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    Name:
    Brefeldin A Solution 1 000X
    Description:
    Brefeldin A Solution 1 000X Apps ICFC Size 1 ml
    Catalog Number:
    420601
    Price:
    55
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    Buffer Solution Chemical
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    ICFC
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    Structured Review

    BioLegend brefeldin
    Curcusomes inhibit oxidative burst, cytokine production, and NF-κB in peritoneal macrophages from ob/ob mice. A : Cells isolated from peritoneal exudates from ob/ob mice were cultured for 30 min with or without zymosan in the presence of 1 μg/mL final concentration of empty liposomes or curcusomes. Oxidative burst was measured by chemiluminescence and was expressed as relative light units (RLUs). * P ≤ 0.05 (one-way ANOVA followed by Bonferroni post hoc test). Data are mean ± SEM of two separate experiments analyzing peritoneal exudates pooled from three mice. B : Cells isolated from peritoneal exudates from ob/ob mice were cultured in media for 6 h with or without LPS in the presence of 1 μg/mL final concentration of empty liposomes or curcusomes. Shown is IL-6 concentration in supernatants measured by cytokine bead array. *** P ≤ 0.001 (one-way ANOVA followed by Bonferroni post hoc test). Data are mean ± SEM of two separate experiments analyzing peritoneal exudates pooled from three mice. C : Ob/ob mice were injected intraperitoneally with DiI-curcusomes, and 24 h later, cells from peritoneal exudates were harvested and stained for MHC class II and B220. Shown is DiI uptake by both macrophages and B cells. Data representative of five separate experiments. D : Cells isolated from peritoneal exudates from ob/ob mice injected intraperitoneally with DiI-labeled empty liposomes or curcumin liposomes were cultured with or without LPS for 24 h in the presence of <t>brefeldin</t> and then stained for surface B220 and intracellular IL-6. Shown are flow cytometry plots with B220-negative macrophages as the main source of IL-6. Data representative of two separate experiments. E : Cells in C were adhered to chamber slides for 1 h in the presence of LPS and then stained for RelA (green) or p50 (green), CD11c (blue), and DAPI (cyan). DiI staining is shown in red; images acquired by immunofluorescence microscopy. Original magnification ×25. Data representative of three separate experiments. F : Nuclear extracts from cells in C were analyzed for DNA binding of RelA by chemiluminescence. Shown is the mean of duplicates from two separate experiments. Ip, intraperitoneally. (A high-quality digital representation of this figure is available in the online issue.)
    Brefeldin A Solution 1 000X Apps ICFC Size 1 ml
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    Images

    1) Product Images from "Targeting Curcusomes to Inflammatory Dendritic Cells Inhibits NF-?B and Improves Insulin Resistance in Obese Mice"

    Article Title: Targeting Curcusomes to Inflammatory Dendritic Cells Inhibits NF-?B and Improves Insulin Resistance in Obese Mice

    Journal: Diabetes

    doi: 10.2337/db11-0275

    Curcusomes inhibit oxidative burst, cytokine production, and NF-κB in peritoneal macrophages from ob/ob mice. A : Cells isolated from peritoneal exudates from ob/ob mice were cultured for 30 min with or without zymosan in the presence of 1 μg/mL final concentration of empty liposomes or curcusomes. Oxidative burst was measured by chemiluminescence and was expressed as relative light units (RLUs). * P ≤ 0.05 (one-way ANOVA followed by Bonferroni post hoc test). Data are mean ± SEM of two separate experiments analyzing peritoneal exudates pooled from three mice. B : Cells isolated from peritoneal exudates from ob/ob mice were cultured in media for 6 h with or without LPS in the presence of 1 μg/mL final concentration of empty liposomes or curcusomes. Shown is IL-6 concentration in supernatants measured by cytokine bead array. *** P ≤ 0.001 (one-way ANOVA followed by Bonferroni post hoc test). Data are mean ± SEM of two separate experiments analyzing peritoneal exudates pooled from three mice. C : Ob/ob mice were injected intraperitoneally with DiI-curcusomes, and 24 h later, cells from peritoneal exudates were harvested and stained for MHC class II and B220. Shown is DiI uptake by both macrophages and B cells. Data representative of five separate experiments. D : Cells isolated from peritoneal exudates from ob/ob mice injected intraperitoneally with DiI-labeled empty liposomes or curcumin liposomes were cultured with or without LPS for 24 h in the presence of brefeldin and then stained for surface B220 and intracellular IL-6. Shown are flow cytometry plots with B220-negative macrophages as the main source of IL-6. Data representative of two separate experiments. E : Cells in C were adhered to chamber slides for 1 h in the presence of LPS and then stained for RelA (green) or p50 (green), CD11c (blue), and DAPI (cyan). DiI staining is shown in red; images acquired by immunofluorescence microscopy. Original magnification ×25. Data representative of three separate experiments. F : Nuclear extracts from cells in C were analyzed for DNA binding of RelA by chemiluminescence. Shown is the mean of duplicates from two separate experiments. Ip, intraperitoneally. (A high-quality digital representation of this figure is available in the online issue.)
    Figure Legend Snippet: Curcusomes inhibit oxidative burst, cytokine production, and NF-κB in peritoneal macrophages from ob/ob mice. A : Cells isolated from peritoneal exudates from ob/ob mice were cultured for 30 min with or without zymosan in the presence of 1 μg/mL final concentration of empty liposomes or curcusomes. Oxidative burst was measured by chemiluminescence and was expressed as relative light units (RLUs). * P ≤ 0.05 (one-way ANOVA followed by Bonferroni post hoc test). Data are mean ± SEM of two separate experiments analyzing peritoneal exudates pooled from three mice. B : Cells isolated from peritoneal exudates from ob/ob mice were cultured in media for 6 h with or without LPS in the presence of 1 μg/mL final concentration of empty liposomes or curcusomes. Shown is IL-6 concentration in supernatants measured by cytokine bead array. *** P ≤ 0.001 (one-way ANOVA followed by Bonferroni post hoc test). Data are mean ± SEM of two separate experiments analyzing peritoneal exudates pooled from three mice. C : Ob/ob mice were injected intraperitoneally with DiI-curcusomes, and 24 h later, cells from peritoneal exudates were harvested and stained for MHC class II and B220. Shown is DiI uptake by both macrophages and B cells. Data representative of five separate experiments. D : Cells isolated from peritoneal exudates from ob/ob mice injected intraperitoneally with DiI-labeled empty liposomes or curcumin liposomes were cultured with or without LPS for 24 h in the presence of brefeldin and then stained for surface B220 and intracellular IL-6. Shown are flow cytometry plots with B220-negative macrophages as the main source of IL-6. Data representative of two separate experiments. E : Cells in C were adhered to chamber slides for 1 h in the presence of LPS and then stained for RelA (green) or p50 (green), CD11c (blue), and DAPI (cyan). DiI staining is shown in red; images acquired by immunofluorescence microscopy. Original magnification ×25. Data representative of three separate experiments. F : Nuclear extracts from cells in C were analyzed for DNA binding of RelA by chemiluminescence. Shown is the mean of duplicates from two separate experiments. Ip, intraperitoneally. (A high-quality digital representation of this figure is available in the online issue.)

    Techniques Used: Mouse Assay, Isolation, Cell Culture, Concentration Assay, Injection, Staining, Labeling, Flow Cytometry, Cytometry, Immunofluorescence, Microscopy, Binding Assay

    Inflammatory liver DCs in ob/ob mice. A : Hepatic mRNA was purified from 12-week-old ob/ob mice or 12-week-old nonobese littermate controls (NOB) and analyzed by qPCR for relative expression of cytokines. Shown is the fold increase in cytokine expression comparing RNA from ob/ob liver with NOB liver. Data are mean ± SEM of two separate experiments analyzing individual mice. B : Gradient-purified cells isolated from the livers of 12-week-old ob/ob mice or NOB mice were stained for surface markers against leukocyte subsets and analyzed by flow cytometry. ** P ≤ 0.01, *** P ≤ 0.001 (one-way ANOVA followed by Bonferroni post hoc test). Data are mean ± SEM of two separate experiments analyzing individual mice. pDC, plasmacytoid DC. C : CD11c+ cells were purified by magnetic bead selection from gradient-enriched liver leukocytes from 12-week-old ob/ob mice or NOB mice. Cells were cultured with or without LPS for 24 h in the presence of brefeldin and then stained for intracellular TNF and iNOS. Shown are flow cytometry analysis of cells and percentage of cells staining positive for TNF and iNOS. Data are mean ± SEM of two separate experiments analyzing individual mice. D : CD11c purified liver cells from ob/ob mice were stained for surface markers, costimulatory molecules, and MHC class II and analyzed by flow cytometry. E : CD11c+ DCs purified from livers of ob/ob mice were cultured for 48 h, stained for costimulatory molecules and MHC class II, and analyzed by flow cytometry. F : Livers from 12-week-old ob/ob mice or NOB mice were harvested, frozen in optimal cutting temperature media, sectioned, and stained with RelA (red) and CD11c (blue) antibodies. Arrows indicate nuclear RelA in CD11c+ cells. Original magnification ×25. (A high-quality digital representation of this figure is available in the online issue.)
    Figure Legend Snippet: Inflammatory liver DCs in ob/ob mice. A : Hepatic mRNA was purified from 12-week-old ob/ob mice or 12-week-old nonobese littermate controls (NOB) and analyzed by qPCR for relative expression of cytokines. Shown is the fold increase in cytokine expression comparing RNA from ob/ob liver with NOB liver. Data are mean ± SEM of two separate experiments analyzing individual mice. B : Gradient-purified cells isolated from the livers of 12-week-old ob/ob mice or NOB mice were stained for surface markers against leukocyte subsets and analyzed by flow cytometry. ** P ≤ 0.01, *** P ≤ 0.001 (one-way ANOVA followed by Bonferroni post hoc test). Data are mean ± SEM of two separate experiments analyzing individual mice. pDC, plasmacytoid DC. C : CD11c+ cells were purified by magnetic bead selection from gradient-enriched liver leukocytes from 12-week-old ob/ob mice or NOB mice. Cells were cultured with or without LPS for 24 h in the presence of brefeldin and then stained for intracellular TNF and iNOS. Shown are flow cytometry analysis of cells and percentage of cells staining positive for TNF and iNOS. Data are mean ± SEM of two separate experiments analyzing individual mice. D : CD11c purified liver cells from ob/ob mice were stained for surface markers, costimulatory molecules, and MHC class II and analyzed by flow cytometry. E : CD11c+ DCs purified from livers of ob/ob mice were cultured for 48 h, stained for costimulatory molecules and MHC class II, and analyzed by flow cytometry. F : Livers from 12-week-old ob/ob mice or NOB mice were harvested, frozen in optimal cutting temperature media, sectioned, and stained with RelA (red) and CD11c (blue) antibodies. Arrows indicate nuclear RelA in CD11c+ cells. Original magnification ×25. (A high-quality digital representation of this figure is available in the online issue.)

    Techniques Used: Mouse Assay, Purification, Real-time Polymerase Chain Reaction, Expressing, Isolation, Staining, Flow Cytometry, Cytometry, Selection, Cell Culture

    Related Articles

    Inhibition:

    Article Title: CD36 Ecto-domain Phosphorylation Blocks Thrombospondin-1 Binding: Structure - Function Relationships and Regulation by Protein Kinase C
    Article Snippet: We thus hypothesized that CD36 phosphorylation occurs on newly synthesized protein and indeed found that phosphorylation was blocked by inhibiting new protein synthesis. .. Furthermore, inhibition by brefeldin A suggests that phosphorylation occurs in an intracellular post-ER compartment. .. In summary, we showed with in vitro assays that CD36 phosphorylation directly inhibits binding of TSR domains to CD36 CLESH domains and provide convincing in vivo evidence that low basal levels of cellular CD36 phosphorylation can be enhanced in the setting of robust CD36 synthesis by activating an intracellular PKC – mediated signaling pathway.

    Cell Culture:

    Article Title: Peripheral Blood Invariant Natural Killer T Cells of Pig-Tailed Macaques
    Article Snippet: .. PBMCs Stimulation by α-GalCer PBMCs were cultured in a 96-well U-bottom plate at 1×106 cells/well in the presence of 5 µg/ml or 0.1 µg/ml of α-GalCer for 6 hours at 37°C followed by the addition of Brefeldin A (BioLegend) at 5 µg/ml for the last 4 hours of incubation. .. In a negative control group, cells were stimulated with medium containing 0.1% of DMSO vehicle.

    Incubation:

    Article Title: Peripheral Blood Invariant Natural Killer T Cells of Pig-Tailed Macaques
    Article Snippet: .. PBMCs Stimulation by α-GalCer PBMCs were cultured in a 96-well U-bottom plate at 1×106 cells/well in the presence of 5 µg/ml or 0.1 µg/ml of α-GalCer for 6 hours at 37°C followed by the addition of Brefeldin A (BioLegend) at 5 µg/ml for the last 4 hours of incubation. .. In a negative control group, cells were stimulated with medium containing 0.1% of DMSO vehicle.

    Purification:

    Article Title: Decrease in the proportion of CD24hiCD38hi B cells and impairment of their regulatory capacity in type 1 diabetes patients
    Article Snippet: Purified B and CD4+ CD25− T cells were cocultured in 96‐well U‐bottomed plates in RPMI‐1640 medium ( gibco , Carlsbad, CA, USA) containing 10% fetal calf serum (FCS) ( gibco ), penicillin (200 µg/ml; Sigma, St Louis, MO, USA), streptomycin (200 U/ml, Sigma) and 4 mM L‐glutamine. .. For cytokine detection, CD4+ CD25− T cells in co‐cultures with B cells were stimulated with purified plate‐bound CD3 mAb (0·5 µg/ml, BD) for 72 h. Brefeldin A (BFA) (10 µg/ml; Biolegend) was added for the last 5 h together with phorbol 12‐myristate 13‐acetate (PMA) (50 ng/ml; Sigma‐Aldrich, St Louis, MO, USA) and ionomycin (1 µg/ml, Sigma‐Aldrich). .. Alternatively, anti‐IL‐10 (5 µg/ml) (JES3‐9D7), anti‐IL‐10 receptor (3F9), anti‐CD80 (10 µg/ml) (2D10.4) or anti‐CD86 (10 µg/ml) (IT2.2) or with their matching irrelevant isotype control was added to the co‐culture of CD3 mAb‐stimulated B and T cells.

    In Vitro:

    Article Title: 1,25-Dihydroxyvitamin D3 Ameliorates Collagen-Induced Arthritis via Suppression of Th17 Cells Through miR-124 Mediated Inhibition of IL-6 Signaling
    Article Snippet: T Cell Subsets Analysis in CIA Model Cells were isolated from draining lymph nodes of arthritic mice at day 30 and day 60 after CII immunization. .. For Th1 cells and Th17 cells detection, cells were stimulated in vitro with PMA (50 ng/ml) and ionomycin (500 ng/ml) (all from Sigma) for 5 h, with brefeldin A (10 μg/ml, biolegend) added in the last 4 h, and intracellular IL-17A, IFN-γ expression on CD4+ T cells was analyzed by flow cytometry. .. For Tregs, total cells from draining lymph nodes or synovial fluid of knee joint were stained with Foxp3 (GFP), Nrp-1 and CD4 antibodies and then analyzed by flow cytometry.

    Article Title: CD4+ T cells promote humoral immunity and viral control during Zika virus infection
    Article Snippet: LysMCre + Ifnar1 fl/fl C57BL/6 mice were infected retro-orbitally with 104 FFU of ZIKV strains MR766 or FSS13025. .. At day 7 post-infection, splenocytes were prepared and stimulated in vitro with one of the indicated immunodominant ZIKV epitopes in the presence of brefeldin A for 5 h. The frequency of cells producing ( A ) IL-4, ( B ) IL-5, or ( C ) IL-17A was assessed by ICS. ..

    Expressing:

    Article Title: 1,25-Dihydroxyvitamin D3 Ameliorates Collagen-Induced Arthritis via Suppression of Th17 Cells Through miR-124 Mediated Inhibition of IL-6 Signaling
    Article Snippet: T Cell Subsets Analysis in CIA Model Cells were isolated from draining lymph nodes of arthritic mice at day 30 and day 60 after CII immunization. .. For Th1 cells and Th17 cells detection, cells were stimulated in vitro with PMA (50 ng/ml) and ionomycin (500 ng/ml) (all from Sigma) for 5 h, with brefeldin A (10 μg/ml, biolegend) added in the last 4 h, and intracellular IL-17A, IFN-γ expression on CD4+ T cells was analyzed by flow cytometry. .. For Tregs, total cells from draining lymph nodes or synovial fluid of knee joint were stained with Foxp3 (GFP), Nrp-1 and CD4 antibodies and then analyzed by flow cytometry.

    Flow Cytometry:

    Article Title: 1,25-Dihydroxyvitamin D3 Ameliorates Collagen-Induced Arthritis via Suppression of Th17 Cells Through miR-124 Mediated Inhibition of IL-6 Signaling
    Article Snippet: T Cell Subsets Analysis in CIA Model Cells were isolated from draining lymph nodes of arthritic mice at day 30 and day 60 after CII immunization. .. For Th1 cells and Th17 cells detection, cells were stimulated in vitro with PMA (50 ng/ml) and ionomycin (500 ng/ml) (all from Sigma) for 5 h, with brefeldin A (10 μg/ml, biolegend) added in the last 4 h, and intracellular IL-17A, IFN-γ expression on CD4+ T cells was analyzed by flow cytometry. .. For Tregs, total cells from draining lymph nodes or synovial fluid of knee joint were stained with Foxp3 (GFP), Nrp-1 and CD4 antibodies and then analyzed by flow cytometry.

    Cytometry:

    Article Title: 1,25-Dihydroxyvitamin D3 Ameliorates Collagen-Induced Arthritis via Suppression of Th17 Cells Through miR-124 Mediated Inhibition of IL-6 Signaling
    Article Snippet: T Cell Subsets Analysis in CIA Model Cells were isolated from draining lymph nodes of arthritic mice at day 30 and day 60 after CII immunization. .. For Th1 cells and Th17 cells detection, cells were stimulated in vitro with PMA (50 ng/ml) and ionomycin (500 ng/ml) (all from Sigma) for 5 h, with brefeldin A (10 μg/ml, biolegend) added in the last 4 h, and intracellular IL-17A, IFN-γ expression on CD4+ T cells was analyzed by flow cytometry. .. For Tregs, total cells from draining lymph nodes or synovial fluid of knee joint were stained with Foxp3 (GFP), Nrp-1 and CD4 antibodies and then analyzed by flow cytometry.

    Concentration Assay:

    Article Title: Selective targeting of pro-inflammatory Th1 cells by microRNA-148a-specific antagomirs in vivo
    Article Snippet: In order to distinguish splenocytes from BM cells, splenocytes were labeled with CFSE (1 μM, 10 min at 37 °C, 5% CO2 ) prior to stimulation with NP-CGG. .. After the first 2 h of stimulation, Brefeldin A (5 μg/ml final concentration) was added to the cultures. .. Subsequently, cells were stained intracellularly for cytokines and CD40L as described for cells stimulated with PMA/ionomycin above.

    other:

    Article Title: Regulatory B Cells Are Decreased and Impaired in Their Function in Peripheral Maternal Blood in Pre-term Birth
    Article Snippet: PMA (50 ng/ml), ionomycin (500 ng/ml) and Brefeldin A was added for the last 5 h.

    Cell Stimulation:

    Article Title: Human intestinal tissue-resident memory CD8+ T cells comprise transcriptionally and functionally distinct subsets
    Article Snippet: In brief, purified intestine-derived mononuclear cells were plated at approximately 1×106 /well in a U-bottom 96-well plate. .. Cell stimulation cocktail containing phorbol 12-myristate 13-acetate (PMA) and ionomycin (Biolegend) was added in accordance with manufacturer’s instructions in the presence of brefeldin A and monensin (both Biolegend). ..

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    BioLegend brefeldin a
    PMA-induced CD36 phosphorylation inhibits TSR-mediated Src recruitment to CD36.  A.  CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA or vehicle control. Some cells were then treated for 1h with 40u/ml alkaline phosphatase (AP) or vehicle control. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. CD36 was then immunoprecipitated and the precipitates were analyzed by western blot with an antibody to Src family proteins. Blots were re-probed with anti-CD36 antibodies. Blots were scanned and the amount of co-precipitated Src was normalized to total CD36.  B.  CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA along with either 100μg/ml cycloheximide or 5ng/ml Brefeldin A. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. Src family association was then assessed by western blot and analyzed as in panel A. C) Cells prepared as in Panel B were analyzed by western blot using antibodies to phospho-PKCα (upper blot). Blots were then stripped and re-probed with an antibody to total PKC (lower blot). All blots are representative of n=3.
    Brefeldin A, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/brefeldin a/product/BioLegend
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    brefeldin a - by Bioz Stars, 2021-06
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    PMA-induced CD36 phosphorylation inhibits TSR-mediated Src recruitment to CD36.  A.  CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA or vehicle control. Some cells were then treated for 1h with 40u/ml alkaline phosphatase (AP) or vehicle control. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. CD36 was then immunoprecipitated and the precipitates were analyzed by western blot with an antibody to Src family proteins. Blots were re-probed with anti-CD36 antibodies. Blots were scanned and the amount of co-precipitated Src was normalized to total CD36.  B.  CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA along with either 100μg/ml cycloheximide or 5ng/ml Brefeldin A. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. Src family association was then assessed by western blot and analyzed as in panel A. C) Cells prepared as in Panel B were analyzed by western blot using antibodies to phospho-PKCα (upper blot). Blots were then stripped and re-probed with an antibody to total PKC (lower blot). All blots are representative of n=3.

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: CD36 Ecto-domain Phosphorylation Blocks Thrombospondin-1 Binding: Structure - Function Relationships and Regulation by Protein Kinase C

    doi: 10.1161/ATVBAHA.111.242511

    Figure Lengend Snippet: PMA-induced CD36 phosphorylation inhibits TSR-mediated Src recruitment to CD36. A. CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA or vehicle control. Some cells were then treated for 1h with 40u/ml alkaline phosphatase (AP) or vehicle control. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. CD36 was then immunoprecipitated and the precipitates were analyzed by western blot with an antibody to Src family proteins. Blots were re-probed with anti-CD36 antibodies. Blots were scanned and the amount of co-precipitated Src was normalized to total CD36. B. CD36 transfected Bowes melanoma cells were incubated for 16h in low serum media and then treated for 4h with 0.1μg/ml PMA along with either 100μg/ml cycloheximide or 5ng/ml Brefeldin A. Cells were then exposed to 100nM recombinant TSR2/3 or thioredoxin control for 15min. Src family association was then assessed by western blot and analyzed as in panel A. C) Cells prepared as in Panel B were analyzed by western blot using antibodies to phospho-PKCα (upper blot). Blots were then stripped and re-probed with an antibody to total PKC (lower blot). All blots are representative of n=3.

    Article Snippet: Furthermore, inhibition by brefeldin A suggests that phosphorylation occurs in an intracellular post-ER compartment.

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

    VD treatment inhibited T helper cells differentiation while promoted Tregs induction in vivo . MACS-sorted CD4 + CD62L + T cells (5 × 10 5 ) from spleen of C57BL6 Foxp3 gfp reporter mice were transferred to C57BL6 Rag1 −/− mice through i.p . injection and VD was given every other day for 15 days starting 1 day before cells transfer or just the same volume of 0.1% ethanol as control. (A,B) Representative flow data (A) and statistical analysis (B) for the percentages of cells expressing IL-17A in the CD4 + population of spleen (SP) and lymph nodes (LN) at day 30 after cells transfer. Fresh cells taken from spleens and LNs of mice in the VD-treated group and control groups were stimulated with phorbol12-myristate13-acetate (PMA) and Ionomycin for 5 h and with brefeldin A (BFA) for 4 h, followed by staining for IL-17A. Cells were gated on CD4 + T cells. Data were presented as the mean ± SEM from two independent experiments ( n = 4). * P

    Journal: Frontiers in Immunology

    Article Title: 1,25-Dihydroxyvitamin D3 Ameliorates Collagen-Induced Arthritis via Suppression of Th17 Cells Through miR-124 Mediated Inhibition of IL-6 Signaling

    doi: 10.3389/fimmu.2019.00178

    Figure Lengend Snippet: VD treatment inhibited T helper cells differentiation while promoted Tregs induction in vivo . MACS-sorted CD4 + CD62L + T cells (5 × 10 5 ) from spleen of C57BL6 Foxp3 gfp reporter mice were transferred to C57BL6 Rag1 −/− mice through i.p . injection and VD was given every other day for 15 days starting 1 day before cells transfer or just the same volume of 0.1% ethanol as control. (A,B) Representative flow data (A) and statistical analysis (B) for the percentages of cells expressing IL-17A in the CD4 + population of spleen (SP) and lymph nodes (LN) at day 30 after cells transfer. Fresh cells taken from spleens and LNs of mice in the VD-treated group and control groups were stimulated with phorbol12-myristate13-acetate (PMA) and Ionomycin for 5 h and with brefeldin A (BFA) for 4 h, followed by staining for IL-17A. Cells were gated on CD4 + T cells. Data were presented as the mean ± SEM from two independent experiments ( n = 4). * P

    Article Snippet: For Th1 cells and Th17 cells detection, cells were stimulated in vitro with PMA (50 ng/ml) and ionomycin (500 ng/ml) (all from Sigma) for 5 h, with brefeldin A (10 μg/ml, biolegend) added in the last 4 h, and intracellular IL-17A, IFN-γ expression on CD4+ T cells was analyzed by flow cytometry.

    Techniques: In Vivo, Magnetic Cell Separation, Mouse Assay, Injection, Flow Cytometry, Expressing, Staining

    Systemic antagomir-148a treatment of colitic mice depletes IFN-γ-expressing Th1 cells selectively in the inflamed colon.  (A) Schematic overview depicting the experimental procedure for inducing colitis by adoptive transfer of repeatedly activated Th1 cells into  Rag1 −/−  mice and subsequent antagomir treatment. (B) Representative dot plots showing the frequencies of CD3 + CD4 +  Th cells isolated from inflamed colons of colitic mice that were treated with antagomir-Scr or antagomir-148a. Displayed frequencies are percentages of Th cells among isolated viable cells. (C) Total cell numbers of viable CD3 + CD4 +  Th cells that were isolated from the colons and spleens of colitic mice following antagomir-148a or antagomir-Scr treatment as shown in (A). (D–G) Lymphocytes from colitic mice were isolated from the inflamed colons and stimulated with PMA/ionomycin in the presence of Brefeldin A. Shown are the absolute cell numbers of CD3 + CD4 +  Th cells that expressed IFN-γ (D), IL-10 (E), IL-17A (F) or IL-22 (G). (H) Quantification of different myeloid subsets isolated from inflamed colonic mucosae of colitic mice. Depicted data are pooled from two independent experiments with n = 12 and n = 13 for antagomir-148a- and antagomir-Scr-treated mice, respectively.

    Journal: Journal of Autoimmunity

    Article Title: Selective targeting of pro-inflammatory Th1 cells by microRNA-148a-specific antagomirs in vivo

    doi: 10.1016/j.jaut.2017.11.005

    Figure Lengend Snippet: Systemic antagomir-148a treatment of colitic mice depletes IFN-γ-expressing Th1 cells selectively in the inflamed colon. (A) Schematic overview depicting the experimental procedure for inducing colitis by adoptive transfer of repeatedly activated Th1 cells into Rag1 −/− mice and subsequent antagomir treatment. (B) Representative dot plots showing the frequencies of CD3 + CD4 + Th cells isolated from inflamed colons of colitic mice that were treated with antagomir-Scr or antagomir-148a. Displayed frequencies are percentages of Th cells among isolated viable cells. (C) Total cell numbers of viable CD3 + CD4 + Th cells that were isolated from the colons and spleens of colitic mice following antagomir-148a or antagomir-Scr treatment as shown in (A). (D–G) Lymphocytes from colitic mice were isolated from the inflamed colons and stimulated with PMA/ionomycin in the presence of Brefeldin A. Shown are the absolute cell numbers of CD3 + CD4 + Th cells that expressed IFN-γ (D), IL-10 (E), IL-17A (F) or IL-22 (G). (H) Quantification of different myeloid subsets isolated from inflamed colonic mucosae of colitic mice. Depicted data are pooled from two independent experiments with n = 12 and n = 13 for antagomir-148a- and antagomir-Scr-treated mice, respectively.

    Article Snippet: After the first 2 h of stimulation, Brefeldin A (5 μg/ml final concentration) was added to the cultures.

    Techniques: Mouse Assay, Expressing, Adoptive Transfer Assay, Isolation

    Host dysbiosis negatively impacts intestinal IL-9-producing T cells. Specific pathogen-free C57BL/6 mice received an antibiotic cocktail twice a day by gavage for 4 weeks (ABX), or were left untreated (control). Microbiota depletion in faecal pellets was evaluated by thioglycolate culture ( a ) and bacterial 16 S V2 DNA load ( b ) at the end of treatment. The total RNA was extracted from the colon, and relative expression of TGF-β ( c ) and IL-4 ( d ) was determined by real-time qPCR. Colonic lamina propria cells from control and ABX mice were extracted and stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml) and Brefeldin A (5 μg/ml) for 4 h at 37 °C and 5% CO 2 , and analysed by flow cytometry ( e ) to determine the frequency of IL-9-producing CD4 + ( f ) and CD8 + ( g ) T cells. Data are shown as mean ± SD. Unpaired Student’s t test was used for statistical analysis.

    Journal: British Journal of Cancer

    Article Title: Host dysbiosis negatively impacts IL-9-producing T-cell differentiation and antitumour immunity

    doi: 10.1038/s41416-020-0915-6

    Figure Lengend Snippet: Host dysbiosis negatively impacts intestinal IL-9-producing T cells. Specific pathogen-free C57BL/6 mice received an antibiotic cocktail twice a day by gavage for 4 weeks (ABX), or were left untreated (control). Microbiota depletion in faecal pellets was evaluated by thioglycolate culture ( a ) and bacterial 16 S V2 DNA load ( b ) at the end of treatment. The total RNA was extracted from the colon, and relative expression of TGF-β ( c ) and IL-4 ( d ) was determined by real-time qPCR. Colonic lamina propria cells from control and ABX mice were extracted and stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml) and Brefeldin A (5 μg/ml) for 4 h at 37 °C and 5% CO 2 , and analysed by flow cytometry ( e ) to determine the frequency of IL-9-producing CD4 + ( f ) and CD8 + ( g ) T cells. Data are shown as mean ± SD. Unpaired Student’s t test was used for statistical analysis.

    Article Snippet: Cells from lamina propria and lungs were stimulated with PMA (Sigma-Aldrich) 50 ng/ml, Ionomycin (Sigma-Aldrich) 500 ng/ml and Brefeldin A (Biolegend, San Diego, CA, USA) 5 µg/ml for 4 h at 37 °C and 5% CO2.

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

    Faecal microbiota transplant restores lung-infiltrating IL-9-producing T cells and protects antibiotic-treated mice from tumour development. C57BL/6 mice received an antibiotic cocktail twice a day by gavage for 4 weeks (ABX), or were left untreated (control). After 4 weeks of treatment, a group of ABX-treated mice received faecal microbiota transplant (FMT), while the other group was kept under treatment. All mice were intravenously injected with 5 × 10 4 B16F10 cells, and tumour foci number in the lungs was determined 14 days after injection ( a ). Lungs were then digested, and cells stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml) and Brefeldin A (5 μg/ml) for 4 h at 37 °C and 5% CO 2 . The frequency of IL-9-producing CD4 + ( b ) and CD8 + ( c ) T cells was evaluated by flow cytometry. Metagenomic analysis of the gut microbiota was performed in faecal samples collected at the day of and 2 weeks after injection of B16F10 cells ( d ). Microbiota alpha-diversity was determined by the Shannon index ( e ). Principal coordinate analysis (PCoA) based on weighted Unifrac metric of faecal microbiota among all samples was also performed ( f ). Data are shown as mean ± SD. One-way ANOVA followed by Tukey’s multiple-comparison test and Kruskal–Wallis test was used for statistical analysis. Control animals before (C1) and 15 days after tumour injection (C2). ABX-treated animals before (A1) and 15 days after tumour injection (A2). Microbiota-reconstituted animals before (R1) and 15 days after tumour injection (R2). * p = 0.009 compared with Groups 1 and 2. # p = 0.009 compared with Groups 5 and 6.

    Journal: British Journal of Cancer

    Article Title: Host dysbiosis negatively impacts IL-9-producing T-cell differentiation and antitumour immunity

    doi: 10.1038/s41416-020-0915-6

    Figure Lengend Snippet: Faecal microbiota transplant restores lung-infiltrating IL-9-producing T cells and protects antibiotic-treated mice from tumour development. C57BL/6 mice received an antibiotic cocktail twice a day by gavage for 4 weeks (ABX), or were left untreated (control). After 4 weeks of treatment, a group of ABX-treated mice received faecal microbiota transplant (FMT), while the other group was kept under treatment. All mice were intravenously injected with 5 × 10 4 B16F10 cells, and tumour foci number in the lungs was determined 14 days after injection ( a ). Lungs were then digested, and cells stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml) and Brefeldin A (5 μg/ml) for 4 h at 37 °C and 5% CO 2 . The frequency of IL-9-producing CD4 + ( b ) and CD8 + ( c ) T cells was evaluated by flow cytometry. Metagenomic analysis of the gut microbiota was performed in faecal samples collected at the day of and 2 weeks after injection of B16F10 cells ( d ). Microbiota alpha-diversity was determined by the Shannon index ( e ). Principal coordinate analysis (PCoA) based on weighted Unifrac metric of faecal microbiota among all samples was also performed ( f ). Data are shown as mean ± SD. One-way ANOVA followed by Tukey’s multiple-comparison test and Kruskal–Wallis test was used for statistical analysis. Control animals before (C1) and 15 days after tumour injection (C2). ABX-treated animals before (A1) and 15 days after tumour injection (A2). Microbiota-reconstituted animals before (R1) and 15 days after tumour injection (R2). * p = 0.009 compared with Groups 1 and 2. # p = 0.009 compared with Groups 5 and 6.

    Article Snippet: Cells from lamina propria and lungs were stimulated with PMA (Sigma-Aldrich) 50 ng/ml, Ionomycin (Sigma-Aldrich) 500 ng/ml and Brefeldin A (Biolegend, San Diego, CA, USA) 5 µg/ml for 4 h at 37 °C and 5% CO2.

    Techniques: Mouse Assay, Injection, Flow Cytometry

    Antibiotic-treated mice have a higher number of tumour foci and lower lung-infiltrating IL-9-producing T cells. Antibiotic-treated (ABX) and -untreated (control) C57BL/6 mice were intravenously injected with 5 × 10 4 B16F10 cells. Tumour foci number in the lungs was determined 14 days after injection ( a ). Lungs were then digested, and cells stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml) and Brefeldin A (5 μg/ml) for 4 h at 37 °C and 5% CO 2 . The frequency of IL-9-producing CD4 + ( b ) and CD8 + ( c ) T cells and IL-9-producing non-T cells ( d ) was evaluated by flow cytometry. Metagenomic analysis of the gut microbiota was performed in faecal samples collected at the day of and 2 weeks after injection of B16F10 cells ( e ). Mice from the ABX group were kept under treatment throughout the experiment. Data are shown as mean ± SD. Unpaired Student’s t test was used for statistical analysis.

    Journal: British Journal of Cancer

    Article Title: Host dysbiosis negatively impacts IL-9-producing T-cell differentiation and antitumour immunity

    doi: 10.1038/s41416-020-0915-6

    Figure Lengend Snippet: Antibiotic-treated mice have a higher number of tumour foci and lower lung-infiltrating IL-9-producing T cells. Antibiotic-treated (ABX) and -untreated (control) C57BL/6 mice were intravenously injected with 5 × 10 4 B16F10 cells. Tumour foci number in the lungs was determined 14 days after injection ( a ). Lungs were then digested, and cells stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml) and Brefeldin A (5 μg/ml) for 4 h at 37 °C and 5% CO 2 . The frequency of IL-9-producing CD4 + ( b ) and CD8 + ( c ) T cells and IL-9-producing non-T cells ( d ) was evaluated by flow cytometry. Metagenomic analysis of the gut microbiota was performed in faecal samples collected at the day of and 2 weeks after injection of B16F10 cells ( e ). Mice from the ABX group were kept under treatment throughout the experiment. Data are shown as mean ± SD. Unpaired Student’s t test was used for statistical analysis.

    Article Snippet: Cells from lamina propria and lungs were stimulated with PMA (Sigma-Aldrich) 50 ng/ml, Ionomycin (Sigma-Aldrich) 500 ng/ml and Brefeldin A (Biolegend, San Diego, CA, USA) 5 µg/ml for 4 h at 37 °C and 5% CO2.

    Techniques: Mouse Assay, Injection, Flow Cytometry

    Host microbiota play a key role in the development of intestinal IL-9-producing T cells. Colonic lamina propria cells from conventional Swiss, germ-free or germ-free conventionalised (CNV) mice were extracted and stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml) and Brefeldin A (5 μg/ml) for 4 h at 37 °C and 5% CO 2 , and analysed by flow cytometry ( a ) to determine the frequency of IL-9-producing CD4 + ( b ) and CD8 + ( c ) T cells. Data are shown as mean ± SD. One-way ANOVA followed by Tukey’s multiple-comparison test was used for statistical analysis.

    Journal: British Journal of Cancer

    Article Title: Host dysbiosis negatively impacts IL-9-producing T-cell differentiation and antitumour immunity

    doi: 10.1038/s41416-020-0915-6

    Figure Lengend Snippet: Host microbiota play a key role in the development of intestinal IL-9-producing T cells. Colonic lamina propria cells from conventional Swiss, germ-free or germ-free conventionalised (CNV) mice were extracted and stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml) and Brefeldin A (5 μg/ml) for 4 h at 37 °C and 5% CO 2 , and analysed by flow cytometry ( a ) to determine the frequency of IL-9-producing CD4 + ( b ) and CD8 + ( c ) T cells. Data are shown as mean ± SD. One-way ANOVA followed by Tukey’s multiple-comparison test was used for statistical analysis.

    Article Snippet: Cells from lamina propria and lungs were stimulated with PMA (Sigma-Aldrich) 50 ng/ml, Ionomycin (Sigma-Aldrich) 500 ng/ml and Brefeldin A (Biolegend, San Diego, CA, USA) 5 µg/ml for 4 h at 37 °C and 5% CO2.

    Techniques: Mouse Assay, Flow Cytometry

    IL-9 treatment improves tumour control in dysbiotic animals. C57BL/6 mice received an antibiotic cocktail twice a day by gavage for 4 weeks (ABX) or were left untreated (control). All mice were intravenously injected with 5 × 10 4 B16F10 cells. ABX-treated mice were intraperitoneally injected either with IL-9 or PBS every other day throughout tumour development. Tumour foci number in the lungs was determined 14 days after injection ( a ). Lungs were then digested, and cells stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml) and Brefeldin A (5 μg/ml) for 4 h at 37 °C and 5% CO 2 . The frequency of IFN-γ-producing CD8 + T cells ( b ) and mast cells ( c ) was evaluated by flow cytometry. Data are shown as mean ± SD. One-way ANOVA followed by Tukey’s multiple-comparison test was used for statistical analysis.

    Journal: British Journal of Cancer

    Article Title: Host dysbiosis negatively impacts IL-9-producing T-cell differentiation and antitumour immunity

    doi: 10.1038/s41416-020-0915-6

    Figure Lengend Snippet: IL-9 treatment improves tumour control in dysbiotic animals. C57BL/6 mice received an antibiotic cocktail twice a day by gavage for 4 weeks (ABX) or were left untreated (control). All mice were intravenously injected with 5 × 10 4 B16F10 cells. ABX-treated mice were intraperitoneally injected either with IL-9 or PBS every other day throughout tumour development. Tumour foci number in the lungs was determined 14 days after injection ( a ). Lungs were then digested, and cells stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml) and Brefeldin A (5 μg/ml) for 4 h at 37 °C and 5% CO 2 . The frequency of IFN-γ-producing CD8 + T cells ( b ) and mast cells ( c ) was evaluated by flow cytometry. Data are shown as mean ± SD. One-way ANOVA followed by Tukey’s multiple-comparison test was used for statistical analysis.

    Article Snippet: Cells from lamina propria and lungs were stimulated with PMA (Sigma-Aldrich) 50 ng/ml, Ionomycin (Sigma-Aldrich) 500 ng/ml and Brefeldin A (Biolegend, San Diego, CA, USA) 5 µg/ml for 4 h at 37 °C and 5% CO2.

    Techniques: Mouse Assay, Injection, Flow Cytometry