6; TNFR2 −/− mice, n =9. ( g , h ) Wound-induced anagen HFs in mice constitutively expressing TNF-α (Tg-TNF-α) were significantly increased compared with WT mice, and the number of anagen HFs in the two different groups was quantified ( h ). Control mice (WT), n =6; Tg-TNF-α mice, n =5. ( i ) Bioluminescent imaging of Tnf-Luc-eGFP mice at different days (D) after wounding showed changes in the TNF-α level in the wound. Areas with high TNF-α levels (green/red) shifted from the wound (W) periphery to the wound centre with the progression of wound healing. n =15 for Tnf-Luc-eGFP mice, and n =10 for wild-type (WT) mice. ( j ) IF analysis of sections from the PWD-14 wound showed the presence of F4/80 + macrophages in the wound, which largely co-localized with TNF-α. Scale bar, 50 μm. ( k , l ) WIHN analysis in WT, TNFA −/− and Tg-TNF-α mice at PWD-30 ( k ), and the number of neogenic HFs in wounds was quantified ( l ). n =6 for both wild-type and TNFA −/− mice; n =12 for Tg-TNF-α mice. Scale bars, 2 mm. Data are expressed as the mean±s.e.m. * P

    Journal: Nature Communications

    Article Title: Macrophages induce AKT/β-catenin-dependent Lgr5+ stem cell activation and hair follicle regeneration through TNF

    doi: 10.1038/ncomms14091

    Figure Lengend Snippet: TNF-α is sufficient to induce HF TAT and is crucial for WIHN. ( a , b ) Intracutaneous injection of TNF-α can induce the HF telogen–anagen transition at the injection site in 7-week (W)-old mice (refractory phase) and 9-week-old mice (competent phase) ( a ), and the number of TNF-α-induced anagen hair follicles in the two different group was quantified ( b ). Seven-week-old mice, n =6 for each group; 9-week-old mice, n =8 for each group. ( c , d ) Wounding to the skin induced more anagen HFs in 9-week-old mice than in 7-week-old mice ( c ), and the number of anagen HFs in the two different groups was quantified ( d ). n =7 for each age group. ( e , f ) WIH-A analysis in TNFR1 −/− mice, TNFR2 −/− mice and WT mice ( e ), and the number of anagen HFs in different groups was quantified ( f ). Wild-type (C57BL/6) mice, n =7; TNFR1 −/− mice, n =6; TNFR2 −/− mice, n =9. ( g , h ) Wound-induced anagen HFs in mice constitutively expressing TNF-α (Tg-TNF-α) were significantly increased compared with WT mice, and the number of anagen HFs in the two different groups was quantified ( h ). Control mice (WT), n =6; Tg-TNF-α mice, n =5. ( i ) Bioluminescent imaging of Tnf-Luc-eGFP mice at different days (D) after wounding showed changes in the TNF-α level in the wound. Areas with high TNF-α levels (green/red) shifted from the wound (W) periphery to the wound centre with the progression of wound healing. n =15 for Tnf-Luc-eGFP mice, and n =10 for wild-type (WT) mice. ( j ) IF analysis of sections from the PWD-14 wound showed the presence of F4/80 + macrophages in the wound, which largely co-localized with TNF-α. Scale bar, 50 μm. ( k , l ) WIHN analysis in WT, TNFA −/− and Tg-TNF-α mice at PWD-30 ( k ), and the number of neogenic HFs in wounds was quantified ( l ). n =6 for both wild-type and TNFA −/− mice; n =12 for Tg-TNF-α mice. Scale bars, 2 mm. Data are expressed as the mean±s.e.m. * P

    Article Snippet: Immunostaining Frozen skin tissue sections were incubated with different primary antibodies at 4 °C overnight; antibodies included anti-CD49f-biotin (GoH3, 1:150, BioLegend), anti-Ki67 (20Raj1, 1:100, eBioscience), anti-Akt (GTX28932, phospho Ser473, 1:150, GeneTex), anti-Akt (Akt 1+2+3) (GTX121937, 1:150, GeneTex), anti-CD34-biotin (RAM34, 1:150, eBioscience), anti-Lgr-5 (ab137484, 1:200, Abcam), anti-CD45 (30-F11, 1:150, BioLegend), anti-CD45-Biotin (30-F11, 1:150, BioLegend), anti-F4/80-Biotin (BM8, 1:150, BioLegend), anti-F4/80 (BM8, 1:150, BioLegend), anti-Ly6C-Biotin (RB6-8C5, 1:200, BioLegend), anti-Ly6C (HK1.4, 1:200, BioLegend), anti-MHC II (14-4-4S, 1:200, eBioscience), anti-TNF-α (1F3F3D4, 1:100, eBioscience), anti-TNFR1-Biotin (55R-170, 1:100, BioLegend), anti-β-catenin (C2206, 1:150, Sigma) and anti-Phospho-β-Catenin (Ser552) (5651S, 1:100, CST Signaling).

    Techniques: Injection, Mouse Assay, Expressing, Imaging

    TNF is a crucial mediator to induce the HF TAT. ( a ) Gene expression profile of Ly6C + /F4/80 + macrophages (M), CX3CR1 + /F4/80 + macrophages and Ly6G + /F4/80 − neutrophils (Neu), which were sorted from wound tissue at PWD-3. Total RNA was extracted from samples of three mice. ( b ) Differentially expressed cytokine genes from microarray analysis were further validated by real-time PCR analysis. (For all real-time PCR analyses, gene expression was normalized to GAPDH with 40 cycles, data are represented as the mean±s.d., and n =3.) ( c , d ) WIH-A analysis in WT and deficient for IL6 (IL6-KO) or TNFA (TNFα-KO) gene mice, and the number of anagen HFs in different mice was quantified ( d ). IL6 -knockout mice, n =5; TNFA -knockout mice, n =6; WT mice, n =6. ( e ) Real-time PCR analysis showed TNFA expression levels in the tissue surrounding the wounds (2 mm in width) at different times post-wounding. ( f ) Bioluminescent imaging of Tnf-Luc-eGFP mice at different times after wounding showed changes of the TNF-α level in the wound. Data are representative of 7–9 independent experiments at each time point. ( g , h ) Lenalidomide and QNZ treatment resulted in decreased anagen HFs as assessed at PWD-15 ( g ), and the number of anagen HFs in different groups was quantified ( h ). Control mice, n =9; lenalidomide-treated mice, n =7; QNZ-treated mice, n =8. ( i , j ) Wound-induced anagen HFs was significantly decreased in Lysm cre/+ :TNF flox/flox mice ( i ), and the number of anagen HFs in two groups was quantified ( j ). Control mice ( Lysm cre/+ :TNF flox/+ ), n =6; Lysm cre/+ :TNF flox/flox mice, n =4. Data are expressed as the mean±s.e.m. ** P

    Journal: Nature Communications

    Article Title: Macrophages induce AKT/β-catenin-dependent Lgr5+ stem cell activation and hair follicle regeneration through TNF

    doi: 10.1038/ncomms14091

    Figure Lengend Snippet: TNF is a crucial mediator to induce the HF TAT. ( a ) Gene expression profile of Ly6C + /F4/80 + macrophages (M), CX3CR1 + /F4/80 + macrophages and Ly6G + /F4/80 − neutrophils (Neu), which were sorted from wound tissue at PWD-3. Total RNA was extracted from samples of three mice. ( b ) Differentially expressed cytokine genes from microarray analysis were further validated by real-time PCR analysis. (For all real-time PCR analyses, gene expression was normalized to GAPDH with 40 cycles, data are represented as the mean±s.d., and n =3.) ( c , d ) WIH-A analysis in WT and deficient for IL6 (IL6-KO) or TNFA (TNFα-KO) gene mice, and the number of anagen HFs in different mice was quantified ( d ). IL6 -knockout mice, n =5; TNFA -knockout mice, n =6; WT mice, n =6. ( e ) Real-time PCR analysis showed TNFA expression levels in the tissue surrounding the wounds (2 mm in width) at different times post-wounding. ( f ) Bioluminescent imaging of Tnf-Luc-eGFP mice at different times after wounding showed changes of the TNF-α level in the wound. Data are representative of 7–9 independent experiments at each time point. ( g , h ) Lenalidomide and QNZ treatment resulted in decreased anagen HFs as assessed at PWD-15 ( g ), and the number of anagen HFs in different groups was quantified ( h ). Control mice, n =9; lenalidomide-treated mice, n =7; QNZ-treated mice, n =8. ( i , j ) Wound-induced anagen HFs was significantly decreased in Lysm cre/+ :TNF flox/flox mice ( i ), and the number of anagen HFs in two groups was quantified ( j ). Control mice ( Lysm cre/+ :TNF flox/+ ), n =6; Lysm cre/+ :TNF flox/flox mice, n =4. Data are expressed as the mean±s.e.m. ** P

    Article Snippet: Immunostaining Frozen skin tissue sections were incubated with different primary antibodies at 4 °C overnight; antibodies included anti-CD49f-biotin (GoH3, 1:150, BioLegend), anti-Ki67 (20Raj1, 1:100, eBioscience), anti-Akt (GTX28932, phospho Ser473, 1:150, GeneTex), anti-Akt (Akt 1+2+3) (GTX121937, 1:150, GeneTex), anti-CD34-biotin (RAM34, 1:150, eBioscience), anti-Lgr-5 (ab137484, 1:200, Abcam), anti-CD45 (30-F11, 1:150, BioLegend), anti-CD45-Biotin (30-F11, 1:150, BioLegend), anti-F4/80-Biotin (BM8, 1:150, BioLegend), anti-F4/80 (BM8, 1:150, BioLegend), anti-Ly6C-Biotin (RB6-8C5, 1:200, BioLegend), anti-Ly6C (HK1.4, 1:200, BioLegend), anti-MHC II (14-4-4S, 1:200, eBioscience), anti-TNF-α (1F3F3D4, 1:100, eBioscience), anti-TNFR1-Biotin (55R-170, 1:100, BioLegend), anti-β-catenin (C2206, 1:150, Sigma) and anti-Phospho-β-Catenin (Ser552) (5651S, 1:100, CST Signaling).

    Techniques: Expressing, Mouse Assay, Microarray, Real-time Polymerase Chain Reaction, Knock-Out, Imaging

    TNF activates the PI3K/AKT pathway in HF stem cells. ( a ) Using TMT labelling and affinity enrichment followed by high-resolution LC–MS/MS and quantitative phosphor-proteomics, functional enrichment-based cluster analysis identified up-regulated signals in cultured epidermal stem cells after TNF-α treatment. ( b ) Western blot analysis of p-AKT (at Serine 473) and total AKT in cultured murine epidermal stem cells in the presence of different concentrations of TNF-α for 0.5 h. For all western blot analysis, data are representative of 3–5 independent experiments. ( c ) TNF-α treatment (8 h) resulted in no obvious changes in p-GSK-3β(ser9) and p-β-catenin (Ser33/37/Thr45, Ser675) but significantly increased the level of p-β-catenin (Ser552), which also showed in a TNF-dose-dependent manner, quite similar to p-AKT. ( d ) Perifosine diminished p-β-catenin (Ser552) levels that were elevated by TNF-α in cultured epidermal stem cells. ( e ) Western blot analysis indicated that TNF-α increased the level of β-catenin, and the effect was attenuated by Perifosine or LY294002. ( f ) TCF/LEF Dual-luciferase reporter analysis showed the relative transcription activity in differently treated groups. ( g ) At PWD-3, both p-AKT (Ser473) and p-β-catenin (Ser552) were detected in the wound adjacent to Lgr5 + hair follicle stem cells, and the p-AKT (Ser473) and p-β-catenin (Ser552) were highly co-localized. ( h ) At PWD-3, accumulated β-catenin was detected in the wound adjacent to Lgr5 + hair follicle stem cells, and the accumulated β-catenin was highly co-localized with p-AKT (Ser473). Scale bars, 30 μm.

    Journal: Nature Communications

    Article Title: Macrophages induce AKT/β-catenin-dependent Lgr5+ stem cell activation and hair follicle regeneration through TNF

    doi: 10.1038/ncomms14091

    Figure Lengend Snippet: TNF activates the PI3K/AKT pathway in HF stem cells. ( a ) Using TMT labelling and affinity enrichment followed by high-resolution LC–MS/MS and quantitative phosphor-proteomics, functional enrichment-based cluster analysis identified up-regulated signals in cultured epidermal stem cells after TNF-α treatment. ( b ) Western blot analysis of p-AKT (at Serine 473) and total AKT in cultured murine epidermal stem cells in the presence of different concentrations of TNF-α for 0.5 h. For all western blot analysis, data are representative of 3–5 independent experiments. ( c ) TNF-α treatment (8 h) resulted in no obvious changes in p-GSK-3β(ser9) and p-β-catenin (Ser33/37/Thr45, Ser675) but significantly increased the level of p-β-catenin (Ser552), which also showed in a TNF-dose-dependent manner, quite similar to p-AKT. ( d ) Perifosine diminished p-β-catenin (Ser552) levels that were elevated by TNF-α in cultured epidermal stem cells. ( e ) Western blot analysis indicated that TNF-α increased the level of β-catenin, and the effect was attenuated by Perifosine or LY294002. ( f ) TCF/LEF Dual-luciferase reporter analysis showed the relative transcription activity in differently treated groups. ( g ) At PWD-3, both p-AKT (Ser473) and p-β-catenin (Ser552) were detected in the wound adjacent to Lgr5 + hair follicle stem cells, and the p-AKT (Ser473) and p-β-catenin (Ser552) were highly co-localized. ( h ) At PWD-3, accumulated β-catenin was detected in the wound adjacent to Lgr5 + hair follicle stem cells, and the accumulated β-catenin was highly co-localized with p-AKT (Ser473). Scale bars, 30 μm.

    Article Snippet: Immunostaining Frozen skin tissue sections were incubated with different primary antibodies at 4 °C overnight; antibodies included anti-CD49f-biotin (GoH3, 1:150, BioLegend), anti-Ki67 (20Raj1, 1:100, eBioscience), anti-Akt (GTX28932, phospho Ser473, 1:150, GeneTex), anti-Akt (Akt 1+2+3) (GTX121937, 1:150, GeneTex), anti-CD34-biotin (RAM34, 1:150, eBioscience), anti-Lgr-5 (ab137484, 1:200, Abcam), anti-CD45 (30-F11, 1:150, BioLegend), anti-CD45-Biotin (30-F11, 1:150, BioLegend), anti-F4/80-Biotin (BM8, 1:150, BioLegend), anti-F4/80 (BM8, 1:150, BioLegend), anti-Ly6C-Biotin (RB6-8C5, 1:200, BioLegend), anti-Ly6C (HK1.4, 1:200, BioLegend), anti-MHC II (14-4-4S, 1:200, eBioscience), anti-TNF-α (1F3F3D4, 1:100, eBioscience), anti-TNFR1-Biotin (55R-170, 1:100, BioLegend), anti-β-catenin (C2206, 1:150, Sigma) and anti-Phospho-β-Catenin (Ser552) (5651S, 1:100, CST Signaling).

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Functional Assay, Cell Culture, Western Blot, Luciferase, Activity Assay

    PP242 alleviated the change of autophagy flux caused by TNF-α. ( A , B ) Western blot analysis of P62 and LC3B-II after TNF-α (10 ng/mL, 48 h) and/or PP242 (1 μm, 24 h) treatment. Data were shown as mean ± SD and replicated three times. ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Inhibition of Autophagic Degradation Process Contributes to Claudin-2 Expression Increase and Epithelial Tight Junction Dysfunction in TNF-α Treated Cell Monolayers

    doi: 10.3390/ijms18010157

    Figure Lengend Snippet: PP242 alleviated the change of autophagy flux caused by TNF-α. ( A , B ) Western blot analysis of P62 and LC3B-II after TNF-α (10 ng/mL, 48 h) and/or PP242 (1 μm, 24 h) treatment. Data were shown as mean ± SD and replicated three times. ** p

    Article Snippet: Recombinant TNF-α was obtained from R & D Systems (catalog no. 510-RT-010, Minneapolis, MN, USA).

    Techniques: Western Blot

    Lysosomal acidic environment was impaired after TNF-α treatment and PP242 rescued it. ( A ) Images of Caco-2 cells labeled with Lysotracker Red DND-99 dye after TNF-α (10 ng/mL, 48 h) and/or PP242 (1 μm, 24 h) treatment. The red and blue fluorescence were represented the lysosome and nucleus, respectively. Scale bar: 30 μm. ( B ) Lysosomal pH changes of Caco-2 cells labeled with Lysosensor Yellow/Blue DND-160 after TNF-α and/or PP242 treatment. Data were shown as mean ± SD and replicated three times. *** p

    Journal: International Journal of Molecular Sciences

    Article Title: Inhibition of Autophagic Degradation Process Contributes to Claudin-2 Expression Increase and Epithelial Tight Junction Dysfunction in TNF-α Treated Cell Monolayers

    doi: 10.3390/ijms18010157

    Figure Lengend Snippet: Lysosomal acidic environment was impaired after TNF-α treatment and PP242 rescued it. ( A ) Images of Caco-2 cells labeled with Lysotracker Red DND-99 dye after TNF-α (10 ng/mL, 48 h) and/or PP242 (1 μm, 24 h) treatment. The red and blue fluorescence were represented the lysosome and nucleus, respectively. Scale bar: 30 μm. ( B ) Lysosomal pH changes of Caco-2 cells labeled with Lysosensor Yellow/Blue DND-160 after TNF-α and/or PP242 treatment. Data were shown as mean ± SD and replicated three times. *** p

    Article Snippet: Recombinant TNF-α was obtained from R & D Systems (catalog no. 510-RT-010, Minneapolis, MN, USA).

    Techniques: Labeling, Fluorescence

    PP242 alleviated the change of claudin-2 expression and intestinal epithelial tight junction function in TNF-α treated Caco-2 cells. ( A ) Western blot analysis of claudin-2 after TNF-α (10 ng/mL, 48 h) and/or PP242 (1 μm, 24 h) treatment. ( B ) Immunofluorescence staining of claudin-2 after TNF-α and/or PP242 treatment in Caco-2 cell monolayers. The green and blue fluorescence were represented the claudin-2 protein and nucleus, respectively. Scale bar: 20 μm. ( C ) The effect of PP242 (1 μm, 24 h) on TER of Caco-2 cell monolayers treated with TNF-α (10 ng/mL, 48 h). ( D ) The effect of PP242 on the permeability of FITC-dextran in TNF-α treated Caco-2 cell monolayers. Data were shown as mean ± SD and replicated three times. ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Inhibition of Autophagic Degradation Process Contributes to Claudin-2 Expression Increase and Epithelial Tight Junction Dysfunction in TNF-α Treated Cell Monolayers

    doi: 10.3390/ijms18010157

    Figure Lengend Snippet: PP242 alleviated the change of claudin-2 expression and intestinal epithelial tight junction function in TNF-α treated Caco-2 cells. ( A ) Western blot analysis of claudin-2 after TNF-α (10 ng/mL, 48 h) and/or PP242 (1 μm, 24 h) treatment. ( B ) Immunofluorescence staining of claudin-2 after TNF-α and/or PP242 treatment in Caco-2 cell monolayers. The green and blue fluorescence were represented the claudin-2 protein and nucleus, respectively. Scale bar: 20 μm. ( C ) The effect of PP242 (1 μm, 24 h) on TER of Caco-2 cell monolayers treated with TNF-α (10 ng/mL, 48 h). ( D ) The effect of PP242 on the permeability of FITC-dextran in TNF-α treated Caco-2 cell monolayers. Data were shown as mean ± SD and replicated three times. ** p

    Article Snippet: Recombinant TNF-α was obtained from R & D Systems (catalog no. 510-RT-010, Minneapolis, MN, USA).

    Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence, Permeability

    The inhibition of autophagic degradation in TNF-α treated cells. ( A , B ) Western blotting analysis of LC3B-II in Caco-2 cells treated with TNF-α (10 ng/mL) in the presence or absence of 3-MA (5 mM, 24 h) or Baf A1 (100 nM, 4 h) pretreatment. ( C ) Representative images of Caco-2 cells transfected with ad-mCherry-GFP-LC3B adenovirus after TNF-α (10 ng/mL, 48 h) or Baf A1 treatment (100 nM, 4 h). Scale bar: 15 μm. ( D ) The number of red and yellow LC3 dots per cell were counted under confocal microscope ( > 30 cells/group). Data were shown as mean ± SD and replicated three times. ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Inhibition of Autophagic Degradation Process Contributes to Claudin-2 Expression Increase and Epithelial Tight Junction Dysfunction in TNF-α Treated Cell Monolayers

    doi: 10.3390/ijms18010157

    Figure Lengend Snippet: The inhibition of autophagic degradation in TNF-α treated cells. ( A , B ) Western blotting analysis of LC3B-II in Caco-2 cells treated with TNF-α (10 ng/mL) in the presence or absence of 3-MA (5 mM, 24 h) or Baf A1 (100 nM, 4 h) pretreatment. ( C ) Representative images of Caco-2 cells transfected with ad-mCherry-GFP-LC3B adenovirus after TNF-α (10 ng/mL, 48 h) or Baf A1 treatment (100 nM, 4 h). Scale bar: 15 μm. ( D ) The number of red and yellow LC3 dots per cell were counted under confocal microscope ( > 30 cells/group). Data were shown as mean ± SD and replicated three times. ** p

    Article Snippet: Recombinant TNF-α was obtained from R & D Systems (catalog no. 510-RT-010, Minneapolis, MN, USA).

    Techniques: Inhibition, Western Blot, Transfection, Microscopy

    Change of claudin-2, LC3B-II, and P62 protein in TNF-α treated Caco-2 cell monolayers. ( A ) Western blotting analysis of caludin-2 in Caco-2 cell monolayer after TNF-α (10 ng/mL) administration or 3-MA (5 mM) treatment for 48 h. ( B , C ) Western blotting analysis of LC3B-II and P62 in Caco-2 cells after TNF-α (10 ng/mL) administration for 48 h. β-actin was used as an internal control. Data were shown as mean ± SD and replicated three times. ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Inhibition of Autophagic Degradation Process Contributes to Claudin-2 Expression Increase and Epithelial Tight Junction Dysfunction in TNF-α Treated Cell Monolayers

    doi: 10.3390/ijms18010157

    Figure Lengend Snippet: Change of claudin-2, LC3B-II, and P62 protein in TNF-α treated Caco-2 cell monolayers. ( A ) Western blotting analysis of caludin-2 in Caco-2 cell monolayer after TNF-α (10 ng/mL) administration or 3-MA (5 mM) treatment for 48 h. ( B , C ) Western blotting analysis of LC3B-II and P62 in Caco-2 cells after TNF-α (10 ng/mL) administration for 48 h. β-actin was used as an internal control. Data were shown as mean ± SD and replicated three times. ** p

    Article Snippet: Recombinant TNF-α was obtained from R & D Systems (catalog no. 510-RT-010, Minneapolis, MN, USA).

    Techniques: Western Blot