human il 6 elisa max deluxe  (BioLegend)

 
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    Name:
    Human IL 6 ELISA MAX Deluxe
    Description:
    Human IL 6 ELISA MAX Deluxe Reactivity Human Apps ELISA Size 5 Plates
    Catalog Number:
    430504
    Price:
    295
    Applications:
    ELISA
    Conjugate:
    Deluxe
    Size:
    5 Plates
    Category:
    ELISA KIT
    Source:
    Rat
    Quantity:
    1
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    BioLegend human il 6 elisa max deluxe
    Human IL 6 ELISA MAX Deluxe
    Human IL 6 ELISA MAX Deluxe Reactivity Human Apps ELISA Size 5 Plates
    https://www.bioz.com/result/human il 6 elisa max deluxe/product/BioLegend
    Average 90 stars, based on 97 article reviews
    Price from $9.99 to $1999.99
    human il 6 elisa max deluxe - by Bioz Stars, 2020-01
    90/100 stars

    Images

    1) Product Images from "CD38 Is Robustly Induced in Human Macrophages and Monocytes in Inflammatory Conditions"

    Article Title: CD38 Is Robustly Induced in Human Macrophages and Monocytes in Inflammatory Conditions

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01593

    CD38 promotes inflammatory cytokine secretion in human macrophages. Human monocyte-derived macrophages (MDMs) were treated with 15 µM rhein or DMSO control (A) , 25 µM apigenin or DMSO (B) , or transfected with 100 µM CD38 siRNA cocktail or siRNA control (C) on day 5. On day 6, they were activated with LPS + IFN-γ for an additional 24 h prior to analysis. (A–C) IL-6 and IL-12p40 secretion was analyzed by ELISA from MDM supernatants. Graphs pool normalized (relative to corresponding experiment vehicle control or nonsense siRNA) data from three (A) , five (B) , or six (C) independent experiments/donors, with at least two technical replicates for each sample. Data are expressed as mean cytokine secretion ± SD relative to vehicle condition. (D) Quantification of CD38 + cells analyzed by flow cytometry from MDM transfected with control or CD38 siRNAs for data shown in panel (C) is expressed as percent of cells ± SD, n = 6 independent experiments/donors and two technical replicates per sample. (E) Quantification of IL-1β + MDMs analyzed by flow cytometry for data shown in panel (C) is expressed as percent of cells ± SD, n = 6 independent experiments/donors and two technical replicates per sample. (F) l -Lactate assays run using supernatants from panel (C) , n = 5 independent experiments/donors, with at least three technical replicates per sample. All data were analyzed by unpaired t tests. ** p
    Figure Legend Snippet: CD38 promotes inflammatory cytokine secretion in human macrophages. Human monocyte-derived macrophages (MDMs) were treated with 15 µM rhein or DMSO control (A) , 25 µM apigenin or DMSO (B) , or transfected with 100 µM CD38 siRNA cocktail or siRNA control (C) on day 5. On day 6, they were activated with LPS + IFN-γ for an additional 24 h prior to analysis. (A–C) IL-6 and IL-12p40 secretion was analyzed by ELISA from MDM supernatants. Graphs pool normalized (relative to corresponding experiment vehicle control or nonsense siRNA) data from three (A) , five (B) , or six (C) independent experiments/donors, with at least two technical replicates for each sample. Data are expressed as mean cytokine secretion ± SD relative to vehicle condition. (D) Quantification of CD38 + cells analyzed by flow cytometry from MDM transfected with control or CD38 siRNAs for data shown in panel (C) is expressed as percent of cells ± SD, n = 6 independent experiments/donors and two technical replicates per sample. (E) Quantification of IL-1β + MDMs analyzed by flow cytometry for data shown in panel (C) is expressed as percent of cells ± SD, n = 6 independent experiments/donors and two technical replicates per sample. (F) l -Lactate assays run using supernatants from panel (C) , n = 5 independent experiments/donors, with at least three technical replicates per sample. All data were analyzed by unpaired t tests. ** p

    Techniques Used: Derivative Assay, Transfection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    2) Product Images from "Bidirectional role of IL-6 signal in pathogenesis of lung fibrosis"

    Article Title: Bidirectional role of IL-6 signal in pathogenesis of lung fibrosis

    Journal: Respiratory Research

    doi: 10.1186/s12931-015-0261-z

    Time course of BLM-induced IL-6 level in BALF. BALF from mice treated with BLM were subjected to ELISA for mouse IL-6 at the indicated dpi. IL-6 level in BALF prepared from mice with PBS treatment is shown as negative control (PBS). Data are shown as mean ± S.E.M. ( n = 4). * P
    Figure Legend Snippet: Time course of BLM-induced IL-6 level in BALF. BALF from mice treated with BLM were subjected to ELISA for mouse IL-6 at the indicated dpi. IL-6 level in BALF prepared from mice with PBS treatment is shown as negative control (PBS). Data are shown as mean ± S.E.M. ( n = 4). * P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Negative Control

    3) Product Images from "Treatment of diabetic mice with the SGLT2 inhibitor TA-1887 antagonizes diabetic cachexia and decreases mortality"

    Article Title: Treatment of diabetic mice with the SGLT2 inhibitor TA-1887 antagonizes diabetic cachexia and decreases mortality

    Journal: NPJ Aging and Mechanisms of Disease

    doi: 10.1038/s41514-017-0012-0

    Effects of insulin on tissue composition, pancreatic beta cell, glucose metabolism and inflammation in db/db mice fed a high-fat (HF) diet. a Volume of lean body mass, total fat, visceral fat (v fat) and subcutaneous fat (s fat), as measured by Computed Tomography after 3 months of indicated treatment ( n = 5). b Analysis of INS1 and INS2 mRNAs in samples of pancreatic tissue after 4 months of indicated treatment ( n = 5–7). c Immunostaining for insulin in representative pancreatic tissue samples after 4 months of indicated treatment. Squares in upper panels are magnified in corresponding lower panels. Scale bars: 200 μm. d Double immunostaining for insulin and active caspase 3 in representative pancreatic tissues after 4 months of treatment. Scale bars: 100 μm. e , f Blood glucose and plasma insulin levels at 0, 1, and 2 months of drug treatment ( n = 8). g levels of mRNAs encoding glycolytic enzymes in eWAT, iWAT, MG and MS after 4 months of indicated treatment ( n = 5–7). h Plasma IL-6 concentrations as measured by ELISA after 2 months of drug treatment ( n = 8). i levels of inflammatory mRNAs in eWAT, iWAT, MG and MS after 4 months of indicated treatment ( n = 5–7). DAPI, 4',6-diamidino-2-phenylindole; INS1, insulin I; INS2, insulin II; HK2, hexokinase 2; Eno1, enolase 1; PFKFB1, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1; Gpi1, glucose phosphate isomerase 1; IL-6, interleukin-6; IL-1b, interleukin-1b; PAI-1, plasminogen activator inhibitor-1; MCP1, monocyte chemoattractant protein-1; mmp9, matrix metalloproteinase 9. Values shown are means ± SEM. * p
    Figure Legend Snippet: Effects of insulin on tissue composition, pancreatic beta cell, glucose metabolism and inflammation in db/db mice fed a high-fat (HF) diet. a Volume of lean body mass, total fat, visceral fat (v fat) and subcutaneous fat (s fat), as measured by Computed Tomography after 3 months of indicated treatment ( n = 5). b Analysis of INS1 and INS2 mRNAs in samples of pancreatic tissue after 4 months of indicated treatment ( n = 5–7). c Immunostaining for insulin in representative pancreatic tissue samples after 4 months of indicated treatment. Squares in upper panels are magnified in corresponding lower panels. Scale bars: 200 μm. d Double immunostaining for insulin and active caspase 3 in representative pancreatic tissues after 4 months of treatment. Scale bars: 100 μm. e , f Blood glucose and plasma insulin levels at 0, 1, and 2 months of drug treatment ( n = 8). g levels of mRNAs encoding glycolytic enzymes in eWAT, iWAT, MG and MS after 4 months of indicated treatment ( n = 5–7). h Plasma IL-6 concentrations as measured by ELISA after 2 months of drug treatment ( n = 8). i levels of inflammatory mRNAs in eWAT, iWAT, MG and MS after 4 months of indicated treatment ( n = 5–7). DAPI, 4',6-diamidino-2-phenylindole; INS1, insulin I; INS2, insulin II; HK2, hexokinase 2; Eno1, enolase 1; PFKFB1, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1; Gpi1, glucose phosphate isomerase 1; IL-6, interleukin-6; IL-1b, interleukin-1b; PAI-1, plasminogen activator inhibitor-1; MCP1, monocyte chemoattractant protein-1; mmp9, matrix metalloproteinase 9. Values shown are means ± SEM. * p

    Techniques Used: Mouse Assay, Computed Tomography, Immunostaining, Double Immunostaining, Mass Spectrometry, Enzyme-linked Immunosorbent Assay

    Expression of inflammatory and senescence markers in TA-1887-treated db/db mice fed a high-fat diet. a mRNA levels of inflammatory mediators in eWAT, iWAT, MG and MS after 4 months of indicated treatment ( n = 5-9). b Plasma IL-6 concentration after 2 months of drug treatment, as determined by ELISA ( n = 8). c mRNA levels of the senescence markers p21 and p16 INK4a in eWAT, iWAT, MG and MS after 4 months of indicated treatment ( n = 5–9). d Senescence-associated staining for SPiDER beta-Gal in frozen sections of eWAT, iWAT and MG tissues after 4 months of treatment. Scale bars: 200 μm. IL-6, interleukin-6; IL-1b, interleukin-1b; PAI-1, plasminogen activator inhibitor-1; MCP1, monocyte chemoattractant protein-1; mmp9, matrix metalloproteinase 9. Values shown are means ± SEM. * p
    Figure Legend Snippet: Expression of inflammatory and senescence markers in TA-1887-treated db/db mice fed a high-fat diet. a mRNA levels of inflammatory mediators in eWAT, iWAT, MG and MS after 4 months of indicated treatment ( n = 5-9). b Plasma IL-6 concentration after 2 months of drug treatment, as determined by ELISA ( n = 8). c mRNA levels of the senescence markers p21 and p16 INK4a in eWAT, iWAT, MG and MS after 4 months of indicated treatment ( n = 5–9). d Senescence-associated staining for SPiDER beta-Gal in frozen sections of eWAT, iWAT and MG tissues after 4 months of treatment. Scale bars: 200 μm. IL-6, interleukin-6; IL-1b, interleukin-1b; PAI-1, plasminogen activator inhibitor-1; MCP1, monocyte chemoattractant protein-1; mmp9, matrix metalloproteinase 9. Values shown are means ± SEM. * p

    Techniques Used: Expressing, Mouse Assay, Mass Spectrometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining

    4) Product Images from "Essential Role of HDAC6 in the Regulation of PD-L1 in melanoma"

    Article Title: Essential Role of HDAC6 in the Regulation of PD-L1 in melanoma

    Journal: Molecular Oncology

    doi: 10.1016/j.molonc.2015.12.012

    Constitutive active STAT3 rescue PD‐L1 expression in the absence of HDAC6. NT, HDAC6KD and STAT3KD WM164 melanoma cells were transfected with either STAT3C‐flag plasmid (B) or empty vector (A) and then stimulated with IL‐6 (30 ng/mL)
    Figure Legend Snippet: Constitutive active STAT3 rescue PD‐L1 expression in the absence of HDAC6. NT, HDAC6KD and STAT3KD WM164 melanoma cells were transfected with either STAT3C‐flag plasmid (B) or empty vector (A) and then stimulated with IL‐6 (30 ng/mL)

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    HDAC6 modulates the expression of PD‐L1 in melanoma cells. (A) NT and STAT3KD WM164, WM983A and WM793 melanoma cells were treated with IL‐6 (30 ng/mL) or left untreated. The presence of STAT3, PD‐L1 and GAPDH was evaluated
    Figure Legend Snippet: HDAC6 modulates the expression of PD‐L1 in melanoma cells. (A) NT and STAT3KD WM164, WM983A and WM793 melanoma cells were treated with IL‐6 (30 ng/mL) or left untreated. The presence of STAT3, PD‐L1 and GAPDH was evaluated

    Techniques Used: Expressing

    The absence of HDAC6 impairs the STAT3 activation in melanoma cells. (A) NT and HDAC6KD WM164, WM983A and WM793 melanoma cells were treated with IL‐6 (30 ng/mL) or left untreated. Then, the presence of HDAC6, acetylated tubulin, STAT3,
    Figure Legend Snippet: The absence of HDAC6 impairs the STAT3 activation in melanoma cells. (A) NT and HDAC6KD WM164, WM983A and WM793 melanoma cells were treated with IL‐6 (30 ng/mL) or left untreated. Then, the presence of HDAC6, acetylated tubulin, STAT3,

    Techniques Used: Activation Assay

    5) Product Images from "Immunostimulatory effect of dried bonito extract on mouse macrophage cell lines and mouse primary peritoneal macrophages"

    Article Title: Immunostimulatory effect of dried bonito extract on mouse macrophage cell lines and mouse primary peritoneal macrophages

    Journal: Cytotechnology

    doi: 10.1007/s10616-016-0053-4

    Effects of Toll-like receptor 4 (TLR4) or lipopolysaccharides (LPS) inhibitors on macrophage activation induced by dried bonito extract (DBE). a J774.1 cells were treated with 0.2 µM TAK-242 for 1 h. The cells were subsequently treated with RPMI-1640 medium containing 10% fetal bovine serum and 8.0 mg protein/mL of DBE, 10 mM sodium phosphate buffer (pH 7.4) as control, or 10 ng/mL of LPS as a positive control at 37 °C for 6 h. After incubation, the concentrations of TNF-α and IL-6 in culture media were measured by enzyme-linked immunosorbent assays. Solid bars , non-treatment with TAK-242; open bars , treatment with TAK-242. b DBE (8.0 mg/mL) and LPS (10 ng/mL) were treated with 10 µg/mL of polymixin B at room temperature for 30 min. Then, J774.1 cells were incubated in RPMI-1640 medium containing 10% fetal bovine serum and esach polymyxin B-treated sample at 37 °C for 6 h. After incubation, the concentrations of TNF-α and IL-6 in culture media were measured by enzyme-linked immunosorbent assays. Solid bars , non-treatment with polymyxin B; open bars , treatment with polymyxin B. Data are expressed as the mean ± SD ( n = 3). ** P
    Figure Legend Snippet: Effects of Toll-like receptor 4 (TLR4) or lipopolysaccharides (LPS) inhibitors on macrophage activation induced by dried bonito extract (DBE). a J774.1 cells were treated with 0.2 µM TAK-242 for 1 h. The cells were subsequently treated with RPMI-1640 medium containing 10% fetal bovine serum and 8.0 mg protein/mL of DBE, 10 mM sodium phosphate buffer (pH 7.4) as control, or 10 ng/mL of LPS as a positive control at 37 °C for 6 h. After incubation, the concentrations of TNF-α and IL-6 in culture media were measured by enzyme-linked immunosorbent assays. Solid bars , non-treatment with TAK-242; open bars , treatment with TAK-242. b DBE (8.0 mg/mL) and LPS (10 ng/mL) were treated with 10 µg/mL of polymixin B at room temperature for 30 min. Then, J774.1 cells were incubated in RPMI-1640 medium containing 10% fetal bovine serum and esach polymyxin B-treated sample at 37 °C for 6 h. After incubation, the concentrations of TNF-α and IL-6 in culture media were measured by enzyme-linked immunosorbent assays. Solid bars , non-treatment with polymyxin B; open bars , treatment with polymyxin B. Data are expressed as the mean ± SD ( n = 3). ** P

    Techniques Used: Activation Assay, Positive Control, Incubation

    Effect of proteolytic enzyme-treated dried bonito extract (DBE) on cytokine production by J774.1 cells. DBE was treated with 500 µg/mL of proteinase K at 37 °C for 15 h. After heating the samples to inactivate the protease, J774.1 cells were treated with RPMI-1640 medium containing 10% fetal bovine serum and the proteinase K-treated DBE or 10 mM sodium phosphate buffer (pH 7.4) as control. After incubation at 37 °C for 6 h, the concentrations of TNF-α and IL-6 in culture media were measured by enzyme-linked immunosorbent assays. Solid bars , 10 mM sodium phosphate buffer as control; open bars , DBE. Data are expressed as the mean ± SD ( n = 3). ** P
    Figure Legend Snippet: Effect of proteolytic enzyme-treated dried bonito extract (DBE) on cytokine production by J774.1 cells. DBE was treated with 500 µg/mL of proteinase K at 37 °C for 15 h. After heating the samples to inactivate the protease, J774.1 cells were treated with RPMI-1640 medium containing 10% fetal bovine serum and the proteinase K-treated DBE or 10 mM sodium phosphate buffer (pH 7.4) as control. After incubation at 37 °C for 6 h, the concentrations of TNF-α and IL-6 in culture media were measured by enzyme-linked immunosorbent assays. Solid bars , 10 mM sodium phosphate buffer as control; open bars , DBE. Data are expressed as the mean ± SD ( n = 3). ** P

    Techniques Used: Incubation

    Effect of dried bonito extract (DBE) on cytokine production by J774.1 cells and peritoneal macrophages. J774.1 cells ( a ) or peritoneal macrophages ( b ) were treated with RPMI-1640 medium containing 10% fetal bovine serum and various concentrations of DBE or 10 mM sodium phosphate buffer (pH 7.4) as control. After incubation at 37 °C for 6 h, the concentrations of TNF-α and IL-6 in culture media were measured by enzyme-linked immunosorbent assays. Data are expressed as the mean ± SD ( n = 3). ** P
    Figure Legend Snippet: Effect of dried bonito extract (DBE) on cytokine production by J774.1 cells and peritoneal macrophages. J774.1 cells ( a ) or peritoneal macrophages ( b ) were treated with RPMI-1640 medium containing 10% fetal bovine serum and various concentrations of DBE or 10 mM sodium phosphate buffer (pH 7.4) as control. After incubation at 37 °C for 6 h, the concentrations of TNF-α and IL-6 in culture media were measured by enzyme-linked immunosorbent assays. Data are expressed as the mean ± SD ( n = 3). ** P

    Techniques Used: Incubation

    6) Product Images from "Ginsenoside Rh2 Ameliorates Lipopolysaccharide-Induced Acute Lung Injury by Regulating the TLR4/PI3K/Akt/mTOR, Raf-1/MEK/ERK, and Keap1/Nrf2/HO-1 Signaling Pathways in Mice"

    Article Title: Ginsenoside Rh2 Ameliorates Lipopolysaccharide-Induced Acute Lung Injury by Regulating the TLR4/PI3K/Akt/mTOR, Raf-1/MEK/ERK, and Keap1/Nrf2/HO-1 Signaling Pathways in Mice

    Journal: Nutrients

    doi: 10.3390/nu10091208

    ( A ) GRh2 and Raf-1 inhibitor (GW-5074) reduced NO; ( B ) TNF-α; ( C ) IL-1β; ( D ) IL-6; and ( E ) IL-10 in BALF. BALF was collected. NO, TNF-α, IL-1β, IL-6, and IL-10 were detected at 6 h after LPS challenge by ELISA. Data are represented as mean ± S.E.M. ( n = 5). ### p
    Figure Legend Snippet: ( A ) GRh2 and Raf-1 inhibitor (GW-5074) reduced NO; ( B ) TNF-α; ( C ) IL-1β; ( D ) IL-6; and ( E ) IL-10 in BALF. BALF was collected. NO, TNF-α, IL-1β, IL-6, and IL-10 were detected at 6 h after LPS challenge by ELISA. Data are represented as mean ± S.E.M. ( n = 5). ### p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    GRh2 downregulated ( A ) nitric oxide (NO); ( B ) tumor necrosis factor-alpha (TNF-α); ( C ) IL-1β; ( D ) IL-4; and ( E ) IL-6; and ( F ) increased IL-10 in BALF. BALF was collected. NO, TNF-α, IL-1β, IL-4, IL-6, and IL-10 were detected at 6 h after LPS challenge by enzyme-linked immunosorbent assay (ELISA). Data are represented as mean ± S.E.M. ( n = 5). ### denotes p
    Figure Legend Snippet: GRh2 downregulated ( A ) nitric oxide (NO); ( B ) tumor necrosis factor-alpha (TNF-α); ( C ) IL-1β; ( D ) IL-4; and ( E ) IL-6; and ( F ) increased IL-10 in BALF. BALF was collected. NO, TNF-α, IL-1β, IL-4, IL-6, and IL-10 were detected at 6 h after LPS challenge by enzyme-linked immunosorbent assay (ELISA). Data are represented as mean ± S.E.M. ( n = 5). ### denotes p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    7) Product Images from "Ginsenoside Rh2 Ameliorates Lipopolysaccharide-Induced Acute Lung Injury by Regulating the TLR4/PI3K/Akt/mTOR, Raf-1/MEK/ERK, and Keap1/Nrf2/HO-1 Signaling Pathways in Mice"

    Article Title: Ginsenoside Rh2 Ameliorates Lipopolysaccharide-Induced Acute Lung Injury by Regulating the TLR4/PI3K/Akt/mTOR, Raf-1/MEK/ERK, and Keap1/Nrf2/HO-1 Signaling Pathways in Mice

    Journal: Nutrients

    doi: 10.3390/nu10091208

    ( A ) GRh2 and Raf-1 inhibitor (GW-5074) reduced NO; ( B ) TNF-α; ( C ) IL-1β; ( D ) IL-6; and ( E ) IL-10 in BALF. BALF was collected. NO, TNF-α, IL-1β, IL-6, and IL-10 were detected at 6 h after LPS challenge by ELISA. Data are represented as mean ± S.E.M. ( n = 5). ### p
    Figure Legend Snippet: ( A ) GRh2 and Raf-1 inhibitor (GW-5074) reduced NO; ( B ) TNF-α; ( C ) IL-1β; ( D ) IL-6; and ( E ) IL-10 in BALF. BALF was collected. NO, TNF-α, IL-1β, IL-6, and IL-10 were detected at 6 h after LPS challenge by ELISA. Data are represented as mean ± S.E.M. ( n = 5). ### p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    GRh2 downregulated ( A ) nitric oxide (NO); ( B ) tumor necrosis factor-alpha (TNF-α); ( C ) IL-1β; ( D ) IL-4; and ( E ) IL-6; and ( F ) increased IL-10 in BALF. BALF was collected. NO, TNF-α, IL-1β, IL-4, IL-6, and IL-10 were detected at 6 h after LPS challenge by enzyme-linked immunosorbent assay (ELISA). Data are represented as mean ± S.E.M. ( n = 5). ### denotes p
    Figure Legend Snippet: GRh2 downregulated ( A ) nitric oxide (NO); ( B ) tumor necrosis factor-alpha (TNF-α); ( C ) IL-1β; ( D ) IL-4; and ( E ) IL-6; and ( F ) increased IL-10 in BALF. BALF was collected. NO, TNF-α, IL-1β, IL-4, IL-6, and IL-10 were detected at 6 h after LPS challenge by enzyme-linked immunosorbent assay (ELISA). Data are represented as mean ± S.E.M. ( n = 5). ### denotes p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    8) Product Images from "NDR1 protein kinase promotes IL‐17‐ and TNF‐α‐mediated inflammation by competitively binding TRAF3"

    Article Title: NDR1 protein kinase promotes IL‐17‐ and TNF‐α‐mediated inflammation by competitively binding TRAF3

    Journal: EMBO Reports

    doi: 10.15252/embr.201642140

    NDR1 promotes TNF‐α signaling via targeting TRAF3 Wild‐type ( Traf3 +/+ ) and TRAF3‐deficient ( Traf3 −/− ) MEFs were transfected with NDR1 siRNA or control siRNA and then stimulated with TNF‐α (20 ng/ml) for the indicated times. Whole lysates were subjected to Western blot. Real‐time PCR analysis of IL‐6 , CXCL2, and CCL20 mRNA expression in MEFs treated as described in (A). * P
    Figure Legend Snippet: NDR1 promotes TNF‐α signaling via targeting TRAF3 Wild‐type ( Traf3 +/+ ) and TRAF3‐deficient ( Traf3 −/− ) MEFs were transfected with NDR1 siRNA or control siRNA and then stimulated with TNF‐α (20 ng/ml) for the indicated times. Whole lysates were subjected to Western blot. Real‐time PCR analysis of IL‐6 , CXCL2, and CCL20 mRNA expression in MEFs treated as described in (A). * P

    Techniques Used: Transfection, Western Blot, Real-time Polymerase Chain Reaction, Expressing

    NDR1 deficiency restricts MOG‐induced EAE by its promotion of IL‐17 signaling A Mice with reconstituted bone marrow were immunized with MOG (35‐55) to induce EAE. The mice ( n = 5/group) were treated with intraperitoneal injection of an anti‐IL‐17A antibody (100 μg/mouse each time) or appropriate isotype controls on days 7, 9, 11, and 13 after the second MOG immunization. Mean clinical scores were calculated every other day according to the standards described in the Materials and Methods section (*WT‐WT isotype versus WT‐KO isotype; °WT‐WT isotype versus WT‐WT α‐IL‐17). B, C IL‐6, TNF‐α, CXCL1, and CXCL2 mRNA in the spinal cords (B) or in the brains (C) were measured by real‐time PCR on day 14 after the second MOG immunization. D Histology of the spinal cord was analyzed by hematoxylin and eosin (HE) or Luxol fast blue (LFB) staining on day 14 after the second MOG immunization. Scale bars for the left panel, 200 μm; scale bars for the right panel, 50 μm. Data information: ns, not significant, * P
    Figure Legend Snippet: NDR1 deficiency restricts MOG‐induced EAE by its promotion of IL‐17 signaling A Mice with reconstituted bone marrow were immunized with MOG (35‐55) to induce EAE. The mice ( n = 5/group) were treated with intraperitoneal injection of an anti‐IL‐17A antibody (100 μg/mouse each time) or appropriate isotype controls on days 7, 9, 11, and 13 after the second MOG immunization. Mean clinical scores were calculated every other day according to the standards described in the Materials and Methods section (*WT‐WT isotype versus WT‐KO isotype; °WT‐WT isotype versus WT‐WT α‐IL‐17). B, C IL‐6, TNF‐α, CXCL1, and CXCL2 mRNA in the spinal cords (B) or in the brains (C) were measured by real‐time PCR on day 14 after the second MOG immunization. D Histology of the spinal cord was analyzed by hematoxylin and eosin (HE) or Luxol fast blue (LFB) staining on day 14 after the second MOG immunization. Scale bars for the left panel, 200 μm; scale bars for the right panel, 50 μm. Data information: ns, not significant, * P

    Techniques Used: Mouse Assay, Injection, Real-time Polymerase Chain Reaction, Staining

    NDR1 deficiency in a non‐hematopoietic system restricts MOG‐induced EAE A Mice with reconstituted bone marrow were immunized with MOG (35‐55) to induce EAE. Mean clinical scores were calculated every other day according to the standards described in the Materials and Methods section (*WT‐WT versus WT‐KO; °KO‐WT versus WT‐KO). B, C IL‐6, TNF‐α, CXCL1, and CXCL2 mRNA in the spinal cords (B) or in the brains (C) were measured by real‐time PCR on day 14 after the second MOG immunization. D Histology of the spinal cord was analyzed by hematoxylin and eosin (HE) or Luxol fast blue (LFB) staining on day 14 after the second MOG immunization. Scale bars for the left panel, 200 μm; scale bars for the right panel, 50 μm. Data information: ns, not significant, * P
    Figure Legend Snippet: NDR1 deficiency in a non‐hematopoietic system restricts MOG‐induced EAE A Mice with reconstituted bone marrow were immunized with MOG (35‐55) to induce EAE. Mean clinical scores were calculated every other day according to the standards described in the Materials and Methods section (*WT‐WT versus WT‐KO; °KO‐WT versus WT‐KO). B, C IL‐6, TNF‐α, CXCL1, and CXCL2 mRNA in the spinal cords (B) or in the brains (C) were measured by real‐time PCR on day 14 after the second MOG immunization. D Histology of the spinal cord was analyzed by hematoxylin and eosin (HE) or Luxol fast blue (LFB) staining on day 14 after the second MOG immunization. Scale bars for the left panel, 200 μm; scale bars for the right panel, 50 μm. Data information: ns, not significant, * P

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Staining

    NDR1 promotes IL‐17‐induced inflammation in vivo and in vitro A, B Real‐time PCR (A) and ELISA (B) analysis of IL‐6 , CXCL2, CCL20, and CXCL1 mRNA expression and production in wild‐type (WT) and NDR1 ‐deficient homozygous ( Ndr1 ‐KO) MEFs following stimulation with IL‐17 (100 ng/ml) for the indicated times. C WT and Ndr1 ‐KO MEFs transfected with a retrovirus encoding mock, Flag‐NDR1, or Flag‐NDR1/K118A were treated with IL‐17 (100 ng/ml) for 0, 6, or 12 h. The protein levels of IL‐6 , CCL20 , and CXCL2 were analyzed by ELISA. D, E Real‐time PCR (D) and ELISA (E) analysis IL‐6 , CXCL2, CCL20, and CXCL1 mRNA expression and production in WT and Ndr1 ‐KO primary astrocytes following stimulation with IL‐17 (100 ng/ml) for the indicated times. F Ndr1 ‐KO mice ( n = 5) and WT mice ( n = 5) were treated by intraperitoneal injection of PBS or IL‐17 (0.5 μg in 200 μl PBS) for 24 h, and then, the peritoneal mesothelial cells were isolated to detect IL‐6, CXCL2, and CXCL1 mRNA expression. Data information: Data were normalized to a reference gene, Actin . * P
    Figure Legend Snippet: NDR1 promotes IL‐17‐induced inflammation in vivo and in vitro A, B Real‐time PCR (A) and ELISA (B) analysis of IL‐6 , CXCL2, CCL20, and CXCL1 mRNA expression and production in wild‐type (WT) and NDR1 ‐deficient homozygous ( Ndr1 ‐KO) MEFs following stimulation with IL‐17 (100 ng/ml) for the indicated times. C WT and Ndr1 ‐KO MEFs transfected with a retrovirus encoding mock, Flag‐NDR1, or Flag‐NDR1/K118A were treated with IL‐17 (100 ng/ml) for 0, 6, or 12 h. The protein levels of IL‐6 , CCL20 , and CXCL2 were analyzed by ELISA. D, E Real‐time PCR (D) and ELISA (E) analysis IL‐6 , CXCL2, CCL20, and CXCL1 mRNA expression and production in WT and Ndr1 ‐KO primary astrocytes following stimulation with IL‐17 (100 ng/ml) for the indicated times. F Ndr1 ‐KO mice ( n = 5) and WT mice ( n = 5) were treated by intraperitoneal injection of PBS or IL‐17 (0.5 μg in 200 μl PBS) for 24 h, and then, the peritoneal mesothelial cells were isolated to detect IL‐6, CXCL2, and CXCL1 mRNA expression. Data information: Data were normalized to a reference gene, Actin . * P

    Techniques Used: In Vivo, In Vitro, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Mouse Assay, Injection, Isolation

    NDR1 promotes the IL‐17F‐induced expression of pro‐inflammatory cytokines A WT and Ndr1 ‐KO MEFs transfected with a retrovirus encoding mock, Flag‐NDR1, or Flag‐NDR1/K118A were treated with IL‐17 (100 ng/ml) for 0 or 6 h. The mRNA levels of IL‐6 , CXCL2 , and CCL20 were analyzed by real‐time PCR. B, C WT and Ndr1 ‐KO MEFs (B) or primary astrocytes (C) were treated with IL‐17F (100 ng/ml) for the indicated times, and the induction of IL‐6 , CXCL2, CCL20, and CXCL1 mRNA expression was analyzed by real‐time PCR. D HeLa cells were transfected with NDR1 siRNA or control siRNA and then were treated with IL‐17F (50 ng/ml) for 0, 1, or 3 h, and the induction of IL‐6 , CXCL2 , and CCL20 mRNA expression was analyzed by real‐time PCR. E, F WT and Ndr1 ‐KO MEFs were treated with TNF‐α (20 ng/ml) (D) or IL‐1β (10 ng/ml) (E) for the indicated times, and the induction of IL‐6 , CXCL2, CCL20, and CXCL1 mRNA expression was analyzed by real‐time PCR. Data information: * P
    Figure Legend Snippet: NDR1 promotes the IL‐17F‐induced expression of pro‐inflammatory cytokines A WT and Ndr1 ‐KO MEFs transfected with a retrovirus encoding mock, Flag‐NDR1, or Flag‐NDR1/K118A were treated with IL‐17 (100 ng/ml) for 0 or 6 h. The mRNA levels of IL‐6 , CXCL2 , and CCL20 were analyzed by real‐time PCR. B, C WT and Ndr1 ‐KO MEFs (B) or primary astrocytes (C) were treated with IL‐17F (100 ng/ml) for the indicated times, and the induction of IL‐6 , CXCL2, CCL20, and CXCL1 mRNA expression was analyzed by real‐time PCR. D HeLa cells were transfected with NDR1 siRNA or control siRNA and then were treated with IL‐17F (50 ng/ml) for 0, 1, or 3 h, and the induction of IL‐6 , CXCL2 , and CCL20 mRNA expression was analyzed by real‐time PCR. E, F WT and Ndr1 ‐KO MEFs were treated with TNF‐α (20 ng/ml) (D) or IL‐1β (10 ng/ml) (E) for the indicated times, and the induction of IL‐6 , CXCL2, CCL20, and CXCL1 mRNA expression was analyzed by real‐time PCR. Data information: * P

    Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction

    NDR1 promotes the IL‐17‐induced expression of pro‐inflammatory cytokines in HeLa cells in a kinase activity‐independent manner A–C HeLa cells were transfected with NDR1 siRNA, Act1 siRNA (positive control) or luciferase siRNA (negative control) and then stimulated with IL‐17 (50 ng/ml) for 0, 1, 3, or 12 h. The mRNA levels and production of IL‐6 , CXCL2 , and CCL20 were analyzed by real‐time PCR (A) and ELISA (B), respectively. The efficiency of knockdown was detected by Western blot (C). D, E HeLa cells were transfected with mock, NDR1, or NDR1/K118A plasmids and then stimulated with IL‐17 (50 ng/ml) for the indicated times. The induction of IL‐6 , CXCL2 , and CCL20 mRNA expression were analyzed by real‐time PCR (D) and ELISA (E), respectively. Data information: * P
    Figure Legend Snippet: NDR1 promotes the IL‐17‐induced expression of pro‐inflammatory cytokines in HeLa cells in a kinase activity‐independent manner A–C HeLa cells were transfected with NDR1 siRNA, Act1 siRNA (positive control) or luciferase siRNA (negative control) and then stimulated with IL‐17 (50 ng/ml) for 0, 1, 3, or 12 h. The mRNA levels and production of IL‐6 , CXCL2 , and CCL20 were analyzed by real‐time PCR (A) and ELISA (B), respectively. The efficiency of knockdown was detected by Western blot (C). D, E HeLa cells were transfected with mock, NDR1, or NDR1/K118A plasmids and then stimulated with IL‐17 (50 ng/ml) for the indicated times. The induction of IL‐6 , CXCL2 , and CCL20 mRNA expression were analyzed by real‐time PCR (D) and ELISA (E), respectively. Data information: * P

    Techniques Used: Expressing, Activity Assay, Transfection, Positive Control, Luciferase, Negative Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot

    NDR1 promotes IL‐17 signaling by targeting TRAF3 Wild‐type ( Traf3 +/+ ) and TRAF3‐deficient ( Traf3 −/− ) MEFs were transfected with control siRNA or NDR1 siRNA and stimulated with IL‐17 (100 ng/ml) for the indicated times. Whole lysates were subjected to Western blot. Numbers between two blots indicate densitometry of phospho‐p65 relative to that of total p65. Real‐time PCR analysis of IL‐6 , CXCL2, CXCL1, and CCL20 mRNA expression in MEFs treated as described in (A). ELISA assay of IL‐6 and CCL20 production in MEFs from (B). WT and Ndr1 ‐KO MEFs were treated with TNF‐α (20 ng/ml) for 1 h. Fresh medium containing ActD alone or with IL‐17 was then added. Total RNA was collected at the indicated times, the levels of CXCL1 and actin mRNA were detected by real‐time PCR, and the percentage of remaining CXCL1 mRNA relative to actin is shown for each experimental condition. The mRNA level for CXCL1 was normalized to the Actin mRNA level and plotted as log of the percentage of remaining mRNA versus time. The best fit to liner decay was determined, and the t 1/2 was calculate from the intersection at the point corresponding to 50% residual RNA. Data information: * P
    Figure Legend Snippet: NDR1 promotes IL‐17 signaling by targeting TRAF3 Wild‐type ( Traf3 +/+ ) and TRAF3‐deficient ( Traf3 −/− ) MEFs were transfected with control siRNA or NDR1 siRNA and stimulated with IL‐17 (100 ng/ml) for the indicated times. Whole lysates were subjected to Western blot. Numbers between two blots indicate densitometry of phospho‐p65 relative to that of total p65. Real‐time PCR analysis of IL‐6 , CXCL2, CXCL1, and CCL20 mRNA expression in MEFs treated as described in (A). ELISA assay of IL‐6 and CCL20 production in MEFs from (B). WT and Ndr1 ‐KO MEFs were treated with TNF‐α (20 ng/ml) for 1 h. Fresh medium containing ActD alone or with IL‐17 was then added. Total RNA was collected at the indicated times, the levels of CXCL1 and actin mRNA were detected by real‐time PCR, and the percentage of remaining CXCL1 mRNA relative to actin is shown for each experimental condition. The mRNA level for CXCL1 was normalized to the Actin mRNA level and plotted as log of the percentage of remaining mRNA versus time. The best fit to liner decay was determined, and the t 1/2 was calculate from the intersection at the point corresponding to 50% residual RNA. Data information: * P

    Techniques Used: Transfection, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

    9) Product Images from "The Transcription Factor ZNF395 Is Required for the Maximal Hypoxic Induction of Proinflammatory Cytokines in U87-MG Cells"

    Article Title: The Transcription Factor ZNF395 Is Required for the Maximal Hypoxic Induction of Proinflammatory Cytokines in U87-MG Cells

    Journal: Mediators of Inflammation

    doi: 10.1155/2015/804264

    U87-MG cells were transfected with control siRNA or siRNA against ZNF395 and total RNA was isolated 48 hours after transfection (normoxia) or the cells were transferred 36 hours after transfection to 2% O 2 for additional 12 hours (hypoxia) before total RNA was isolated. cDNA was prepared and quantitative real time-PCRs were performed with primers specific for IL-6, IL-8, IL-1 β , and LIF (a) and for MCP-1/CCL2, CA IX, and ZNF395 (b) RT-PCR of HPRT serving as internal control. The values obtained by dividing the Ct values for the cytokine and HPRT, respectively, from the siControl cells grown under normoxia were set as 1, and the fold activations were calculated. The standard deviations are given. Each PCR was performed six times in duplicate except that for ZNF395. (c) U87-MG cells were transfected with siControl or siZNF395; 24 hours later, one set of cells was incubated in the presence of DMOG, while the other set of cells obtained the equivalent amount of ETOH (which served as solvent for DMOG) as control. Another 24 hours later, the supernatant was used for ELISA to measure the level of IL-6 and IL-8. Each ELISA was performed two times in triplicate. QRT-PCR with RNA isolated from these cells was used to investigate the effect of DMOG on the expression of ZNF395. ∗ p
    Figure Legend Snippet: U87-MG cells were transfected with control siRNA or siRNA against ZNF395 and total RNA was isolated 48 hours after transfection (normoxia) or the cells were transferred 36 hours after transfection to 2% O 2 for additional 12 hours (hypoxia) before total RNA was isolated. cDNA was prepared and quantitative real time-PCRs were performed with primers specific for IL-6, IL-8, IL-1 β , and LIF (a) and for MCP-1/CCL2, CA IX, and ZNF395 (b) RT-PCR of HPRT serving as internal control. The values obtained by dividing the Ct values for the cytokine and HPRT, respectively, from the siControl cells grown under normoxia were set as 1, and the fold activations were calculated. The standard deviations are given. Each PCR was performed six times in duplicate except that for ZNF395. (c) U87-MG cells were transfected with siControl or siZNF395; 24 hours later, one set of cells was incubated in the presence of DMOG, while the other set of cells obtained the equivalent amount of ETOH (which served as solvent for DMOG) as control. Another 24 hours later, the supernatant was used for ELISA to measure the level of IL-6 and IL-8. Each ELISA was performed two times in triplicate. QRT-PCR with RNA isolated from these cells was used to investigate the effect of DMOG on the expression of ZNF395. ∗ p

    Techniques Used: Transfection, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Incubation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing

    10) Product Images from "Essential Role of HDAC6 in the Regulation of PD-L1 in melanoma"

    Article Title: Essential Role of HDAC6 in the Regulation of PD-L1 in melanoma

    Journal: Molecular Oncology

    doi: 10.1016/j.molonc.2015.12.012

    Constitutive active STAT3 rescue PD‐L1 expression in the absence of HDAC6. NT, HDAC6KD and STAT3KD WM164 melanoma cells were transfected with either STAT3C‐flag plasmid (B) or empty vector (A) and then stimulated with IL‐6 (30 ng/mL)
    Figure Legend Snippet: Constitutive active STAT3 rescue PD‐L1 expression in the absence of HDAC6. NT, HDAC6KD and STAT3KD WM164 melanoma cells were transfected with either STAT3C‐flag plasmid (B) or empty vector (A) and then stimulated with IL‐6 (30 ng/mL)

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    HDAC6 modulates the expression of PD‐L1 in melanoma cells. (A) NT and STAT3KD WM164, WM983A and WM793 melanoma cells were treated with IL‐6 (30 ng/mL) or left untreated. The presence of STAT3, PD‐L1 and GAPDH was evaluated
    Figure Legend Snippet: HDAC6 modulates the expression of PD‐L1 in melanoma cells. (A) NT and STAT3KD WM164, WM983A and WM793 melanoma cells were treated with IL‐6 (30 ng/mL) or left untreated. The presence of STAT3, PD‐L1 and GAPDH was evaluated

    Techniques Used: Expressing

    The absence of HDAC6 impairs the STAT3 activation in melanoma cells. (A) NT and HDAC6KD WM164, WM983A and WM793 melanoma cells were treated with IL‐6 (30 ng/mL) or left untreated. Then, the presence of HDAC6, acetylated tubulin, STAT3,
    Figure Legend Snippet: The absence of HDAC6 impairs the STAT3 activation in melanoma cells. (A) NT and HDAC6KD WM164, WM983A and WM793 melanoma cells were treated with IL‐6 (30 ng/mL) or left untreated. Then, the presence of HDAC6, acetylated tubulin, STAT3,

    Techniques Used: Activation Assay

    11) Product Images from "Platelets convert peripheral blood circulating monocytes to regulatory cells via immunoglobulin G and activating-type Fcγ receptors"

    Article Title: Platelets convert peripheral blood circulating monocytes to regulatory cells via immunoglobulin G and activating-type Fcγ receptors

    Journal: BMC Immunology

    doi: 10.1186/s12865-015-0086-z

    Augmented serum IL-10 in a murine in vivo setting. (A, B) Wild-type B6 mice were administered intravenously an anti-human/mouse CD61 or isotype control mAb and subsequently with LPS intraperitoneally. The IL-10 and IL-6 levels in serum samples collected at 3 h after LPS stimulation were measured by ELISA (A) . IL-10 upregulation in sera was observed also in FcγRIIB-deficient but not FcRγ-deficient mice ( n = 4 mice per group) (B) . The results are representative of more than three independent experiments with similar results. * P
    Figure Legend Snippet: Augmented serum IL-10 in a murine in vivo setting. (A, B) Wild-type B6 mice were administered intravenously an anti-human/mouse CD61 or isotype control mAb and subsequently with LPS intraperitoneally. The IL-10 and IL-6 levels in serum samples collected at 3 h after LPS stimulation were measured by ELISA (A) . IL-10 upregulation in sera was observed also in FcγRIIB-deficient but not FcRγ-deficient mice ( n = 4 mice per group) (B) . The results are representative of more than three independent experiments with similar results. * P

    Techniques Used: In Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay

    12) Product Images from "Cardiac myosin-Th17 responses promote heart failure in human myocarditis"

    Article Title: Cardiac myosin-Th17 responses promote heart failure in human myocarditis

    Journal: JCI Insight

    doi: 10.1172/jci.insight.85851

    Significantly elevated IL-6 levels were progressive and associated with heart failure in men.
    Figure Legend Snippet: Significantly elevated IL-6 levels were progressive and associated with heart failure in men.

    Techniques Used:

    In myocarditis/dilated cardiomyopathy (DCM), low Tregs correlated with high TGF-β1 and IL-6 levels produced from peripheral blood mononuclear cells (PBMCs)/CD14 + monocytes stimulated with human cardiac myosin (HCM) TLR ligands.
    Figure Legend Snippet: In myocarditis/dilated cardiomyopathy (DCM), low Tregs correlated with high TGF-β1 and IL-6 levels produced from peripheral blood mononuclear cells (PBMCs)/CD14 + monocytes stimulated with human cardiac myosin (HCM) TLR ligands.

    Techniques Used: Produced

    13) Product Images from "Cardiac myosin-Th17 responses promote heart failure in human myocarditis"

    Article Title: Cardiac myosin-Th17 responses promote heart failure in human myocarditis

    Journal: JCI Insight

    doi: 10.1172/jci.insight.85851

    Significantly elevated IL-6 levels were progressive and associated with heart failure in men.
    Figure Legend Snippet: Significantly elevated IL-6 levels were progressive and associated with heart failure in men.

    Techniques Used:

    In myocarditis/dilated cardiomyopathy (DCM), low Tregs correlated with high TGF-β1 and IL-6 levels produced from peripheral blood mononuclear cells (PBMCs)/CD14 + monocytes stimulated with human cardiac myosin (HCM) TLR ligands.
    Figure Legend Snippet: In myocarditis/dilated cardiomyopathy (DCM), low Tregs correlated with high TGF-β1 and IL-6 levels produced from peripheral blood mononuclear cells (PBMCs)/CD14 + monocytes stimulated with human cardiac myosin (HCM) TLR ligands.

    Techniques Used: Produced

    14) Product Images from "Microbial signals drive pre–leukaemic myeloproliferation in a Tet2–deficient host"

    Article Title: Microbial signals drive pre–leukaemic myeloproliferation in a Tet2–deficient host

    Journal: Nature

    doi: 10.1038/s41586-018-0125-z

    Bacteria-induced IL-6 is required for PMP in Tet2 −/− mice. a , Gene expression of Il6 in the spleen of SPF-housed Tet2 −/− mice, littermates and germ-free Tet2 −/− mice ( n = 7 ( Tet2 +/+ , SPF), 10 ( Tet2 −/− , SPF) or 4 ( Tet2 −/− , GF) mice). b , IL-6 cytokine levels in blood plasma of Tet2 −/− mice with PMP treated with antibiotics (ABX) for four weeks ( n = 5 mice). Lines connect values obtained from the same mouse sampled before and after antibiotics treatment. Two-tailed paired t -test. c , IL-6 cytokine levels in blood plasma of DSS-treated symptom-free Tet2 f/f Vav cre mice that are over 20 weeks old, and littermate controls (see schematic in ( n = 6 ( Tet2 +/+ ) or 9 ( Tet2 −/− ) mice). d , IL-6 cytokine levels in blood plasma of Tet2 f/f Vav cre ( n = 6 ( Tet2 +/+ ) or 8 ( Tet2 −/− ) mice). e , Correlation between IL-6 cytokine levels in blood plasma of Tet2 −/− mice and numbers of peripheral-blood CD11b+ monocytes ( n = 49 mice). Pearson correlation test. f , g , In vitro HSC self-renewal colony-forming assay of haematopoietic progenitors of the spleen from Tet2 −/− mice (red lines) and littermate controls (blue lines) in the presence of anti-IL-6 antibody or isotype control (ISO) after the first replating ( n = 3 mice). g , Representative images of colonies after the 5th replating. Scale bars, 100 μm. h , Representative histogram (left) and quantification of (right) mean fluorescence intensity (MFI) of IL-6Rα + c-Kit + Sca-1 − (LK gated) CD34 + Fc γ RIII/II + GMPs from the bone marrow, ( n = 5 ( Tet2 +/+ SPF), 6 ( Tet2 −/− , SPF), 4 ( Tet2 +/+ GF) or 3 ( Tet2 −/− , GF) mice). i , Representative flow cytometry plot of Stat3 phosphorylation (pY705) response after 30-min stimulation with 10 ng ml −1 IL-6 in splenic c-Kit + Sca-1 − (LK gated) CD34 + and CD34 − myeloid progenitors (MP). j , Numbers of CD11b + myeloid cells ( n = 7 ( Tet2 +/+ ) or 6 ( Tet2 −/− ) mice). k , l , CD11b + F4/80 + macrophages and CD11b + Gr1 + myeloid cells of the spleen, from Tet2 −/− mice and littermate controls, were FACS sorted. Gene expression of Il6 in macrophages ( k ) and CD11b + Gr1 + cells ( l ) ( n = 7 ( Tet2 +/+ ) or 6 ( Tet2 −/− ) mice). Centre is median. m , Representative histogram (left) and quantification of (right) MFI of IL-6Rα + CD11b + Gr1 + myeloid cells in the spleen ( n = 5 ( Tet2 +/+ , SPF), 6 ( Tet2 −/− SPF), 4 ( Tet2 +/+ GF) or 4 ( Tet2 −/− , GF) mice). n , Representative histogram (left) and quantification of (right) MFI of IL-6Rα + c-Kit + Sca-1 − (LK gated) CD34 + Fc γ RIII/II + GMPs from the spleen of Tet2 f/f LysM cre mice and littermate controls ( n = 5 mice). Centre is mean. o , p , anti-IL-6 antibody (+) or ISO treatment (−) of Tet2 −/− . o , Percentage of CD11b + Gr1 + myeloid cells (left; n = 10 ( Tet2 +/+ , with anti-IL-6), 6 ( Tet2 −/− , with anti-IL-6) or 7 ( Tet2 −/− , without anti-IL-6) mice) and numbers of GMPs (right; n = 11 ( Tet2 +/+ , with anti-IL-6), 6 ( Tet2 −/− , with anti-IL-6) or 8 ( Tet2 −/− , without anti-IL-6) mice) in the spleen. p , Intestinal permeability was assessed by blood plasma FITC–dextran concentrations ( n = 6 mice in all cases). q , IL-6 in supernatants from intestinal explants: jejunum (left; n = 6 ( Tet2 +/+ ) or 5 ( Tet2 −/− ) mice) and colon (right; n = 6 mice in both cases). Centre is mean. a , h , m , p , Centre is mean, one-way ANOVA, Sidak’s post hoc test. c , d , j , Centre is median, two-tailed Mann–Whitney U -test. o , Centre is median, Kruskal–Wallis, Dunn’s post hoc test. Data are representative of at least three independent experiments; * P
    Figure Legend Snippet: Bacteria-induced IL-6 is required for PMP in Tet2 −/− mice. a , Gene expression of Il6 in the spleen of SPF-housed Tet2 −/− mice, littermates and germ-free Tet2 −/− mice ( n = 7 ( Tet2 +/+ , SPF), 10 ( Tet2 −/− , SPF) or 4 ( Tet2 −/− , GF) mice). b , IL-6 cytokine levels in blood plasma of Tet2 −/− mice with PMP treated with antibiotics (ABX) for four weeks ( n = 5 mice). Lines connect values obtained from the same mouse sampled before and after antibiotics treatment. Two-tailed paired t -test. c , IL-6 cytokine levels in blood plasma of DSS-treated symptom-free Tet2 f/f Vav cre mice that are over 20 weeks old, and littermate controls (see schematic in ( n = 6 ( Tet2 +/+ ) or 9 ( Tet2 −/− ) mice). d , IL-6 cytokine levels in blood plasma of Tet2 f/f Vav cre ( n = 6 ( Tet2 +/+ ) or 8 ( Tet2 −/− ) mice). e , Correlation between IL-6 cytokine levels in blood plasma of Tet2 −/− mice and numbers of peripheral-blood CD11b+ monocytes ( n = 49 mice). Pearson correlation test. f , g , In vitro HSC self-renewal colony-forming assay of haematopoietic progenitors of the spleen from Tet2 −/− mice (red lines) and littermate controls (blue lines) in the presence of anti-IL-6 antibody or isotype control (ISO) after the first replating ( n = 3 mice). g , Representative images of colonies after the 5th replating. Scale bars, 100 μm. h , Representative histogram (left) and quantification of (right) mean fluorescence intensity (MFI) of IL-6Rα + c-Kit + Sca-1 − (LK gated) CD34 + Fc γ RIII/II + GMPs from the bone marrow, ( n = 5 ( Tet2 +/+ SPF), 6 ( Tet2 −/− , SPF), 4 ( Tet2 +/+ GF) or 3 ( Tet2 −/− , GF) mice). i , Representative flow cytometry plot of Stat3 phosphorylation (pY705) response after 30-min stimulation with 10 ng ml −1 IL-6 in splenic c-Kit + Sca-1 − (LK gated) CD34 + and CD34 − myeloid progenitors (MP). j , Numbers of CD11b + myeloid cells ( n = 7 ( Tet2 +/+ ) or 6 ( Tet2 −/− ) mice). k , l , CD11b + F4/80 + macrophages and CD11b + Gr1 + myeloid cells of the spleen, from Tet2 −/− mice and littermate controls, were FACS sorted. Gene expression of Il6 in macrophages ( k ) and CD11b + Gr1 + cells ( l ) ( n = 7 ( Tet2 +/+ ) or 6 ( Tet2 −/− ) mice). Centre is median. m , Representative histogram (left) and quantification of (right) MFI of IL-6Rα + CD11b + Gr1 + myeloid cells in the spleen ( n = 5 ( Tet2 +/+ , SPF), 6 ( Tet2 −/− SPF), 4 ( Tet2 +/+ GF) or 4 ( Tet2 −/− , GF) mice). n , Representative histogram (left) and quantification of (right) MFI of IL-6Rα + c-Kit + Sca-1 − (LK gated) CD34 + Fc γ RIII/II + GMPs from the spleen of Tet2 f/f LysM cre mice and littermate controls ( n = 5 mice). Centre is mean. o , p , anti-IL-6 antibody (+) or ISO treatment (−) of Tet2 −/− . o , Percentage of CD11b + Gr1 + myeloid cells (left; n = 10 ( Tet2 +/+ , with anti-IL-6), 6 ( Tet2 −/− , with anti-IL-6) or 7 ( Tet2 −/− , without anti-IL-6) mice) and numbers of GMPs (right; n = 11 ( Tet2 +/+ , with anti-IL-6), 6 ( Tet2 −/− , with anti-IL-6) or 8 ( Tet2 −/− , without anti-IL-6) mice) in the spleen. p , Intestinal permeability was assessed by blood plasma FITC–dextran concentrations ( n = 6 mice in all cases). q , IL-6 in supernatants from intestinal explants: jejunum (left; n = 6 ( Tet2 +/+ ) or 5 ( Tet2 −/− ) mice) and colon (right; n = 6 mice in both cases). Centre is mean. a , h , m , p , Centre is mean, one-way ANOVA, Sidak’s post hoc test. c , d , j , Centre is median, two-tailed Mann–Whitney U -test. o , Centre is median, Kruskal–Wallis, Dunn’s post hoc test. Data are representative of at least three independent experiments; * P

    Techniques Used: Mouse Assay, Expressing, Two Tailed Test, In Vitro, Fluorescence, Flow Cytometry, Cytometry, FACS, Permeability, MANN-WHITNEY

    15) Product Images from "Vascular niche IL-6 induces alternative macrophage activation in glioblastoma through HIF-2α"

    Article Title: Vascular niche IL-6 induces alternative macrophage activation in glioblastoma through HIF-2α

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03050-0

    A schematic model. In glioma microenvironment, endothelial cell-derived IL-6 and microenvironmental CSF-1 synergistically activate downstream Akt1/mTOR pathway and induces transcriptional activation of PPARγ in macrophages (Mϕ), in turn leading to HIF-2α-mediated arginase-1 expression, and inducing macrophage alternative polarization. The activation of mTOR also induces cell proliferation, contributing to cell survival and growth of alternatively activated macrophages, eventually leading to glioma progression
    Figure Legend Snippet: A schematic model. In glioma microenvironment, endothelial cell-derived IL-6 and microenvironmental CSF-1 synergistically activate downstream Akt1/mTOR pathway and induces transcriptional activation of PPARγ in macrophages (Mϕ), in turn leading to HIF-2α-mediated arginase-1 expression, and inducing macrophage alternative polarization. The activation of mTOR also induces cell proliferation, contributing to cell survival and growth of alternatively activated macrophages, eventually leading to glioma progression

    Techniques Used: Derivative Assay, Activation Assay, Expressing

    HIF-2α is critical for IL-6-mediated arginase-1 expression and alternative macrophage activation. a Mouse BM-derived macrophages were treated with CSF-1 and IL-6 for 3 days. Nuclei proteins were subjected to multiplex profiling analysis for transcriptional factor activation. Activity was normalized with transcription factor IID, and expressed as the folds of control. b Mouse BM-derived macrophages were transduced with lentivirus that expresses CMV promoter-driven renilla luciferase (CMV-rLuc), hypoxia response element-driven firefly luciferase (HRE-fLuc), and mutated HRE-fLuc (muHRE-fLuc), followed by treatment with CSF-1 and IL-6 for 2 days. Reporter activity radio of fLuc versus rLuc was determined by bioluminescence. Results were expressed as the percentage of muHRE ( n = 3, mean ± SEM). P value was determined by Student’s t test. c – e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence, HIF-1α (#49 and #52) and HIF-2α (#5 and #7), followed by treatment with CSF-1 and IL-6 for 10 days. c Cells were lysed and subjected to immunoblot analysis. d Arginase-1 mRNA was analyzed by real-time RT-PCR. Shown are quantified data (normalized with GAPDH expression, n = 3, mean ± SEM). e Cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, followed by flow cytometry analysis ( n = 3, mean ± SEM)
    Figure Legend Snippet: HIF-2α is critical for IL-6-mediated arginase-1 expression and alternative macrophage activation. a Mouse BM-derived macrophages were treated with CSF-1 and IL-6 for 3 days. Nuclei proteins were subjected to multiplex profiling analysis for transcriptional factor activation. Activity was normalized with transcription factor IID, and expressed as the folds of control. b Mouse BM-derived macrophages were transduced with lentivirus that expresses CMV promoter-driven renilla luciferase (CMV-rLuc), hypoxia response element-driven firefly luciferase (HRE-fLuc), and mutated HRE-fLuc (muHRE-fLuc), followed by treatment with CSF-1 and IL-6 for 2 days. Reporter activity radio of fLuc versus rLuc was determined by bioluminescence. Results were expressed as the percentage of muHRE ( n = 3, mean ± SEM). P value was determined by Student’s t test. c – e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence, HIF-1α (#49 and #52) and HIF-2α (#5 and #7), followed by treatment with CSF-1 and IL-6 for 10 days. c Cells were lysed and subjected to immunoblot analysis. d Arginase-1 mRNA was analyzed by real-time RT-PCR. Shown are quantified data (normalized with GAPDH expression, n = 3, mean ± SEM). e Cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, followed by flow cytometry analysis ( n = 3, mean ± SEM)

    Techniques Used: Expressing, Activation Assay, Derivative Assay, Multiplex Assay, Activity Assay, Transduction, Luciferase, Sequencing, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry

    Endothelial IL-6 is critical for macrophage alternative activation and GBM growth and progression. The genetically engineered GBM model was induced in Ntv-a ; Ink4a-Arf −/− ; Pten −/− ; LSL-Luc donor mice, followed by orthotopic tumor implantation into Cdh5 - Cre ERT2 ; Il6 fl/fl mice that were treated with (IL-6-ΔEC) or without (Control) tamoxifen. a Animal survival was monitored for 50 days post-injection ( n = 5–6 mice, one representative result from three independent experiments). P values were determined by log-rank (Mantel–Cox) tests. MS, median survival. b Tumor growth was analyzed by bioluminescence. Left, representative images. Right, quantitative analysis of integrated luminescence in tumors at day 12 (mean ± SEM, n = 5–6, one representative result from three independent experiments). P value was determined by Student’s t test. c Tumor sections were stained with hematoxylin and eosin (H E). Representative images are shown ( n = 10 mice). P pseudopalisades, MP microvascular proliferation, EN extensive necrosis, LI leukocyte infiltration. Bar represents 100 μm. Zoom-in factor: 3. e , f Tumors were excised. Single-cell suspensions were prepared and subjected to flow cytometry analysis. d , e Single-cell suspensions were probed with anti-F4/80, anti-CD86, and anti-CD206 antibodies. CD206 and CD86 expression were analyzed in sorted F4/80 + cells. d Representative sorting. e Quantified results (mean ± SEM, n = 10–14 mice). f Single-cell suspensions were probed with anti-F4/80, anti-IL-10, and anti-IL-12 antibodies. IL-10 and IL-12 expression was analyzed in sorted F4/80 + cells. Show are quantified results (mean ± SEM, n = 8–13 mice). g Tumor sections were stained and analyzed by immunofluorescence. Tumor sections were probed with anti-iNOS, anti-arginase-1, anti-F4/80 antibodies ( n = 10 mice). Bar represents 100 μm
    Figure Legend Snippet: Endothelial IL-6 is critical for macrophage alternative activation and GBM growth and progression. The genetically engineered GBM model was induced in Ntv-a ; Ink4a-Arf −/− ; Pten −/− ; LSL-Luc donor mice, followed by orthotopic tumor implantation into Cdh5 - Cre ERT2 ; Il6 fl/fl mice that were treated with (IL-6-ΔEC) or without (Control) tamoxifen. a Animal survival was monitored for 50 days post-injection ( n = 5–6 mice, one representative result from three independent experiments). P values were determined by log-rank (Mantel–Cox) tests. MS, median survival. b Tumor growth was analyzed by bioluminescence. Left, representative images. Right, quantitative analysis of integrated luminescence in tumors at day 12 (mean ± SEM, n = 5–6, one representative result from three independent experiments). P value was determined by Student’s t test. c Tumor sections were stained with hematoxylin and eosin (H E). Representative images are shown ( n = 10 mice). P pseudopalisades, MP microvascular proliferation, EN extensive necrosis, LI leukocyte infiltration. Bar represents 100 μm. Zoom-in factor: 3. e , f Tumors were excised. Single-cell suspensions were prepared and subjected to flow cytometry analysis. d , e Single-cell suspensions were probed with anti-F4/80, anti-CD86, and anti-CD206 antibodies. CD206 and CD86 expression were analyzed in sorted F4/80 + cells. d Representative sorting. e Quantified results (mean ± SEM, n = 10–14 mice). f Single-cell suspensions were probed with anti-F4/80, anti-IL-10, and anti-IL-12 antibodies. IL-10 and IL-12 expression was analyzed in sorted F4/80 + cells. Show are quantified results (mean ± SEM, n = 8–13 mice). g Tumor sections were stained and analyzed by immunofluorescence. Tumor sections were probed with anti-iNOS, anti-arginase-1, anti-F4/80 antibodies ( n = 10 mice). Bar represents 100 μm

    Techniques Used: Activation Assay, Mouse Assay, Tumor Implantation, Injection, Mass Spectrometry, Staining, Flow Cytometry, Cytometry, Expressing, Immunofluorescence

    PPARγ induces HIF-2α and arginase-1 expression in macrophages. a , b Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 5 days. a Cell lysates were immunoblotted. b mRNA was extracted and subjected to quantitative RT-PCR analysis. Results were normalized with GAPDH level and expressed as folds of control ( n = 3, mean ± SEM). P value was determined by Student’s t test. c , d Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 3 days. c Nuclei protein was incubated with biotin-labeled synthetic DNAs that encode control scrambled or HIF-2α promoter sequence, followed by immunoprecipitation with streptavidin-conjugated beads. Precipitants and nuclei protein were immunoblotted. d Nuclei extracts were subjected to chromatin immunoprecipitation (ChIP) analysis. Immunoprecipitants with control IgG or anti-PPARγ antibody were analyzed by PCR and electrophoresis (upper) or by quantitative PCR (bottom, n = 3, mean ± SD). e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence and PPARγ (#1657, #1660, and #25967), followed by treatment with CSF-1 and IL-6 for 10 days. Cells were lysed and subjected to immunoblot analysis. f Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6. At different time points post-treatment, cells were lyzed and subjected to immunoblot analysis. Band density was quantified. g Mouse BM cells were pretreated with or without rapamycin, followed by incubation with CSF-1 and IL-6. Cell viability was determined ( n = 3 mice, mean ± SEM)
    Figure Legend Snippet: PPARγ induces HIF-2α and arginase-1 expression in macrophages. a , b Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 5 days. a Cell lysates were immunoblotted. b mRNA was extracted and subjected to quantitative RT-PCR analysis. Results were normalized with GAPDH level and expressed as folds of control ( n = 3, mean ± SEM). P value was determined by Student’s t test. c , d Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 3 days. c Nuclei protein was incubated with biotin-labeled synthetic DNAs that encode control scrambled or HIF-2α promoter sequence, followed by immunoprecipitation with streptavidin-conjugated beads. Precipitants and nuclei protein were immunoblotted. d Nuclei extracts were subjected to chromatin immunoprecipitation (ChIP) analysis. Immunoprecipitants with control IgG or anti-PPARγ antibody were analyzed by PCR and electrophoresis (upper) or by quantitative PCR (bottom, n = 3, mean ± SD). e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence and PPARγ (#1657, #1660, and #25967), followed by treatment with CSF-1 and IL-6 for 10 days. Cells were lysed and subjected to immunoblot analysis. f Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6. At different time points post-treatment, cells were lyzed and subjected to immunoblot analysis. Band density was quantified. g Mouse BM cells were pretreated with or without rapamycin, followed by incubation with CSF-1 and IL-6. Cell viability was determined ( n = 3 mice, mean ± SEM)

    Techniques Used: Expressing, Derivative Assay, Quantitative RT-PCR, Incubation, Labeling, Sequencing, Immunoprecipitation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Electrophoresis, Real-time Polymerase Chain Reaction, Transduction, Mouse Assay

    ECs are a major source for IL-6 expression in GBM. a – c Cdh5 - Cre ERT2 ; Il6 fl/fl mice were generated by crossing Cdh5 - Cre ERT2 mice with Il6 fl/fl mice. Mice (2 weeks old) were injected with tamoxifen for consecutive 5 days to induce EC-specific IL-6 knockout. a Schematic approach. b , c ECs were isolated from mouse aortas. b Brain tissue and ECs were subjected to immunoblot analysis. Band density was quantified. c mRNA was extracted and subjected to quantitative RT-PCR analysis. Results were normalized with GAPDH level and expressed as folds of control ( n = 3, mean ± SEM). d , e The primary GBM in Ntv-a ; Ink4a-Arf −/− ; Pten −/− ; LSL-Luc donor mice was induced by RCAS-mediated somatic gene transfer. Recipient mice were Cdh5 - Cre ERT2 ; Il6 fl/fl mice. d Schematic approach. e Normal brain and tumor tissues were homogenized. Tissue lysates were immunoblotted. Band density was quantified
    Figure Legend Snippet: ECs are a major source for IL-6 expression in GBM. a – c Cdh5 - Cre ERT2 ; Il6 fl/fl mice were generated by crossing Cdh5 - Cre ERT2 mice with Il6 fl/fl mice. Mice (2 weeks old) were injected with tamoxifen for consecutive 5 days to induce EC-specific IL-6 knockout. a Schematic approach. b , c ECs were isolated from mouse aortas. b Brain tissue and ECs were subjected to immunoblot analysis. Band density was quantified. c mRNA was extracted and subjected to quantitative RT-PCR analysis. Results were normalized with GAPDH level and expressed as folds of control ( n = 3, mean ± SEM). d , e The primary GBM in Ntv-a ; Ink4a-Arf −/− ; Pten −/− ; LSL-Luc donor mice was induced by RCAS-mediated somatic gene transfer. Recipient mice were Cdh5 - Cre ERT2 ; Il6 fl/fl mice. d Schematic approach. e Normal brain and tumor tissues were homogenized. Tissue lysates were immunoblotted. Band density was quantified

    Techniques Used: Expressing, Mouse Assay, Generated, Injection, Knock-Out, Isolation, Quantitative RT-PCR

    IL-6 is critical for EC-induced macrophage alternative activation. a , b Mouse microvascular brain ECs were pretreated with the glioma-CM for 24 h. Mouse BM-derived macrophages were co-cultured with pretreated ECs for 5 days in the presence of control IgG, anti-CSF-1 antibody, or anti-IL-6 antibody or both antibodies. The cells were stained with anti-CD11b, anti-CD86, anti-CD206 antibodies, and analyzed by flow cytometry. a Representative sorting for CD206 expression in CD11b + cells. b Quantified data in sorted CD11b + cells ( n = 3–5, mean ± SEM). c – e Mouse BM-derived macrophages were treated with IL-6 and CSF-1 for 5 days. c , d The cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, and analyzed by flow cytometry. c Representative sorting for CD206 and CD86 expression in CD11b + cells. d quantified data in sorted CD11b + cells ( n = 3, mean ± SEM). e Cell lysates were immunoblotted
    Figure Legend Snippet: IL-6 is critical for EC-induced macrophage alternative activation. a , b Mouse microvascular brain ECs were pretreated with the glioma-CM for 24 h. Mouse BM-derived macrophages were co-cultured with pretreated ECs for 5 days in the presence of control IgG, anti-CSF-1 antibody, or anti-IL-6 antibody or both antibodies. The cells were stained with anti-CD11b, anti-CD86, anti-CD206 antibodies, and analyzed by flow cytometry. a Representative sorting for CD206 expression in CD11b + cells. b Quantified data in sorted CD11b + cells ( n = 3–5, mean ± SEM). c – e Mouse BM-derived macrophages were treated with IL-6 and CSF-1 for 5 days. c , d The cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, and analyzed by flow cytometry. c Representative sorting for CD206 and CD86 expression in CD11b + cells. d quantified data in sorted CD11b + cells ( n = 3, mean ± SEM). e Cell lysates were immunoblotted

    Techniques Used: Activation Assay, Derivative Assay, Cell Culture, Staining, Flow Cytometry, Cytometry, Expressing

    GBM ECs express IL-6. a Human brain ECs were treated with glioma-CM for 24 h, and cell lysates were subjected to multiplex cytokine array analysis. Left, a representative blot. Right, quantified dot intensity of most significantly changed cytokines. b Human microvascular brain ECs were treated with glioma-CM that were harvested from different human glioma cells. Cell lysates were immunoblotted. c Human microvascular brain ECs and tumor-associated ECs isolated from different GBM patients were subjected to immunoblot analysis. d Mouse GBM was induced by orthotopic injection of GL26 glioma cells into wild-type mouse. The brain sections that include normal brains and tumors were stained with anti-CD31, anti-IL-6, and anti-CSF-1 antibodies. Representative immunofluorescence images are shown. Right, enlarged area in normal and tumor tissues. Bar represents 50 μm. Zoom-in factor: 4
    Figure Legend Snippet: GBM ECs express IL-6. a Human brain ECs were treated with glioma-CM for 24 h, and cell lysates were subjected to multiplex cytokine array analysis. Left, a representative blot. Right, quantified dot intensity of most significantly changed cytokines. b Human microvascular brain ECs were treated with glioma-CM that were harvested from different human glioma cells. Cell lysates were immunoblotted. c Human microvascular brain ECs and tumor-associated ECs isolated from different GBM patients were subjected to immunoblot analysis. d Mouse GBM was induced by orthotopic injection of GL26 glioma cells into wild-type mouse. The brain sections that include normal brains and tumors were stained with anti-CD31, anti-IL-6, and anti-CSF-1 antibodies. Representative immunofluorescence images are shown. Right, enlarged area in normal and tumor tissues. Bar represents 50 μm. Zoom-in factor: 4

    Techniques Used: Multiplex Assay, Isolation, Injection, Staining, Immunofluorescence

    16) Product Images from "Vascular niche IL-6 induces alternative macrophage activation in glioblastoma through HIF-2α"

    Article Title: Vascular niche IL-6 induces alternative macrophage activation in glioblastoma through HIF-2α

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03050-0

    A schematic model. In glioma microenvironment, endothelial cell-derived IL-6 and microenvironmental CSF-1 synergistically activate downstream Akt1/mTOR pathway and induces transcriptional activation of PPARγ in macrophages (Mϕ), in turn leading to HIF-2α-mediated arginase-1 expression, and inducing macrophage alternative polarization. The activation of mTOR also induces cell proliferation, contributing to cell survival and growth of alternatively activated macrophages, eventually leading to glioma progression
    Figure Legend Snippet: A schematic model. In glioma microenvironment, endothelial cell-derived IL-6 and microenvironmental CSF-1 synergistically activate downstream Akt1/mTOR pathway and induces transcriptional activation of PPARγ in macrophages (Mϕ), in turn leading to HIF-2α-mediated arginase-1 expression, and inducing macrophage alternative polarization. The activation of mTOR also induces cell proliferation, contributing to cell survival and growth of alternatively activated macrophages, eventually leading to glioma progression

    Techniques Used: Derivative Assay, Activation Assay, Expressing

    HIF-2α is critical for IL-6-mediated arginase-1 expression and alternative macrophage activation. a Mouse BM-derived macrophages were treated with CSF-1 and IL-6 for 3 days. Nuclei proteins were subjected to multiplex profiling analysis for transcriptional factor activation. Activity was normalized with transcription factor IID, and expressed as the folds of control. b Mouse BM-derived macrophages were transduced with lentivirus that expresses CMV promoter-driven renilla luciferase (CMV-rLuc), hypoxia response element-driven firefly luciferase (HRE-fLuc), and mutated HRE-fLuc (muHRE-fLuc), followed by treatment with CSF-1 and IL-6 for 2 days. Reporter activity radio of fLuc versus rLuc was determined by bioluminescence. Results were expressed as the percentage of muHRE ( n = 3, mean ± SEM). P value was determined by Student’s t test. c – e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence, HIF-1α (#49 and #52) and HIF-2α (#5 and #7), followed by treatment with CSF-1 and IL-6 for 10 days. c Cells were lysed and subjected to immunoblot analysis. d Arginase-1 mRNA was analyzed by real-time RT-PCR. Shown are quantified data (normalized with GAPDH expression, n = 3, mean ± SEM). e Cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, followed by flow cytometry analysis ( n = 3, mean ± SEM)
    Figure Legend Snippet: HIF-2α is critical for IL-6-mediated arginase-1 expression and alternative macrophage activation. a Mouse BM-derived macrophages were treated with CSF-1 and IL-6 for 3 days. Nuclei proteins were subjected to multiplex profiling analysis for transcriptional factor activation. Activity was normalized with transcription factor IID, and expressed as the folds of control. b Mouse BM-derived macrophages were transduced with lentivirus that expresses CMV promoter-driven renilla luciferase (CMV-rLuc), hypoxia response element-driven firefly luciferase (HRE-fLuc), and mutated HRE-fLuc (muHRE-fLuc), followed by treatment with CSF-1 and IL-6 for 2 days. Reporter activity radio of fLuc versus rLuc was determined by bioluminescence. Results were expressed as the percentage of muHRE ( n = 3, mean ± SEM). P value was determined by Student’s t test. c – e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence, HIF-1α (#49 and #52) and HIF-2α (#5 and #7), followed by treatment with CSF-1 and IL-6 for 10 days. c Cells were lysed and subjected to immunoblot analysis. d Arginase-1 mRNA was analyzed by real-time RT-PCR. Shown are quantified data (normalized with GAPDH expression, n = 3, mean ± SEM). e Cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, followed by flow cytometry analysis ( n = 3, mean ± SEM)

    Techniques Used: Expressing, Activation Assay, Derivative Assay, Multiplex Assay, Activity Assay, Transduction, Luciferase, Sequencing, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry

    Endothelial IL-6 is critical for macrophage alternative activation and GBM growth and progression. The genetically engineered GBM model was induced in Ntv-a ; Ink4a-Arf −/− ; Pten −/− ; LSL-Luc donor mice, followed by orthotopic tumor implantation into Cdh5 - Cre ERT2 ; Il6 fl/fl mice that were treated with (IL-6-ΔEC) or without (Control) tamoxifen. a Animal survival was monitored for 50 days post-injection ( n = 5–6 mice, one representative result from three independent experiments). P values were determined by log-rank (Mantel–Cox) tests. MS, median survival. b Tumor growth was analyzed by bioluminescence. Left, representative images. Right, quantitative analysis of integrated luminescence in tumors at day 12 (mean ± SEM, n = 5–6, one representative result from three independent experiments). P value was determined by Student’s t test. c Tumor sections were stained with hematoxylin and eosin (H E). Representative images are shown ( n = 10 mice). P pseudopalisades, MP microvascular proliferation, EN extensive necrosis, LI leukocyte infiltration. Bar represents 100 μm. Zoom-in factor: 3. e , f Tumors were excised. Single-cell suspensions were prepared and subjected to flow cytometry analysis. d , e Single-cell suspensions were probed with anti-F4/80, anti-CD86, and anti-CD206 antibodies. CD206 and CD86 expression were analyzed in sorted F4/80 + cells. d Representative sorting. e Quantified results (mean ± SEM, n = 10–14 mice). f Single-cell suspensions were probed with anti-F4/80, anti-IL-10, and anti-IL-12 antibodies. IL-10 and IL-12 expression was analyzed in sorted F4/80 + cells. Show are quantified results (mean ± SEM, n = 8–13 mice). g Tumor sections were stained and analyzed by immunofluorescence. Tumor sections were probed with anti-iNOS, anti-arginase-1, anti-F4/80 antibodies ( n = 10 mice). Bar represents 100 μm
    Figure Legend Snippet: Endothelial IL-6 is critical for macrophage alternative activation and GBM growth and progression. The genetically engineered GBM model was induced in Ntv-a ; Ink4a-Arf −/− ; Pten −/− ; LSL-Luc donor mice, followed by orthotopic tumor implantation into Cdh5 - Cre ERT2 ; Il6 fl/fl mice that were treated with (IL-6-ΔEC) or without (Control) tamoxifen. a Animal survival was monitored for 50 days post-injection ( n = 5–6 mice, one representative result from three independent experiments). P values were determined by log-rank (Mantel–Cox) tests. MS, median survival. b Tumor growth was analyzed by bioluminescence. Left, representative images. Right, quantitative analysis of integrated luminescence in tumors at day 12 (mean ± SEM, n = 5–6, one representative result from three independent experiments). P value was determined by Student’s t test. c Tumor sections were stained with hematoxylin and eosin (H E). Representative images are shown ( n = 10 mice). P pseudopalisades, MP microvascular proliferation, EN extensive necrosis, LI leukocyte infiltration. Bar represents 100 μm. Zoom-in factor: 3. e , f Tumors were excised. Single-cell suspensions were prepared and subjected to flow cytometry analysis. d , e Single-cell suspensions were probed with anti-F4/80, anti-CD86, and anti-CD206 antibodies. CD206 and CD86 expression were analyzed in sorted F4/80 + cells. d Representative sorting. e Quantified results (mean ± SEM, n = 10–14 mice). f Single-cell suspensions were probed with anti-F4/80, anti-IL-10, and anti-IL-12 antibodies. IL-10 and IL-12 expression was analyzed in sorted F4/80 + cells. Show are quantified results (mean ± SEM, n = 8–13 mice). g Tumor sections were stained and analyzed by immunofluorescence. Tumor sections were probed with anti-iNOS, anti-arginase-1, anti-F4/80 antibodies ( n = 10 mice). Bar represents 100 μm

    Techniques Used: Activation Assay, Mouse Assay, Tumor Implantation, Injection, Mass Spectrometry, Staining, Flow Cytometry, Cytometry, Expressing, Immunofluorescence

    PPARγ induces HIF-2α and arginase-1 expression in macrophages. a , b Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 5 days. a Cell lysates were immunoblotted. b mRNA was extracted and subjected to quantitative RT-PCR analysis. Results were normalized with GAPDH level and expressed as folds of control ( n = 3, mean ± SEM). P value was determined by Student’s t test. c , d Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 3 days. c Nuclei protein was incubated with biotin-labeled synthetic DNAs that encode control scrambled or HIF-2α promoter sequence, followed by immunoprecipitation with streptavidin-conjugated beads. Precipitants and nuclei protein were immunoblotted. d Nuclei extracts were subjected to chromatin immunoprecipitation (ChIP) analysis. Immunoprecipitants with control IgG or anti-PPARγ antibody were analyzed by PCR and electrophoresis (upper) or by quantitative PCR (bottom, n = 3, mean ± SD). e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence and PPARγ (#1657, #1660, and #25967), followed by treatment with CSF-1 and IL-6 for 10 days. Cells were lysed and subjected to immunoblot analysis. f Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6. At different time points post-treatment, cells were lyzed and subjected to immunoblot analysis. Band density was quantified. g Mouse BM cells were pretreated with or without rapamycin, followed by incubation with CSF-1 and IL-6. Cell viability was determined ( n = 3 mice, mean ± SEM)
    Figure Legend Snippet: PPARγ induces HIF-2α and arginase-1 expression in macrophages. a , b Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 5 days. a Cell lysates were immunoblotted. b mRNA was extracted and subjected to quantitative RT-PCR analysis. Results were normalized with GAPDH level and expressed as folds of control ( n = 3, mean ± SEM). P value was determined by Student’s t test. c , d Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 3 days. c Nuclei protein was incubated with biotin-labeled synthetic DNAs that encode control scrambled or HIF-2α promoter sequence, followed by immunoprecipitation with streptavidin-conjugated beads. Precipitants and nuclei protein were immunoblotted. d Nuclei extracts were subjected to chromatin immunoprecipitation (ChIP) analysis. Immunoprecipitants with control IgG or anti-PPARγ antibody were analyzed by PCR and electrophoresis (upper) or by quantitative PCR (bottom, n = 3, mean ± SD). e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence and PPARγ (#1657, #1660, and #25967), followed by treatment with CSF-1 and IL-6 for 10 days. Cells were lysed and subjected to immunoblot analysis. f Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6. At different time points post-treatment, cells were lyzed and subjected to immunoblot analysis. Band density was quantified. g Mouse BM cells were pretreated with or without rapamycin, followed by incubation with CSF-1 and IL-6. Cell viability was determined ( n = 3 mice, mean ± SEM)

    Techniques Used: Expressing, Derivative Assay, Quantitative RT-PCR, Incubation, Labeling, Sequencing, Immunoprecipitation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Electrophoresis, Real-time Polymerase Chain Reaction, Transduction, Mouse Assay

    ECs are a major source for IL-6 expression in GBM. a – c Cdh5 - Cre ERT2 ; Il6 fl/fl mice were generated by crossing Cdh5 - Cre ERT2 mice with Il6 fl/fl mice. Mice (2 weeks old) were injected with tamoxifen for consecutive 5 days to induce EC-specific IL-6 knockout. a Schematic approach. b , c ECs were isolated from mouse aortas. b Brain tissue and ECs were subjected to immunoblot analysis. Band density was quantified. c mRNA was extracted and subjected to quantitative RT-PCR analysis. Results were normalized with GAPDH level and expressed as folds of control ( n = 3, mean ± SEM). d , e The primary GBM in Ntv-a ; Ink4a-Arf −/− ; Pten −/− ; LSL-Luc donor mice was induced by RCAS-mediated somatic gene transfer. Recipient mice were Cdh5 - Cre ERT2 ; Il6 fl/fl mice. d Schematic approach. e Normal brain and tumor tissues were homogenized. Tissue lysates were immunoblotted. Band density was quantified
    Figure Legend Snippet: ECs are a major source for IL-6 expression in GBM. a – c Cdh5 - Cre ERT2 ; Il6 fl/fl mice were generated by crossing Cdh5 - Cre ERT2 mice with Il6 fl/fl mice. Mice (2 weeks old) were injected with tamoxifen for consecutive 5 days to induce EC-specific IL-6 knockout. a Schematic approach. b , c ECs were isolated from mouse aortas. b Brain tissue and ECs were subjected to immunoblot analysis. Band density was quantified. c mRNA was extracted and subjected to quantitative RT-PCR analysis. Results were normalized with GAPDH level and expressed as folds of control ( n = 3, mean ± SEM). d , e The primary GBM in Ntv-a ; Ink4a-Arf −/− ; Pten −/− ; LSL-Luc donor mice was induced by RCAS-mediated somatic gene transfer. Recipient mice were Cdh5 - Cre ERT2 ; Il6 fl/fl mice. d Schematic approach. e Normal brain and tumor tissues were homogenized. Tissue lysates were immunoblotted. Band density was quantified

    Techniques Used: Expressing, Mouse Assay, Generated, Injection, Knock-Out, Isolation, Quantitative RT-PCR

    IL-6 is critical for EC-induced macrophage alternative activation. a , b Mouse microvascular brain ECs were pretreated with the glioma-CM for 24 h. Mouse BM-derived macrophages were co-cultured with pretreated ECs for 5 days in the presence of control IgG, anti-CSF-1 antibody, or anti-IL-6 antibody or both antibodies. The cells were stained with anti-CD11b, anti-CD86, anti-CD206 antibodies, and analyzed by flow cytometry. a Representative sorting for CD206 expression in CD11b + cells. b Quantified data in sorted CD11b + cells ( n = 3–5, mean ± SEM). c – e Mouse BM-derived macrophages were treated with IL-6 and CSF-1 for 5 days. c , d The cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, and analyzed by flow cytometry. c Representative sorting for CD206 and CD86 expression in CD11b + cells. d quantified data in sorted CD11b + cells ( n = 3, mean ± SEM). e Cell lysates were immunoblotted
    Figure Legend Snippet: IL-6 is critical for EC-induced macrophage alternative activation. a , b Mouse microvascular brain ECs were pretreated with the glioma-CM for 24 h. Mouse BM-derived macrophages were co-cultured with pretreated ECs for 5 days in the presence of control IgG, anti-CSF-1 antibody, or anti-IL-6 antibody or both antibodies. The cells were stained with anti-CD11b, anti-CD86, anti-CD206 antibodies, and analyzed by flow cytometry. a Representative sorting for CD206 expression in CD11b + cells. b Quantified data in sorted CD11b + cells ( n = 3–5, mean ± SEM). c – e Mouse BM-derived macrophages were treated with IL-6 and CSF-1 for 5 days. c , d The cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, and analyzed by flow cytometry. c Representative sorting for CD206 and CD86 expression in CD11b + cells. d quantified data in sorted CD11b + cells ( n = 3, mean ± SEM). e Cell lysates were immunoblotted

    Techniques Used: Activation Assay, Derivative Assay, Cell Culture, Staining, Flow Cytometry, Cytometry, Expressing

    GBM ECs express IL-6. a Human brain ECs were treated with glioma-CM for 24 h, and cell lysates were subjected to multiplex cytokine array analysis. Left, a representative blot. Right, quantified dot intensity of most significantly changed cytokines. b Human microvascular brain ECs were treated with glioma-CM that were harvested from different human glioma cells. Cell lysates were immunoblotted. c Human microvascular brain ECs and tumor-associated ECs isolated from different GBM patients were subjected to immunoblot analysis. d Mouse GBM was induced by orthotopic injection of GL26 glioma cells into wild-type mouse. The brain sections that include normal brains and tumors were stained with anti-CD31, anti-IL-6, and anti-CSF-1 antibodies. Representative immunofluorescence images are shown. Right, enlarged area in normal and tumor tissues. Bar represents 50 μm. Zoom-in factor: 4
    Figure Legend Snippet: GBM ECs express IL-6. a Human brain ECs were treated with glioma-CM for 24 h, and cell lysates were subjected to multiplex cytokine array analysis. Left, a representative blot. Right, quantified dot intensity of most significantly changed cytokines. b Human microvascular brain ECs were treated with glioma-CM that were harvested from different human glioma cells. Cell lysates were immunoblotted. c Human microvascular brain ECs and tumor-associated ECs isolated from different GBM patients were subjected to immunoblot analysis. d Mouse GBM was induced by orthotopic injection of GL26 glioma cells into wild-type mouse. The brain sections that include normal brains and tumors were stained with anti-CD31, anti-IL-6, and anti-CSF-1 antibodies. Representative immunofluorescence images are shown. Right, enlarged area in normal and tumor tissues. Bar represents 50 μm. Zoom-in factor: 4

    Techniques Used: Multiplex Assay, Isolation, Injection, Staining, Immunofluorescence

    17) Product Images from "Vascular niche IL-6 induces alternative macrophage activation in glioblastoma through HIF-2α"

    Article Title: Vascular niche IL-6 induces alternative macrophage activation in glioblastoma through HIF-2α

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03050-0

    A schematic model. In glioma microenvironment, endothelial cell-derived IL-6 and microenvironmental CSF-1 synergistically activate downstream Akt1/mTOR pathway and induces transcriptional activation of PPARγ in macrophages (Mϕ), in turn leading to HIF-2α-mediated arginase-1 expression, and inducing macrophage alternative polarization. The activation of mTOR also induces cell proliferation, contributing to cell survival and growth of alternatively activated macrophages, eventually leading to glioma progression
    Figure Legend Snippet: A schematic model. In glioma microenvironment, endothelial cell-derived IL-6 and microenvironmental CSF-1 synergistically activate downstream Akt1/mTOR pathway and induces transcriptional activation of PPARγ in macrophages (Mϕ), in turn leading to HIF-2α-mediated arginase-1 expression, and inducing macrophage alternative polarization. The activation of mTOR also induces cell proliferation, contributing to cell survival and growth of alternatively activated macrophages, eventually leading to glioma progression

    Techniques Used: Derivative Assay, Activation Assay, Expressing

    HIF-2α is critical for IL-6-mediated arginase-1 expression and alternative macrophage activation. a Mouse BM-derived macrophages were treated with CSF-1 and IL-6 for 3 days. Nuclei proteins were subjected to multiplex profiling analysis for transcriptional factor activation. Activity was normalized with transcription factor IID, and expressed as the folds of control. b Mouse BM-derived macrophages were transduced with lentivirus that expresses CMV promoter-driven renilla luciferase (CMV-rLuc), hypoxia response element-driven firefly luciferase (HRE-fLuc), and mutated HRE-fLuc (muHRE-fLuc), followed by treatment with CSF-1 and IL-6 for 2 days. Reporter activity radio of fLuc versus rLuc was determined by bioluminescence. Results were expressed as the percentage of muHRE ( n = 3, mean ± SEM). P value was determined by Student’s t test. c – e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence, HIF-1α (#49 and #52) and HIF-2α (#5 and #7), followed by treatment with CSF-1 and IL-6 for 10 days. c Cells were lysed and subjected to immunoblot analysis. d Arginase-1 mRNA was analyzed by real-time RT-PCR. Shown are quantified data (normalized with GAPDH expression, n = 3, mean ± SEM). e Cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, followed by flow cytometry analysis ( n = 3, mean ± SEM)
    Figure Legend Snippet: HIF-2α is critical for IL-6-mediated arginase-1 expression and alternative macrophage activation. a Mouse BM-derived macrophages were treated with CSF-1 and IL-6 for 3 days. Nuclei proteins were subjected to multiplex profiling analysis for transcriptional factor activation. Activity was normalized with transcription factor IID, and expressed as the folds of control. b Mouse BM-derived macrophages were transduced with lentivirus that expresses CMV promoter-driven renilla luciferase (CMV-rLuc), hypoxia response element-driven firefly luciferase (HRE-fLuc), and mutated HRE-fLuc (muHRE-fLuc), followed by treatment with CSF-1 and IL-6 for 2 days. Reporter activity radio of fLuc versus rLuc was determined by bioluminescence. Results were expressed as the percentage of muHRE ( n = 3, mean ± SEM). P value was determined by Student’s t test. c – e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence, HIF-1α (#49 and #52) and HIF-2α (#5 and #7), followed by treatment with CSF-1 and IL-6 for 10 days. c Cells were lysed and subjected to immunoblot analysis. d Arginase-1 mRNA was analyzed by real-time RT-PCR. Shown are quantified data (normalized with GAPDH expression, n = 3, mean ± SEM). e Cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, followed by flow cytometry analysis ( n = 3, mean ± SEM)

    Techniques Used: Expressing, Activation Assay, Derivative Assay, Multiplex Assay, Activity Assay, Transduction, Luciferase, Sequencing, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry

    Endothelial IL-6 is critical for macrophage alternative activation and GBM growth and progression. The genetically engineered GBM model was induced in Ntv-a ; Ink4a-Arf −/− ; Pten −/− ; LSL-Luc donor mice, followed by orthotopic tumor implantation into Cdh5 - Cre ERT2 ; Il6 fl/fl mice that were treated with (IL-6-ΔEC) or without (Control) tamoxifen. a Animal survival was monitored for 50 days post-injection ( n = 5–6 mice, one representative result from three independent experiments). P values were determined by log-rank (Mantel–Cox) tests. MS, median survival. b Tumor growth was analyzed by bioluminescence. Left, representative images. Right, quantitative analysis of integrated luminescence in tumors at day 12 (mean ± SEM, n = 5–6, one representative result from three independent experiments). P value was determined by Student’s t test. c Tumor sections were stained with hematoxylin and eosin (H E). Representative images are shown ( n = 10 mice). P pseudopalisades, MP microvascular proliferation, EN extensive necrosis, LI leukocyte infiltration. Bar represents 100 μm. Zoom-in factor: 3. e , f Tumors were excised. Single-cell suspensions were prepared and subjected to flow cytometry analysis. d , e Single-cell suspensions were probed with anti-F4/80, anti-CD86, and anti-CD206 antibodies. CD206 and CD86 expression were analyzed in sorted F4/80 + cells. d Representative sorting. e Quantified results (mean ± SEM, n = 10–14 mice). f Single-cell suspensions were probed with anti-F4/80, anti-IL-10, and anti-IL-12 antibodies. IL-10 and IL-12 expression was analyzed in sorted F4/80 + cells. Show are quantified results (mean ± SEM, n = 8–13 mice). g Tumor sections were stained and analyzed by immunofluorescence. Tumor sections were probed with anti-iNOS, anti-arginase-1, anti-F4/80 antibodies ( n = 10 mice). Bar represents 100 μm
    Figure Legend Snippet: Endothelial IL-6 is critical for macrophage alternative activation and GBM growth and progression. The genetically engineered GBM model was induced in Ntv-a ; Ink4a-Arf −/− ; Pten −/− ; LSL-Luc donor mice, followed by orthotopic tumor implantation into Cdh5 - Cre ERT2 ; Il6 fl/fl mice that were treated with (IL-6-ΔEC) or without (Control) tamoxifen. a Animal survival was monitored for 50 days post-injection ( n = 5–6 mice, one representative result from three independent experiments). P values were determined by log-rank (Mantel–Cox) tests. MS, median survival. b Tumor growth was analyzed by bioluminescence. Left, representative images. Right, quantitative analysis of integrated luminescence in tumors at day 12 (mean ± SEM, n = 5–6, one representative result from three independent experiments). P value was determined by Student’s t test. c Tumor sections were stained with hematoxylin and eosin (H E). Representative images are shown ( n = 10 mice). P pseudopalisades, MP microvascular proliferation, EN extensive necrosis, LI leukocyte infiltration. Bar represents 100 μm. Zoom-in factor: 3. e , f Tumors were excised. Single-cell suspensions were prepared and subjected to flow cytometry analysis. d , e Single-cell suspensions were probed with anti-F4/80, anti-CD86, and anti-CD206 antibodies. CD206 and CD86 expression were analyzed in sorted F4/80 + cells. d Representative sorting. e Quantified results (mean ± SEM, n = 10–14 mice). f Single-cell suspensions were probed with anti-F4/80, anti-IL-10, and anti-IL-12 antibodies. IL-10 and IL-12 expression was analyzed in sorted F4/80 + cells. Show are quantified results (mean ± SEM, n = 8–13 mice). g Tumor sections were stained and analyzed by immunofluorescence. Tumor sections were probed with anti-iNOS, anti-arginase-1, anti-F4/80 antibodies ( n = 10 mice). Bar represents 100 μm

    Techniques Used: Activation Assay, Mouse Assay, Tumor Implantation, Injection, Mass Spectrometry, Staining, Flow Cytometry, Cytometry, Expressing, Immunofluorescence

    PPARγ induces HIF-2α and arginase-1 expression in macrophages. a , b Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 5 days. a Cell lysates were immunoblotted. b mRNA was extracted and subjected to quantitative RT-PCR analysis. Results were normalized with GAPDH level and expressed as folds of control ( n = 3, mean ± SEM). P value was determined by Student’s t test. c , d Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 3 days. c Nuclei protein was incubated with biotin-labeled synthetic DNAs that encode control scrambled or HIF-2α promoter sequence, followed by immunoprecipitation with streptavidin-conjugated beads. Precipitants and nuclei protein were immunoblotted. d Nuclei extracts were subjected to chromatin immunoprecipitation (ChIP) analysis. Immunoprecipitants with control IgG or anti-PPARγ antibody were analyzed by PCR and electrophoresis (upper) or by quantitative PCR (bottom, n = 3, mean ± SD). e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence and PPARγ (#1657, #1660, and #25967), followed by treatment with CSF-1 and IL-6 for 10 days. Cells were lysed and subjected to immunoblot analysis. f Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6. At different time points post-treatment, cells were lyzed and subjected to immunoblot analysis. Band density was quantified. g Mouse BM cells were pretreated with or without rapamycin, followed by incubation with CSF-1 and IL-6. Cell viability was determined ( n = 3 mice, mean ± SEM)
    Figure Legend Snippet: PPARγ induces HIF-2α and arginase-1 expression in macrophages. a , b Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 5 days. a Cell lysates were immunoblotted. b mRNA was extracted and subjected to quantitative RT-PCR analysis. Results were normalized with GAPDH level and expressed as folds of control ( n = 3, mean ± SEM). P value was determined by Student’s t test. c , d Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 3 days. c Nuclei protein was incubated with biotin-labeled synthetic DNAs that encode control scrambled or HIF-2α promoter sequence, followed by immunoprecipitation with streptavidin-conjugated beads. Precipitants and nuclei protein were immunoblotted. d Nuclei extracts were subjected to chromatin immunoprecipitation (ChIP) analysis. Immunoprecipitants with control IgG or anti-PPARγ antibody were analyzed by PCR and electrophoresis (upper) or by quantitative PCR (bottom, n = 3, mean ± SD). e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence and PPARγ (#1657, #1660, and #25967), followed by treatment with CSF-1 and IL-6 for 10 days. Cells were lysed and subjected to immunoblot analysis. f Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6. At different time points post-treatment, cells were lyzed and subjected to immunoblot analysis. Band density was quantified. g Mouse BM cells were pretreated with or without rapamycin, followed by incubation with CSF-1 and IL-6. Cell viability was determined ( n = 3 mice, mean ± SEM)

    Techniques Used: Expressing, Derivative Assay, Quantitative RT-PCR, Incubation, Labeling, Sequencing, Immunoprecipitation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Electrophoresis, Real-time Polymerase Chain Reaction, Transduction, Mouse Assay

    ECs are a major source for IL-6 expression in GBM. a – c Cdh5 - Cre ERT2 ; Il6 fl/fl mice were generated by crossing Cdh5 - Cre ERT2 mice with Il6 fl/fl mice. Mice (2 weeks old) were injected with tamoxifen for consecutive 5 days to induce EC-specific IL-6 knockout. a Schematic approach. b , c ECs were isolated from mouse aortas. b Brain tissue and ECs were subjected to immunoblot analysis. Band density was quantified. c mRNA was extracted and subjected to quantitative RT-PCR analysis. Results were normalized with GAPDH level and expressed as folds of control ( n = 3, mean ± SEM). d , e The primary GBM in Ntv-a ; Ink4a-Arf −/− ; Pten −/− ; LSL-Luc donor mice was induced by RCAS-mediated somatic gene transfer. Recipient mice were Cdh5 - Cre ERT2 ; Il6 fl/fl mice. d Schematic approach. e Normal brain and tumor tissues were homogenized. Tissue lysates were immunoblotted. Band density was quantified
    Figure Legend Snippet: ECs are a major source for IL-6 expression in GBM. a – c Cdh5 - Cre ERT2 ; Il6 fl/fl mice were generated by crossing Cdh5 - Cre ERT2 mice with Il6 fl/fl mice. Mice (2 weeks old) were injected with tamoxifen for consecutive 5 days to induce EC-specific IL-6 knockout. a Schematic approach. b , c ECs were isolated from mouse aortas. b Brain tissue and ECs were subjected to immunoblot analysis. Band density was quantified. c mRNA was extracted and subjected to quantitative RT-PCR analysis. Results were normalized with GAPDH level and expressed as folds of control ( n = 3, mean ± SEM). d , e The primary GBM in Ntv-a ; Ink4a-Arf −/− ; Pten −/− ; LSL-Luc donor mice was induced by RCAS-mediated somatic gene transfer. Recipient mice were Cdh5 - Cre ERT2 ; Il6 fl/fl mice. d Schematic approach. e Normal brain and tumor tissues were homogenized. Tissue lysates were immunoblotted. Band density was quantified

    Techniques Used: Expressing, Mouse Assay, Generated, Injection, Knock-Out, Isolation, Quantitative RT-PCR

    IL-6 is critical for EC-induced macrophage alternative activation. a , b Mouse microvascular brain ECs were pretreated with the glioma-CM for 24 h. Mouse BM-derived macrophages were co-cultured with pretreated ECs for 5 days in the presence of control IgG, anti-CSF-1 antibody, or anti-IL-6 antibody or both antibodies. The cells were stained with anti-CD11b, anti-CD86, anti-CD206 antibodies, and analyzed by flow cytometry. a Representative sorting for CD206 expression in CD11b + cells. b Quantified data in sorted CD11b + cells ( n = 3–5, mean ± SEM). c – e Mouse BM-derived macrophages were treated with IL-6 and CSF-1 for 5 days. c , d The cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, and analyzed by flow cytometry. c Representative sorting for CD206 and CD86 expression in CD11b + cells. d quantified data in sorted CD11b + cells ( n = 3, mean ± SEM). e Cell lysates were immunoblotted
    Figure Legend Snippet: IL-6 is critical for EC-induced macrophage alternative activation. a , b Mouse microvascular brain ECs were pretreated with the glioma-CM for 24 h. Mouse BM-derived macrophages were co-cultured with pretreated ECs for 5 days in the presence of control IgG, anti-CSF-1 antibody, or anti-IL-6 antibody or both antibodies. The cells were stained with anti-CD11b, anti-CD86, anti-CD206 antibodies, and analyzed by flow cytometry. a Representative sorting for CD206 expression in CD11b + cells. b Quantified data in sorted CD11b + cells ( n = 3–5, mean ± SEM). c – e Mouse BM-derived macrophages were treated with IL-6 and CSF-1 for 5 days. c , d The cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, and analyzed by flow cytometry. c Representative sorting for CD206 and CD86 expression in CD11b + cells. d quantified data in sorted CD11b + cells ( n = 3, mean ± SEM). e Cell lysates were immunoblotted

    Techniques Used: Activation Assay, Derivative Assay, Cell Culture, Staining, Flow Cytometry, Cytometry, Expressing

    GBM ECs express IL-6. a Human brain ECs were treated with glioma-CM for 24 h, and cell lysates were subjected to multiplex cytokine array analysis. Left, a representative blot. Right, quantified dot intensity of most significantly changed cytokines. b Human microvascular brain ECs were treated with glioma-CM that were harvested from different human glioma cells. Cell lysates were immunoblotted. c Human microvascular brain ECs and tumor-associated ECs isolated from different GBM patients were subjected to immunoblot analysis. d Mouse GBM was induced by orthotopic injection of GL26 glioma cells into wild-type mouse. The brain sections that include normal brains and tumors were stained with anti-CD31, anti-IL-6, and anti-CSF-1 antibodies. Representative immunofluorescence images are shown. Right, enlarged area in normal and tumor tissues. Bar represents 50 μm. Zoom-in factor: 4
    Figure Legend Snippet: GBM ECs express IL-6. a Human brain ECs were treated with glioma-CM for 24 h, and cell lysates were subjected to multiplex cytokine array analysis. Left, a representative blot. Right, quantified dot intensity of most significantly changed cytokines. b Human microvascular brain ECs were treated with glioma-CM that were harvested from different human glioma cells. Cell lysates were immunoblotted. c Human microvascular brain ECs and tumor-associated ECs isolated from different GBM patients were subjected to immunoblot analysis. d Mouse GBM was induced by orthotopic injection of GL26 glioma cells into wild-type mouse. The brain sections that include normal brains and tumors were stained with anti-CD31, anti-IL-6, and anti-CSF-1 antibodies. Representative immunofluorescence images are shown. Right, enlarged area in normal and tumor tissues. Bar represents 50 μm. Zoom-in factor: 4

    Techniques Used: Multiplex Assay, Isolation, Injection, Staining, Immunofluorescence

    18) Product Images from "Vascular niche IL-6 induces alternative macrophage activation in glioblastoma through HIF-2α"

    Article Title: Vascular niche IL-6 induces alternative macrophage activation in glioblastoma through HIF-2α

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03050-0

    A schematic model. In glioma microenvironment, endothelial cell-derived IL-6 and microenvironmental CSF-1 synergistically activate downstream Akt1/mTOR pathway and induces transcriptional activation of PPARγ in macrophages (Mϕ), in turn leading to HIF-2α-mediated arginase-1 expression, and inducing macrophage alternative polarization. The activation of mTOR also induces cell proliferation, contributing to cell survival and growth of alternatively activated macrophages, eventually leading to glioma progression
    Figure Legend Snippet: A schematic model. In glioma microenvironment, endothelial cell-derived IL-6 and microenvironmental CSF-1 synergistically activate downstream Akt1/mTOR pathway and induces transcriptional activation of PPARγ in macrophages (Mϕ), in turn leading to HIF-2α-mediated arginase-1 expression, and inducing macrophage alternative polarization. The activation of mTOR also induces cell proliferation, contributing to cell survival and growth of alternatively activated macrophages, eventually leading to glioma progression

    Techniques Used: Derivative Assay, Activation Assay, Expressing

    HIF-2α is critical for IL-6-mediated arginase-1 expression and alternative macrophage activation. a Mouse BM-derived macrophages were treated with CSF-1 and IL-6 for 3 days. Nuclei proteins were subjected to multiplex profiling analysis for transcriptional factor activation. Activity was normalized with transcription factor IID, and expressed as the folds of control. b Mouse BM-derived macrophages were transduced with lentivirus that expresses CMV promoter-driven renilla luciferase (CMV-rLuc), hypoxia response element-driven firefly luciferase (HRE-fLuc), and mutated HRE-fLuc (muHRE-fLuc), followed by treatment with CSF-1 and IL-6 for 2 days. Reporter activity radio of fLuc versus rLuc was determined by bioluminescence. Results were expressed as the percentage of muHRE ( n = 3, mean ± SEM). P value was determined by Student’s t test. c – e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence, HIF-1α (#49 and #52) and HIF-2α (#5 and #7), followed by treatment with CSF-1 and IL-6 for 10 days. c Cells were lysed and subjected to immunoblot analysis. d Arginase-1 mRNA was analyzed by real-time RT-PCR. Shown are quantified data (normalized with GAPDH expression, n = 3, mean ± SEM). e Cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, followed by flow cytometry analysis ( n = 3, mean ± SEM)
    Figure Legend Snippet: HIF-2α is critical for IL-6-mediated arginase-1 expression and alternative macrophage activation. a Mouse BM-derived macrophages were treated with CSF-1 and IL-6 for 3 days. Nuclei proteins were subjected to multiplex profiling analysis for transcriptional factor activation. Activity was normalized with transcription factor IID, and expressed as the folds of control. b Mouse BM-derived macrophages were transduced with lentivirus that expresses CMV promoter-driven renilla luciferase (CMV-rLuc), hypoxia response element-driven firefly luciferase (HRE-fLuc), and mutated HRE-fLuc (muHRE-fLuc), followed by treatment with CSF-1 and IL-6 for 2 days. Reporter activity radio of fLuc versus rLuc was determined by bioluminescence. Results were expressed as the percentage of muHRE ( n = 3, mean ± SEM). P value was determined by Student’s t test. c – e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence, HIF-1α (#49 and #52) and HIF-2α (#5 and #7), followed by treatment with CSF-1 and IL-6 for 10 days. c Cells were lysed and subjected to immunoblot analysis. d Arginase-1 mRNA was analyzed by real-time RT-PCR. Shown are quantified data (normalized with GAPDH expression, n = 3, mean ± SEM). e Cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, followed by flow cytometry analysis ( n = 3, mean ± SEM)

    Techniques Used: Expressing, Activation Assay, Derivative Assay, Multiplex Assay, Activity Assay, Transduction, Luciferase, Sequencing, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry

    Endothelial IL-6 is critical for macrophage alternative activation and GBM growth and progression. The genetically engineered GBM model was induced in Ntv-a ; Ink4a-Arf −/− ; Pten −/− ; LSL-Luc donor mice, followed by orthotopic tumor implantation into Cdh5 - Cre ERT2 ; Il6 fl/fl mice that were treated with (IL-6-ΔEC) or without (Control) tamoxifen. a Animal survival was monitored for 50 days post-injection ( n = 5–6 mice, one representative result from three independent experiments). P values were determined by log-rank (Mantel–Cox) tests. MS, median survival. b Tumor growth was analyzed by bioluminescence. Left, representative images. Right, quantitative analysis of integrated luminescence in tumors at day 12 (mean ± SEM, n = 5–6, one representative result from three independent experiments). P value was determined by Student’s t test. c Tumor sections were stained with hematoxylin and eosin (H E). Representative images are shown ( n = 10 mice). P pseudopalisades, MP microvascular proliferation, EN extensive necrosis, LI leukocyte infiltration. Bar represents 100 μm. Zoom-in factor: 3. e , f Tumors were excised. Single-cell suspensions were prepared and subjected to flow cytometry analysis. d , e Single-cell suspensions were probed with anti-F4/80, anti-CD86, and anti-CD206 antibodies. CD206 and CD86 expression were analyzed in sorted F4/80 + cells. d Representative sorting. e Quantified results (mean ± SEM, n = 10–14 mice). f Single-cell suspensions were probed with anti-F4/80, anti-IL-10, and anti-IL-12 antibodies. IL-10 and IL-12 expression was analyzed in sorted F4/80 + cells. Show are quantified results (mean ± SEM, n = 8–13 mice). g Tumor sections were stained and analyzed by immunofluorescence. Tumor sections were probed with anti-iNOS, anti-arginase-1, anti-F4/80 antibodies ( n = 10 mice). Bar represents 100 μm
    Figure Legend Snippet: Endothelial IL-6 is critical for macrophage alternative activation and GBM growth and progression. The genetically engineered GBM model was induced in Ntv-a ; Ink4a-Arf −/− ; Pten −/− ; LSL-Luc donor mice, followed by orthotopic tumor implantation into Cdh5 - Cre ERT2 ; Il6 fl/fl mice that were treated with (IL-6-ΔEC) or without (Control) tamoxifen. a Animal survival was monitored for 50 days post-injection ( n = 5–6 mice, one representative result from three independent experiments). P values were determined by log-rank (Mantel–Cox) tests. MS, median survival. b Tumor growth was analyzed by bioluminescence. Left, representative images. Right, quantitative analysis of integrated luminescence in tumors at day 12 (mean ± SEM, n = 5–6, one representative result from three independent experiments). P value was determined by Student’s t test. c Tumor sections were stained with hematoxylin and eosin (H E). Representative images are shown ( n = 10 mice). P pseudopalisades, MP microvascular proliferation, EN extensive necrosis, LI leukocyte infiltration. Bar represents 100 μm. Zoom-in factor: 3. e , f Tumors were excised. Single-cell suspensions were prepared and subjected to flow cytometry analysis. d , e Single-cell suspensions were probed with anti-F4/80, anti-CD86, and anti-CD206 antibodies. CD206 and CD86 expression were analyzed in sorted F4/80 + cells. d Representative sorting. e Quantified results (mean ± SEM, n = 10–14 mice). f Single-cell suspensions were probed with anti-F4/80, anti-IL-10, and anti-IL-12 antibodies. IL-10 and IL-12 expression was analyzed in sorted F4/80 + cells. Show are quantified results (mean ± SEM, n = 8–13 mice). g Tumor sections were stained and analyzed by immunofluorescence. Tumor sections were probed with anti-iNOS, anti-arginase-1, anti-F4/80 antibodies ( n = 10 mice). Bar represents 100 μm

    Techniques Used: Activation Assay, Mouse Assay, Tumor Implantation, Injection, Mass Spectrometry, Staining, Flow Cytometry, Cytometry, Expressing, Immunofluorescence

    PPARγ induces HIF-2α and arginase-1 expression in macrophages. a , b Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 5 days. a Cell lysates were immunoblotted. b mRNA was extracted and subjected to quantitative RT-PCR analysis. Results were normalized with GAPDH level and expressed as folds of control ( n = 3, mean ± SEM). P value was determined by Student’s t test. c , d Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 3 days. c Nuclei protein was incubated with biotin-labeled synthetic DNAs that encode control scrambled or HIF-2α promoter sequence, followed by immunoprecipitation with streptavidin-conjugated beads. Precipitants and nuclei protein were immunoblotted. d Nuclei extracts were subjected to chromatin immunoprecipitation (ChIP) analysis. Immunoprecipitants with control IgG or anti-PPARγ antibody were analyzed by PCR and electrophoresis (upper) or by quantitative PCR (bottom, n = 3, mean ± SD). e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence and PPARγ (#1657, #1660, and #25967), followed by treatment with CSF-1 and IL-6 for 10 days. Cells were lysed and subjected to immunoblot analysis. f Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6. At different time points post-treatment, cells were lyzed and subjected to immunoblot analysis. Band density was quantified. g Mouse BM cells were pretreated with or without rapamycin, followed by incubation with CSF-1 and IL-6. Cell viability was determined ( n = 3 mice, mean ± SEM)
    Figure Legend Snippet: PPARγ induces HIF-2α and arginase-1 expression in macrophages. a , b Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 5 days. a Cell lysates were immunoblotted. b mRNA was extracted and subjected to quantitative RT-PCR analysis. Results were normalized with GAPDH level and expressed as folds of control ( n = 3, mean ± SEM). P value was determined by Student’s t test. c , d Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6 for 3 days. c Nuclei protein was incubated with biotin-labeled synthetic DNAs that encode control scrambled or HIF-2α promoter sequence, followed by immunoprecipitation with streptavidin-conjugated beads. Precipitants and nuclei protein were immunoblotted. d Nuclei extracts were subjected to chromatin immunoprecipitation (ChIP) analysis. Immunoprecipitants with control IgG or anti-PPARγ antibody were analyzed by PCR and electrophoresis (upper) or by quantitative PCR (bottom, n = 3, mean ± SD). e Mouse BM-derived macrophages were transduced with lentivirus that expresses shRNAs targeting control scrambled sequence and PPARγ (#1657, #1660, and #25967), followed by treatment with CSF-1 and IL-6 for 10 days. Cells were lysed and subjected to immunoblot analysis. f Mouse BM-derived macrophages were treated with or without CSF-1 and IL-6. At different time points post-treatment, cells were lyzed and subjected to immunoblot analysis. Band density was quantified. g Mouse BM cells were pretreated with or without rapamycin, followed by incubation with CSF-1 and IL-6. Cell viability was determined ( n = 3 mice, mean ± SEM)

    Techniques Used: Expressing, Derivative Assay, Quantitative RT-PCR, Incubation, Labeling, Sequencing, Immunoprecipitation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Electrophoresis, Real-time Polymerase Chain Reaction, Transduction, Mouse Assay

    ECs are a major source for IL-6 expression in GBM. a – c Cdh5 - Cre ERT2 ; Il6 fl/fl mice were generated by crossing Cdh5 - Cre ERT2 mice with Il6 fl/fl mice. Mice (2 weeks old) were injected with tamoxifen for consecutive 5 days to induce EC-specific IL-6 knockout. a Schematic approach. b , c ECs were isolated from mouse aortas. b Brain tissue and ECs were subjected to immunoblot analysis. Band density was quantified. c mRNA was extracted and subjected to quantitative RT-PCR analysis. Results were normalized with GAPDH level and expressed as folds of control ( n = 3, mean ± SEM). d , e The primary GBM in Ntv-a ; Ink4a-Arf −/− ; Pten −/− ; LSL-Luc donor mice was induced by RCAS-mediated somatic gene transfer. Recipient mice were Cdh5 - Cre ERT2 ; Il6 fl/fl mice. d Schematic approach. e Normal brain and tumor tissues were homogenized. Tissue lysates were immunoblotted. Band density was quantified
    Figure Legend Snippet: ECs are a major source for IL-6 expression in GBM. a – c Cdh5 - Cre ERT2 ; Il6 fl/fl mice were generated by crossing Cdh5 - Cre ERT2 mice with Il6 fl/fl mice. Mice (2 weeks old) were injected with tamoxifen for consecutive 5 days to induce EC-specific IL-6 knockout. a Schematic approach. b , c ECs were isolated from mouse aortas. b Brain tissue and ECs were subjected to immunoblot analysis. Band density was quantified. c mRNA was extracted and subjected to quantitative RT-PCR analysis. Results were normalized with GAPDH level and expressed as folds of control ( n = 3, mean ± SEM). d , e The primary GBM in Ntv-a ; Ink4a-Arf −/− ; Pten −/− ; LSL-Luc donor mice was induced by RCAS-mediated somatic gene transfer. Recipient mice were Cdh5 - Cre ERT2 ; Il6 fl/fl mice. d Schematic approach. e Normal brain and tumor tissues were homogenized. Tissue lysates were immunoblotted. Band density was quantified

    Techniques Used: Expressing, Mouse Assay, Generated, Injection, Knock-Out, Isolation, Quantitative RT-PCR

    IL-6 is critical for EC-induced macrophage alternative activation. a , b Mouse microvascular brain ECs were pretreated with the glioma-CM for 24 h. Mouse BM-derived macrophages were co-cultured with pretreated ECs for 5 days in the presence of control IgG, anti-CSF-1 antibody, or anti-IL-6 antibody or both antibodies. The cells were stained with anti-CD11b, anti-CD86, anti-CD206 antibodies, and analyzed by flow cytometry. a Representative sorting for CD206 expression in CD11b + cells. b Quantified data in sorted CD11b + cells ( n = 3–5, mean ± SEM). c – e Mouse BM-derived macrophages were treated with IL-6 and CSF-1 for 5 days. c , d The cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, and analyzed by flow cytometry. c Representative sorting for CD206 and CD86 expression in CD11b + cells. d quantified data in sorted CD11b + cells ( n = 3, mean ± SEM). e Cell lysates were immunoblotted
    Figure Legend Snippet: IL-6 is critical for EC-induced macrophage alternative activation. a , b Mouse microvascular brain ECs were pretreated with the glioma-CM for 24 h. Mouse BM-derived macrophages were co-cultured with pretreated ECs for 5 days in the presence of control IgG, anti-CSF-1 antibody, or anti-IL-6 antibody or both antibodies. The cells were stained with anti-CD11b, anti-CD86, anti-CD206 antibodies, and analyzed by flow cytometry. a Representative sorting for CD206 expression in CD11b + cells. b Quantified data in sorted CD11b + cells ( n = 3–5, mean ± SEM). c – e Mouse BM-derived macrophages were treated with IL-6 and CSF-1 for 5 days. c , d The cells were stained with anti-CD11b, anti-CD86, and anti-CD206 antibodies, and analyzed by flow cytometry. c Representative sorting for CD206 and CD86 expression in CD11b + cells. d quantified data in sorted CD11b + cells ( n = 3, mean ± SEM). e Cell lysates were immunoblotted

    Techniques Used: Activation Assay, Derivative Assay, Cell Culture, Staining, Flow Cytometry, Cytometry, Expressing

    GBM ECs express IL-6. a Human brain ECs were treated with glioma-CM for 24 h, and cell lysates were subjected to multiplex cytokine array analysis. Left, a representative blot. Right, quantified dot intensity of most significantly changed cytokines. b Human microvascular brain ECs were treated with glioma-CM that were harvested from different human glioma cells. Cell lysates were immunoblotted. c Human microvascular brain ECs and tumor-associated ECs isolated from different GBM patients were subjected to immunoblot analysis. d Mouse GBM was induced by orthotopic injection of GL26 glioma cells into wild-type mouse. The brain sections that include normal brains and tumors were stained with anti-CD31, anti-IL-6, and anti-CSF-1 antibodies. Representative immunofluorescence images are shown. Right, enlarged area in normal and tumor tissues. Bar represents 50 μm. Zoom-in factor: 4
    Figure Legend Snippet: GBM ECs express IL-6. a Human brain ECs were treated with glioma-CM for 24 h, and cell lysates were subjected to multiplex cytokine array analysis. Left, a representative blot. Right, quantified dot intensity of most significantly changed cytokines. b Human microvascular brain ECs were treated with glioma-CM that were harvested from different human glioma cells. Cell lysates were immunoblotted. c Human microvascular brain ECs and tumor-associated ECs isolated from different GBM patients were subjected to immunoblot analysis. d Mouse GBM was induced by orthotopic injection of GL26 glioma cells into wild-type mouse. The brain sections that include normal brains and tumors were stained with anti-CD31, anti-IL-6, and anti-CSF-1 antibodies. Representative immunofluorescence images are shown. Right, enlarged area in normal and tumor tissues. Bar represents 50 μm. Zoom-in factor: 4

    Techniques Used: Multiplex Assay, Isolation, Injection, Staining, Immunofluorescence

    19) Product Images from "Dopaminergic Stimulation of Myeloid Antigen-Presenting Cells Attenuates Signal Transducer and Activator of Transcription 3-Activation Favouring the Development of Experimental Autoimmune Encephalomyelitis"

    Article Title: Dopaminergic Stimulation of Myeloid Antigen-Presenting Cells Attenuates Signal Transducer and Activator of Transcription 3-Activation Favouring the Development of Experimental Autoimmune Encephalomyelitis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00571

    Signal transducer and activator of transcription 3 (STAT3) phosphorylation is decreased by dopamine in human monocytes. Fresh blood samples obtained from healthy donors were unstimulated or pre-incubated with dopamine (DA) in the presence or absence of interleukin (IL)-6 for 15 min. Afterwards, the extent of phosphorylated STAT3 (pSTAT3) was evaluated by intracellular immunostaining in the CD14 + population and analysed by flow cytometry using the gating strategy indicated in Figure S7 in Supplementary Material. (A) Representative histograms for pSTAT3 in peripheral blood CD14 + cells unstimulated (black lines) or treated with 1 µM DA (green lines) in the absence (left panel) or in the presence of IL-6 (right panel). The extent of pSTAT3 was also determined in peripheral blood CD14 + cells stimulated with IL-6 alone (red line). (B) The extent of pSTAT3 in unstimulated (white symbols) or IL-6 treated (black symbols) CD14 + in the presence of increasing DA concentrations was quantified as the mean fluorescence intensity (MFI) associated with pSTAT3-immunostaining (left panel) or as the ratio of the pSTAT3-associated MFI of stimulated cells to the pSTAT3-associated MFI of unstimulated cells (in-fold, right panel). Values represent mean ± SEM, n = 5. * p
    Figure Legend Snippet: Signal transducer and activator of transcription 3 (STAT3) phosphorylation is decreased by dopamine in human monocytes. Fresh blood samples obtained from healthy donors were unstimulated or pre-incubated with dopamine (DA) in the presence or absence of interleukin (IL)-6 for 15 min. Afterwards, the extent of phosphorylated STAT3 (pSTAT3) was evaluated by intracellular immunostaining in the CD14 + population and analysed by flow cytometry using the gating strategy indicated in Figure S7 in Supplementary Material. (A) Representative histograms for pSTAT3 in peripheral blood CD14 + cells unstimulated (black lines) or treated with 1 µM DA (green lines) in the absence (left panel) or in the presence of IL-6 (right panel). The extent of pSTAT3 was also determined in peripheral blood CD14 + cells stimulated with IL-6 alone (red line). (B) The extent of pSTAT3 in unstimulated (white symbols) or IL-6 treated (black symbols) CD14 + in the presence of increasing DA concentrations was quantified as the mean fluorescence intensity (MFI) associated with pSTAT3-immunostaining (left panel) or as the ratio of the pSTAT3-associated MFI of stimulated cells to the pSTAT3-associated MFI of unstimulated cells (in-fold, right panel). Values represent mean ± SEM, n = 5. * p

    Techniques Used: Incubation, Immunostaining, Flow Cytometry, Cytometry, Fluorescence

    20) Product Images from "Soluble transforming growth factor beta-1 enhances murine mast cell release of Interleukin 6 in IgE-independent and Interleukin 13 in IgE-dependent settings in vitro"

    Article Title: Soluble transforming growth factor beta-1 enhances murine mast cell release of Interleukin 6 in IgE-independent and Interleukin 13 in IgE-dependent settings in vitro

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0207704

    IL-6 and IL-13 secretion by mast cells based on treatment. Raw cytokine production for IL-6 (a) and IL-13 (c) normalized as secretion per 10 6 cells. (b) IL-6 production in resting (no IgE-crosslink) BMMCs only (emphasis from 2a above). * in (a) and (b) indicates significant difference from no IgE-crosslink, no TGF-β1, no SCF control. * in (c) indicates significant difference from IgE-crosslinked, no TGF-β1, no SCF control; † indicates significant difference from IgE-crosslinked, no TGF-β1, SCF-treated control. Significance was noted at p
    Figure Legend Snippet: IL-6 and IL-13 secretion by mast cells based on treatment. Raw cytokine production for IL-6 (a) and IL-13 (c) normalized as secretion per 10 6 cells. (b) IL-6 production in resting (no IgE-crosslink) BMMCs only (emphasis from 2a above). * in (a) and (b) indicates significant difference from no IgE-crosslink, no TGF-β1, no SCF control. * in (c) indicates significant difference from IgE-crosslinked, no TGF-β1, no SCF control; † indicates significant difference from IgE-crosslinked, no TGF-β1, SCF-treated control. Significance was noted at p

    Techniques Used:

    IgE-stimulated LAMP-1 translocation and early phase IL-6 production. Representative cytograms of (a) surface LAMP-1 expression (10 minute activation) and (c) intracellular IL-6 (90 minute activation) analyzed via flow cytometry. (b) Fold change of mean fluorescence intensity compared to baseline (no TGF-β1, no IgE crosslink) for LAMP-1 expression; * indicates significant difference from baseline, † indicates significant difference compared to TGF-β1-treated, IgE-crosslinked groups. (d) Representative histogram of intracellular IL-6. (e) Mean fluorescence intensity for intracellular IL-6. Significance was noted at p
    Figure Legend Snippet: IgE-stimulated LAMP-1 translocation and early phase IL-6 production. Representative cytograms of (a) surface LAMP-1 expression (10 minute activation) and (c) intracellular IL-6 (90 minute activation) analyzed via flow cytometry. (b) Fold change of mean fluorescence intensity compared to baseline (no TGF-β1, no IgE crosslink) for LAMP-1 expression; * indicates significant difference from baseline, † indicates significant difference compared to TGF-β1-treated, IgE-crosslinked groups. (d) Representative histogram of intracellular IL-6. (e) Mean fluorescence intensity for intracellular IL-6. Significance was noted at p

    Techniques Used: Translocation Assay, Expressing, Activation Assay, Flow Cytometry, Cytometry, Fluorescence

    IL-33-stimulated early phase IL-6 production. (a) Representative cytograms of intracellular IL-6 (90 minute activation) analyzed via flow cytometry. (b) Representative histogram of intracellular IL-6. (c) Mean fluorescence intensity for intracellular IL-6; * indicates significant difference (p
    Figure Legend Snippet: IL-33-stimulated early phase IL-6 production. (a) Representative cytograms of intracellular IL-6 (90 minute activation) analyzed via flow cytometry. (b) Representative histogram of intracellular IL-6. (c) Mean fluorescence intensity for intracellular IL-6; * indicates significant difference (p

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

    21) Product Images from "Soluble transforming growth factor beta-1 enhances murine mast cell release of Interleukin 6 in IgE-independent and Interleukin 13 in IgE-dependent settings in vitro"

    Article Title: Soluble transforming growth factor beta-1 enhances murine mast cell release of Interleukin 6 in IgE-independent and Interleukin 13 in IgE-dependent settings in vitro

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0207704

    IL-6 and IL-13 secretion by mast cells based on treatment. Raw cytokine production for IL-6 (a) and IL-13 (c) normalized as secretion per 10 6 cells. (b) IL-6 production in resting (no IgE-crosslink) BMMCs only (emphasis from 2a above). * in (a) and (b) indicates significant difference from no IgE-crosslink, no TGF-β1, no SCF control. * in (c) indicates significant difference from IgE-crosslinked, no TGF-β1, no SCF control; † indicates significant difference from IgE-crosslinked, no TGF-β1, SCF-treated control. Significance was noted at p
    Figure Legend Snippet: IL-6 and IL-13 secretion by mast cells based on treatment. Raw cytokine production for IL-6 (a) and IL-13 (c) normalized as secretion per 10 6 cells. (b) IL-6 production in resting (no IgE-crosslink) BMMCs only (emphasis from 2a above). * in (a) and (b) indicates significant difference from no IgE-crosslink, no TGF-β1, no SCF control. * in (c) indicates significant difference from IgE-crosslinked, no TGF-β1, no SCF control; † indicates significant difference from IgE-crosslinked, no TGF-β1, SCF-treated control. Significance was noted at p

    Techniques Used:

    IgE-stimulated LAMP-1 translocation and early phase IL-6 production. Representative cytograms of (a) surface LAMP-1 expression (10 minute activation) and (c) intracellular IL-6 (90 minute activation) analyzed via flow cytometry. (b) Fold change of mean fluorescence intensity compared to baseline (no TGF-β1, no IgE crosslink) for LAMP-1 expression; * indicates significant difference from baseline, † indicates significant difference compared to TGF-β1-treated, IgE-crosslinked groups. (d) Representative histogram of intracellular IL-6. (e) Mean fluorescence intensity for intracellular IL-6. Significance was noted at p
    Figure Legend Snippet: IgE-stimulated LAMP-1 translocation and early phase IL-6 production. Representative cytograms of (a) surface LAMP-1 expression (10 minute activation) and (c) intracellular IL-6 (90 minute activation) analyzed via flow cytometry. (b) Fold change of mean fluorescence intensity compared to baseline (no TGF-β1, no IgE crosslink) for LAMP-1 expression; * indicates significant difference from baseline, † indicates significant difference compared to TGF-β1-treated, IgE-crosslinked groups. (d) Representative histogram of intracellular IL-6. (e) Mean fluorescence intensity for intracellular IL-6. Significance was noted at p

    Techniques Used: Translocation Assay, Expressing, Activation Assay, Flow Cytometry, Cytometry, Fluorescence

    IL-33-stimulated early phase IL-6 production. (a) Representative cytograms of intracellular IL-6 (90 minute activation) analyzed via flow cytometry. (b) Representative histogram of intracellular IL-6. (c) Mean fluorescence intensity for intracellular IL-6; * indicates significant difference (p
    Figure Legend Snippet: IL-33-stimulated early phase IL-6 production. (a) Representative cytograms of intracellular IL-6 (90 minute activation) analyzed via flow cytometry. (b) Representative histogram of intracellular IL-6. (c) Mean fluorescence intensity for intracellular IL-6; * indicates significant difference (p

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

    22) Product Images from "NMI and IFP35 serve as proinflammatory DAMPs during cellular infection and injury"

    Article Title: NMI and IFP35 serve as proinflammatory DAMPs during cellular infection and injury

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00930-9

    NMI stimulates macrophages through the TLR4 pathway. a Western blot analysis of mNMI in the BMDM cell lysate after 1 h incubation with recombinant mNMI. The BMDM cells were isolated from Nmi −/− mice and pretreated with macrophage colony-stimulating factor (MCSF). b , c TNF and IL-6 released by BMDMs from WT (C57BL/6) mice, pretreated with bafilomycin A1 (10 nM), TAK-242 (100 nM) or dimethyl sulphoxide (DMSO) for 2 h and stimulated with mNMI (5 μg ml −1 ) or LPS (100 ng ml −1 ) for 8 h. d – g TNF and IL-6 levels in the supernatants of BMDMs from WT, Tlr4 −/− and Tlr2 −/− mice were analyzed using ELISA 4 h post activation by different stimulus. h After incubation with mNMI-GFP for 1 h, the percentage of GFP labeled CD11b + F4/80 + cells was determined by Flow cytometric analysis. The cells were isolated from spleen in WT or Tlr4 −/− mice. i NMI in the human THP1 cell (ATCC TIB-202™) lysates interacts with hTLR4. Ni-NTA beads coupled with 2 μg His-hTLR4 and/or His-hMD2 fusion proteins were used as bait. j The luciferase activity of HEK293T cells (ATCC CRL-11268™) are shown after stimulated with 5 μg ml −1 mNMI for 4 h (in the presence or absence of 25 μg ml −1 polymyxin B (PMB)). The cells were pre-transfected with mTLR4-MD2-CD14 and NF-κB promoter with luciferase activity. 100 ng ml −1 LPS was administrated as positive control. In b – e and g , error bars indicate ± s.e.m. from 3 biological replicates. Significance was tested by one-way ANOVA followed by Student–Newman–Keuls test. ** P
    Figure Legend Snippet: NMI stimulates macrophages through the TLR4 pathway. a Western blot analysis of mNMI in the BMDM cell lysate after 1 h incubation with recombinant mNMI. The BMDM cells were isolated from Nmi −/− mice and pretreated with macrophage colony-stimulating factor (MCSF). b , c TNF and IL-6 released by BMDMs from WT (C57BL/6) mice, pretreated with bafilomycin A1 (10 nM), TAK-242 (100 nM) or dimethyl sulphoxide (DMSO) for 2 h and stimulated with mNMI (5 μg ml −1 ) or LPS (100 ng ml −1 ) for 8 h. d – g TNF and IL-6 levels in the supernatants of BMDMs from WT, Tlr4 −/− and Tlr2 −/− mice were analyzed using ELISA 4 h post activation by different stimulus. h After incubation with mNMI-GFP for 1 h, the percentage of GFP labeled CD11b + F4/80 + cells was determined by Flow cytometric analysis. The cells were isolated from spleen in WT or Tlr4 −/− mice. i NMI in the human THP1 cell (ATCC TIB-202™) lysates interacts with hTLR4. Ni-NTA beads coupled with 2 μg His-hTLR4 and/or His-hMD2 fusion proteins were used as bait. j The luciferase activity of HEK293T cells (ATCC CRL-11268™) are shown after stimulated with 5 μg ml −1 mNMI for 4 h (in the presence or absence of 25 μg ml −1 polymyxin B (PMB)). The cells were pre-transfected with mTLR4-MD2-CD14 and NF-κB promoter with luciferase activity. 100 ng ml −1 LPS was administrated as positive control. In b – e and g , error bars indicate ± s.e.m. from 3 biological replicates. Significance was tested by one-way ANOVA followed by Student–Newman–Keuls test. ** P

    Techniques Used: Western Blot, Incubation, Recombinant, Isolation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Labeling, Flow Cytometry, Luciferase, Activity Assay, Transfection, Positive Control

    Recombinant NMI protein induces TNF release and NF-κB activation. a , b Tumor necrosis factor (TNF) and interleukin 6 (IL-6) in the sera of mice ( n = 5) were determined by ELISA, 6 or 24 h after intravenous injection of recombinant mNMI (10 mg kg −1 ) or PBS. c , d TNF and IL-6 released by mouse RAW264.7 cells (ATCC TIB-71™), 4 h post-stimulation with increasing concentrations of mNMI. e , f TNF and IL-6 released by RAW264.7 cells stimulated with 1 μg ml −1 mNMI. g TNF released by RAW264.7 cells stimulated with mNMI (5 μg ml −1 ) or LPS (100 ng ml −1 ) for 8 h, with (+) or without (−) pre-treatment of polymyxin B (PMB) (25 μg ml −1 ), heat (80 °C for 30 min) and trypsin (5 μg ml −1 , 37 °C overnight). h IκB, p-IκB, NF-κB (p50 and p65), phosphorated p65 at Ser536 (p-p65 (S536)) in the cytoplasm and NF-κB (p50 and p65) protein in nucleus extracts of RAW264.7 cells were assessed by immunoblotting, after the cells were treated with 5 μg ml −1 recombinant mNMI protein. β-actin in the cytoplasm and LaminB in the nucleus were used as control. LaminB in the cytoplasm was detected to ensure that there was no contamination during the fractionation. Error bars in c – g indicate ± s.e.m. from three biological replicates. Significance was tested by unpaired Student’s t -test test. * P
    Figure Legend Snippet: Recombinant NMI protein induces TNF release and NF-κB activation. a , b Tumor necrosis factor (TNF) and interleukin 6 (IL-6) in the sera of mice ( n = 5) were determined by ELISA, 6 or 24 h after intravenous injection of recombinant mNMI (10 mg kg −1 ) or PBS. c , d TNF and IL-6 released by mouse RAW264.7 cells (ATCC TIB-71™), 4 h post-stimulation with increasing concentrations of mNMI. e , f TNF and IL-6 released by RAW264.7 cells stimulated with 1 μg ml −1 mNMI. g TNF released by RAW264.7 cells stimulated with mNMI (5 μg ml −1 ) or LPS (100 ng ml −1 ) for 8 h, with (+) or without (−) pre-treatment of polymyxin B (PMB) (25 μg ml −1 ), heat (80 °C for 30 min) and trypsin (5 μg ml −1 , 37 °C overnight). h IκB, p-IκB, NF-κB (p50 and p65), phosphorated p65 at Ser536 (p-p65 (S536)) in the cytoplasm and NF-κB (p50 and p65) protein in nucleus extracts of RAW264.7 cells were assessed by immunoblotting, after the cells were treated with 5 μg ml −1 recombinant mNMI protein. β-actin in the cytoplasm and LaminB in the nucleus were used as control. LaminB in the cytoplasm was detected to ensure that there was no contamination during the fractionation. Error bars in c – g indicate ± s.e.m. from three biological replicates. Significance was tested by unpaired Student’s t -test test. * P

    Techniques Used: Recombinant, Activation Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection, Fractionation

    23) Product Images from "Ultrasensitive Label-Free Sensing of IL-6 Based on PASE Functionalized Carbon Nanotube Micro-Arrays with RNA-Aptamers as Molecular Recognition Elements"

    Article Title: Ultrasensitive Label-Free Sensing of IL-6 Based on PASE Functionalized Carbon Nanotube Micro-Arrays with RNA-Aptamers as Molecular Recognition Elements

    Journal: Biosensors

    doi: 10.3390/bios7020017

    Schematic showing the reaction of 1-Pyrenebutanoic Acid Succinimidyl Ester (PASE) onto IL-6 aptamer forming the IL-6 aptamer PASE complex. Three groups of devices were prepared, Carbon Nanotube (CNT) device functionalized with ( B ) IL-6 aptamer PASE complex, positive control; ( C ) IgG, negative control; and ( D ) tween-20 blocking agent, negative control.
    Figure Legend Snippet: Schematic showing the reaction of 1-Pyrenebutanoic Acid Succinimidyl Ester (PASE) onto IL-6 aptamer forming the IL-6 aptamer PASE complex. Three groups of devices were prepared, Carbon Nanotube (CNT) device functionalized with ( B ) IL-6 aptamer PASE complex, positive control; ( C ) IgG, negative control; and ( D ) tween-20 blocking agent, negative control.

    Techniques Used: Positive Control, Negative Control, Blocking Assay

    Before and after I DS -V GS characterization of IL-6 aptamer functionalized CNT-biosensor with ( A ) blank 1 mM MgCL 2 1× PBS samples; ( B ) 20 nM BSA; and ( C ) 1 pg and 1 ng IL-6 protein injection. I DS -V GS response of tween-20 blocked CNT-biosensor to BSA ( D ); and IL-6 protein ( E ).
    Figure Legend Snippet: Before and after I DS -V GS characterization of IL-6 aptamer functionalized CNT-biosensor with ( A ) blank 1 mM MgCL 2 1× PBS samples; ( B ) 20 nM BSA; and ( C ) 1 pg and 1 ng IL-6 protein injection. I DS -V GS response of tween-20 blocked CNT-biosensor to BSA ( D ); and IL-6 protein ( E ).

    Techniques Used: Injection

    ( A ) Time dependence of normalized source-drain current of the CNT-biosensor at the source-drain bias of 0.1 V and at gate bias of 0 V after the introduction of IL-6 target protein at various concentrations onto the IL-6 aptamer functionalized CNT-biosensor. Arrows indicate the points of IL-6 target protein injection; ( B ) The Sensitivity of IL-6 aptamer CNT-biosensor as a function of IL-6 protein concentration (1 pg to 10 ng /mL). The inner box demonstrates the data in logarithmic scale.
    Figure Legend Snippet: ( A ) Time dependence of normalized source-drain current of the CNT-biosensor at the source-drain bias of 0.1 V and at gate bias of 0 V after the introduction of IL-6 target protein at various concentrations onto the IL-6 aptamer functionalized CNT-biosensor. Arrows indicate the points of IL-6 target protein injection; ( B ) The Sensitivity of IL-6 aptamer CNT-biosensor as a function of IL-6 protein concentration (1 pg to 10 ng /mL). The inner box demonstrates the data in logarithmic scale.

    Techniques Used: Injection, Protein Concentration

    Atomic Force Microscope (AFM) image showing a phase image ( A ); and Z -Axis image ( B ), of bare CNT ( a , b ), functionalized CNT with IL-6 aptamer ( c , d ), and IL-6 protein binding to aptamer/CNT structure ( e , f ). Images were obtained using dry tapping mode (Nanosurf NaioAFM system). Scale bar = 100 nm.
    Figure Legend Snippet: Atomic Force Microscope (AFM) image showing a phase image ( A ); and Z -Axis image ( B ), of bare CNT ( a , b ), functionalized CNT with IL-6 aptamer ( c , d ), and IL-6 protein binding to aptamer/CNT structure ( e , f ). Images were obtained using dry tapping mode (Nanosurf NaioAFM system). Scale bar = 100 nm.

    Techniques Used: Microscopy, Protein Binding

    Sensor response of the CNT-biosensor at source-drain bias of 0.1 V and at the gate bias of 0 V. ( A ) indicates the response after the introduction of 20 nM BSA (red line), 1 mM MgCL 2 1× PBS (gray line), 1 pg IL-6 target protein (blue line) to functionalized CNT-biosensor and ( B ) 1 pg IL-6 target protein to bare unfunctionalized CNT-biosensor (green line); ( C ) Response of tween-20 blocked CNT-biosensor to IL-6 protein (blue line), and BSA (red line). Arrow indicates the point of sample injection.
    Figure Legend Snippet: Sensor response of the CNT-biosensor at source-drain bias of 0.1 V and at the gate bias of 0 V. ( A ) indicates the response after the introduction of 20 nM BSA (red line), 1 mM MgCL 2 1× PBS (gray line), 1 pg IL-6 target protein (blue line) to functionalized CNT-biosensor and ( B ) 1 pg IL-6 target protein to bare unfunctionalized CNT-biosensor (green line); ( C ) Response of tween-20 blocked CNT-biosensor to IL-6 protein (blue line), and BSA (red line). Arrow indicates the point of sample injection.

    Techniques Used: Injection

    Blood experiment results. Response of the CNT-biosensor at source-drain bias of 0.1 V and at the gate bias of 0 V. IL-6 spiked blood samples were tested on two different CNT-biosensor surfaces, IL-6 aptamer ( A ), and IgG ( B ) functionalized surfaces. The response of the IL-6 aptamer functionalized devices to control buffer, 1 mM MgCl 2 1× PBS, is presented in each panel as a reference point (black marks). ( A ) Shows the respond of the IL-6 aptamer functionalized device to IL-6 protein spiked blood (10 pg/mL), ~8–13% drop in the current (diagnostic zone); ( B ) Presents the response to IgG functionalized CNT-biosensor, negative control surface, to IL-6 spiked blood showing no significant change in signal. Arrows indicate the points of IL-6 spiked blood injection, at 60 s.
    Figure Legend Snippet: Blood experiment results. Response of the CNT-biosensor at source-drain bias of 0.1 V and at the gate bias of 0 V. IL-6 spiked blood samples were tested on two different CNT-biosensor surfaces, IL-6 aptamer ( A ), and IgG ( B ) functionalized surfaces. The response of the IL-6 aptamer functionalized devices to control buffer, 1 mM MgCl 2 1× PBS, is presented in each panel as a reference point (black marks). ( A ) Shows the respond of the IL-6 aptamer functionalized device to IL-6 protein spiked blood (10 pg/mL), ~8–13% drop in the current (diagnostic zone); ( B ) Presents the response to IgG functionalized CNT-biosensor, negative control surface, to IL-6 spiked blood showing no significant change in signal. Arrows indicate the points of IL-6 spiked blood injection, at 60 s.

    Techniques Used: Diagnostic Assay, Negative Control, Injection

    ( A ) Time dependence of normalized source-drain current of the CNT-biosensor at the source-drain bias of 0.1 V and at gate bias of 0 V after the introduction of IL-6 spiked blood sample at various concentrations onto the IL-6 aptamer functionalized CNT-biosensor. Arrows indicate the points of IL-6 spiked blood injection; ( B ) The Sensitivity of IL-6 aptamer CNT-biosensor as a function of IL-6 protein concentration in blood sample (10 pg to 1 ng /mL) in logarithmic scale. Diagnostic gray zone for large tumor mass at ~12–40 pg/mL is highlighted in yellow which is higher than our sensor’s lower limit sensing threshold at 10 pg/mL.
    Figure Legend Snippet: ( A ) Time dependence of normalized source-drain current of the CNT-biosensor at the source-drain bias of 0.1 V and at gate bias of 0 V after the introduction of IL-6 spiked blood sample at various concentrations onto the IL-6 aptamer functionalized CNT-biosensor. Arrows indicate the points of IL-6 spiked blood injection; ( B ) The Sensitivity of IL-6 aptamer CNT-biosensor as a function of IL-6 protein concentration in blood sample (10 pg to 1 ng /mL) in logarithmic scale. Diagnostic gray zone for large tumor mass at ~12–40 pg/mL is highlighted in yellow which is higher than our sensor’s lower limit sensing threshold at 10 pg/mL.

    Techniques Used: Injection, Protein Concentration, Diagnostic Assay

    24) Product Images from "Ethyl acetate extract from Asparagus cochinchinensis exerts anti-inflammatory effects in LPS-stimulated RAW264.7 macrophage cells by regulating COX-2/iNOS, inflammatory cytokine expression, MAP kinase pathways, the cell cycle and anti-oxidant activity"

    Article Title: Ethyl acetate extract from Asparagus cochinchinensis exerts anti-inflammatory effects in LPS-stimulated RAW264.7 macrophage cells by regulating COX-2/iNOS, inflammatory cytokine expression, MAP kinase pathways, the cell cycle and anti-oxidant activity

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2017.6166

    Analysis of pro- and anti-inflammatory cytokine expression. Following treatment of RAW264.7 cells with LPS and 0 (vehicle), 100 or 200 µg/ml of EaEAC, (A) IL-1β, TNF-α, IL-6 and IL-10 mRNA levels were assessed by semi-quantitative reverse transcription polymerase chain reaction using transcript-specific primers, and (B) IL-6 concentration was detected using an enzyme-linked immunosorbent assay kit with a minimum detection threshold of 9.3 pg/ml. Values are presented as the mean ± standard deviation of three replicates. *P
    Figure Legend Snippet: Analysis of pro- and anti-inflammatory cytokine expression. Following treatment of RAW264.7 cells with LPS and 0 (vehicle), 100 or 200 µg/ml of EaEAC, (A) IL-1β, TNF-α, IL-6 and IL-10 mRNA levels were assessed by semi-quantitative reverse transcription polymerase chain reaction using transcript-specific primers, and (B) IL-6 concentration was detected using an enzyme-linked immunosorbent assay kit with a minimum detection threshold of 9.3 pg/ml. Values are presented as the mean ± standard deviation of three replicates. *P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

    25) Product Images from "Myosin 1F Regulates M1-Polarization by Stimulating Intercellular Adhesion in Macrophages"

    Article Title: Myosin 1F Regulates M1-Polarization by Stimulating Intercellular Adhesion in Macrophages

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.03118

    Myo1F is required to stimulate a pro-inflammatory phenotype in macrophages. (A) iNOS expression was analyzed by RT-PCR in WT and Myo1F deficient bone marrow-derived macrophages differentiated into M1 phenotype. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation. n = 3. Results are given as mean values ± SD. *** p = 0.0005. (B) iNOS and pro-IL-1β were analyzed by western blotting cell lysates of WT and Myo1F deficient bone marrow-derived macrophages differentiated into M0, M1, or M2 phenotype. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation and M2 was obtained by IL-4 (20 ng/ml) exposition. GAPDH was used as loading control. n = 3. (C) Expression of CD80 and CD86 was analyzed by flow cytometry in WT and Myo1F deficient bone marrow-derived macrophages differentiated into M1 phenotype and expressed as Mean Florescence Intensity. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation. n = 3. Results are given as mean values ± SD. * p = 0.05, ** p = 0.01. (D) NLRP3, Caspase 1, proIL-1β, and IL-1β were analyzed by western blotting cell lysates of WT and Myo1F deficient bone marrow-derived macrophages differentiated into M1 or M2 phenotype. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation and M2 was obtained by IL-4 (20 ng/ml) exposition. GAPDH was used as loading control. n = 3. Densitometric analyses obtained from those results are shown as graphs. ** p = 0.01. (E) Secretion of IL-1β and IL-6 in supernatants of WT and Myo1F −/− derived BMM was performed by ELISA assay. LPS and IFN-γ/LPS stimulation was carried out for 5 h. Graphs are derived from independent experiments carry out by duplicate. n = 3. Results are given as mean values ± SEM. * p = 0.05, ** p = 0.01, *** p = 0.0005. (F) Western blotting for NLRP3, Caspase 1 and IL-1β in J774 cells overexpressing Myo1F or GFP under homeostatic conditions. GAPDH was used as loading control. n = 3. Densitometric analysis obtained from IL-1β is shown as graph. ** p = 0.01. (G) Secretion of IL-1β in supernatants of J774 cells overexpressing Myo1F or GFP was performed by ELISA assay. Graphs are derived from independent experiments carry out by duplicate. n = 3. Results are given as mean values ± SEM. * p = 0.05. (H) Quantification of IL-1β release was analyzed in colonic explants of WT and Myo1F deficient mice stimulated with IFN-γ/LPS. Inflammatory stimulus was administered for 5 h. IL-1β was quantified by ELISA. Graphs are derived from three independent experiments. n = 6. Results are given as mean values ± SEM. * p = 0.05.
    Figure Legend Snippet: Myo1F is required to stimulate a pro-inflammatory phenotype in macrophages. (A) iNOS expression was analyzed by RT-PCR in WT and Myo1F deficient bone marrow-derived macrophages differentiated into M1 phenotype. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation. n = 3. Results are given as mean values ± SD. *** p = 0.0005. (B) iNOS and pro-IL-1β were analyzed by western blotting cell lysates of WT and Myo1F deficient bone marrow-derived macrophages differentiated into M0, M1, or M2 phenotype. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation and M2 was obtained by IL-4 (20 ng/ml) exposition. GAPDH was used as loading control. n = 3. (C) Expression of CD80 and CD86 was analyzed by flow cytometry in WT and Myo1F deficient bone marrow-derived macrophages differentiated into M1 phenotype and expressed as Mean Florescence Intensity. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation. n = 3. Results are given as mean values ± SD. * p = 0.05, ** p = 0.01. (D) NLRP3, Caspase 1, proIL-1β, and IL-1β were analyzed by western blotting cell lysates of WT and Myo1F deficient bone marrow-derived macrophages differentiated into M1 or M2 phenotype. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation and M2 was obtained by IL-4 (20 ng/ml) exposition. GAPDH was used as loading control. n = 3. Densitometric analyses obtained from those results are shown as graphs. ** p = 0.01. (E) Secretion of IL-1β and IL-6 in supernatants of WT and Myo1F −/− derived BMM was performed by ELISA assay. LPS and IFN-γ/LPS stimulation was carried out for 5 h. Graphs are derived from independent experiments carry out by duplicate. n = 3. Results are given as mean values ± SEM. * p = 0.05, ** p = 0.01, *** p = 0.0005. (F) Western blotting for NLRP3, Caspase 1 and IL-1β in J774 cells overexpressing Myo1F or GFP under homeostatic conditions. GAPDH was used as loading control. n = 3. Densitometric analysis obtained from IL-1β is shown as graph. ** p = 0.01. (G) Secretion of IL-1β in supernatants of J774 cells overexpressing Myo1F or GFP was performed by ELISA assay. Graphs are derived from independent experiments carry out by duplicate. n = 3. Results are given as mean values ± SEM. * p = 0.05. (H) Quantification of IL-1β release was analyzed in colonic explants of WT and Myo1F deficient mice stimulated with IFN-γ/LPS. Inflammatory stimulus was administered for 5 h. IL-1β was quantified by ELISA. Graphs are derived from three independent experiments. n = 6. Results are given as mean values ± SEM. * p = 0.05.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Western Blot, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Mouse Assay

    26) Product Images from "UDP-Induced Phagocytosis and ATP-Stimulated Chemotactic Migration Are Impaired in STIM1−/− Microglia In Vitro and In Vivo"

    Article Title: UDP-Induced Phagocytosis and ATP-Stimulated Chemotactic Migration Are Impaired in STIM1−/− Microglia In Vitro and In Vivo

    Journal: Mediators of Inflammation

    doi: 10.1155/2017/8158514

    Lipopolysaccharide-induced phagocytosis and cytokine secretion in wild-type and STIM1 − / − microglia. (a, b) Representative images of image-based phagocytosis measurement using phagocytic engulfment of FITC bead in WT and STIM1 − / − microglia. Summary of five independent phagocytosis measurements. Seven images were used for phagocytosis measurements and then depicted as a bar graph. ∗∗∗ p ≤ 0.0001 in LPS-induced fold-induction of WT; ∗ p ≤ 0.005 in WT mock, n = 7. (c, d) Comparisons of LPS-induced cytokine secretion activity: TNF- α secretion (c) and IL-6 secretion (d) were compared in WT and STIM1 − / − microglia. p ≤ 0.0001, n = 4, where n is independent ELISA experiment; ns, no significant difference from WT mock.
    Figure Legend Snippet: Lipopolysaccharide-induced phagocytosis and cytokine secretion in wild-type and STIM1 − / − microglia. (a, b) Representative images of image-based phagocytosis measurement using phagocytic engulfment of FITC bead in WT and STIM1 − / − microglia. Summary of five independent phagocytosis measurements. Seven images were used for phagocytosis measurements and then depicted as a bar graph. ∗∗∗ p ≤ 0.0001 in LPS-induced fold-induction of WT; ∗ p ≤ 0.005 in WT mock, n = 7. (c, d) Comparisons of LPS-induced cytokine secretion activity: TNF- α secretion (c) and IL-6 secretion (d) were compared in WT and STIM1 − / − microglia. p ≤ 0.0001, n = 4, where n is independent ELISA experiment; ns, no significant difference from WT mock.

    Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay

    27) Product Images from "Myosin 1F Regulates M1-Polarization by Stimulating Intercellular Adhesion in Macrophages"

    Article Title: Myosin 1F Regulates M1-Polarization by Stimulating Intercellular Adhesion in Macrophages

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.03118

    Myo1F is required to stimulate a pro-inflammatory phenotype in macrophages. (A) iNOS expression was analyzed by RT-PCR in WT and Myo1F deficient bone marrow-derived macrophages differentiated into M1 phenotype. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation. n = 3. Results are given as mean values ± SD. *** p = 0.0005. (B) iNOS and pro-IL-1β were analyzed by western blotting cell lysates of WT and Myo1F deficient bone marrow-derived macrophages differentiated into M0, M1, or M2 phenotype. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation and M2 was obtained by IL-4 (20 ng/ml) exposition. GAPDH was used as loading control. n = 3. (C) Expression of CD80 and CD86 was analyzed by flow cytometry in WT and Myo1F deficient bone marrow-derived macrophages differentiated into M1 phenotype and expressed as Mean Florescence Intensity. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation. n = 3. Results are given as mean values ± SD. * p = 0.05, ** p = 0.01. (D) NLRP3, Caspase 1, proIL-1β, and IL-1β were analyzed by western blotting cell lysates of WT and Myo1F deficient bone marrow-derived macrophages differentiated into M1 or M2 phenotype. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation and M2 was obtained by IL-4 (20 ng/ml) exposition. GAPDH was used as loading control. n = 3. Densitometric analyses obtained from those results are shown as graphs. ** p = 0.01. (E) Secretion of IL-1β and IL-6 in supernatants of WT and Myo1F −/− derived BMM was performed by ELISA assay. LPS and IFN-γ/LPS stimulation was carried out for 5 h. Graphs are derived from independent experiments carry out by duplicate. n = 3. Results are given as mean values ± SEM. * p = 0.05, ** p = 0.01, *** p = 0.0005. (F) Western blotting for NLRP3, Caspase 1 and IL-1β in J774 cells overexpressing Myo1F or GFP under homeostatic conditions. GAPDH was used as loading control. n = 3. Densitometric analysis obtained from IL-1β is shown as graph. ** p = 0.01. (G) Secretion of IL-1β in supernatants of J774 cells overexpressing Myo1F or GFP was performed by ELISA assay. Graphs are derived from independent experiments carry out by duplicate. n = 3. Results are given as mean values ± SEM. * p = 0.05. (H) Quantification of IL-1β release was analyzed in colonic explants of WT and Myo1F deficient mice stimulated with IFN-γ/LPS. Inflammatory stimulus was administered for 5 h. IL-1β was quantified by ELISA. Graphs are derived from three independent experiments. n = 6. Results are given as mean values ± SEM. * p = 0.05.
    Figure Legend Snippet: Myo1F is required to stimulate a pro-inflammatory phenotype in macrophages. (A) iNOS expression was analyzed by RT-PCR in WT and Myo1F deficient bone marrow-derived macrophages differentiated into M1 phenotype. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation. n = 3. Results are given as mean values ± SD. *** p = 0.0005. (B) iNOS and pro-IL-1β were analyzed by western blotting cell lysates of WT and Myo1F deficient bone marrow-derived macrophages differentiated into M0, M1, or M2 phenotype. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation and M2 was obtained by IL-4 (20 ng/ml) exposition. GAPDH was used as loading control. n = 3. (C) Expression of CD80 and CD86 was analyzed by flow cytometry in WT and Myo1F deficient bone marrow-derived macrophages differentiated into M1 phenotype and expressed as Mean Florescence Intensity. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation. n = 3. Results are given as mean values ± SD. * p = 0.05, ** p = 0.01. (D) NLRP3, Caspase 1, proIL-1β, and IL-1β were analyzed by western blotting cell lysates of WT and Myo1F deficient bone marrow-derived macrophages differentiated into M1 or M2 phenotype. M1 phenotype was induced by IFN-γ/LPS (20 ng/ml; 1 μg/ml) stimulation and M2 was obtained by IL-4 (20 ng/ml) exposition. GAPDH was used as loading control. n = 3. Densitometric analyses obtained from those results are shown as graphs. ** p = 0.01. (E) Secretion of IL-1β and IL-6 in supernatants of WT and Myo1F −/− derived BMM was performed by ELISA assay. LPS and IFN-γ/LPS stimulation was carried out for 5 h. Graphs are derived from independent experiments carry out by duplicate. n = 3. Results are given as mean values ± SEM. * p = 0.05, ** p = 0.01, *** p = 0.0005. (F) Western blotting for NLRP3, Caspase 1 and IL-1β in J774 cells overexpressing Myo1F or GFP under homeostatic conditions. GAPDH was used as loading control. n = 3. Densitometric analysis obtained from IL-1β is shown as graph. ** p = 0.01. (G) Secretion of IL-1β in supernatants of J774 cells overexpressing Myo1F or GFP was performed by ELISA assay. Graphs are derived from independent experiments carry out by duplicate. n = 3. Results are given as mean values ± SEM. * p = 0.05. (H) Quantification of IL-1β release was analyzed in colonic explants of WT and Myo1F deficient mice stimulated with IFN-γ/LPS. Inflammatory stimulus was administered for 5 h. IL-1β was quantified by ELISA. Graphs are derived from three independent experiments. n = 6. Results are given as mean values ± SEM. * p = 0.05.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Western Blot, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Mouse Assay

    28) Product Images from "Dopaminergic Stimulation of Myeloid Antigen-Presenting Cells Attenuates Signal Transducer and Activator of Transcription 3-Activation Favouring the Development of Experimental Autoimmune Encephalomyelitis"

    Article Title: Dopaminergic Stimulation of Myeloid Antigen-Presenting Cells Attenuates Signal Transducer and Activator of Transcription 3-Activation Favouring the Development of Experimental Autoimmune Encephalomyelitis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00571

    Signal transducer and activator of transcription 3 (STAT3) phosphorylation is decreased by dopamine in human monocytes. Fresh blood samples obtained from healthy donors were unstimulated or pre-incubated with dopamine (DA) in the presence or absence of interleukin (IL)-6 for 15 min. Afterwards, the extent of phosphorylated STAT3 (pSTAT3) was evaluated by intracellular immunostaining in the CD14 + population and analysed by flow cytometry using the gating strategy indicated in Figure S7 in Supplementary Material. (A) Representative histograms for pSTAT3 in peripheral blood CD14 + cells unstimulated (black lines) or treated with 1 µM DA (green lines) in the absence (left panel) or in the presence of IL-6 (right panel). The extent of pSTAT3 was also determined in peripheral blood CD14 + cells stimulated with IL-6 alone (red line). (B) The extent of pSTAT3 in unstimulated (white symbols) or IL-6 treated (black symbols) CD14 + in the presence of increasing DA concentrations was quantified as the mean fluorescence intensity (MFI) associated with pSTAT3-immunostaining (left panel) or as the ratio of the pSTAT3-associated MFI of stimulated cells to the pSTAT3-associated MFI of unstimulated cells (in-fold, right panel). Values represent mean ± SEM, n = 5. * p
    Figure Legend Snippet: Signal transducer and activator of transcription 3 (STAT3) phosphorylation is decreased by dopamine in human monocytes. Fresh blood samples obtained from healthy donors were unstimulated or pre-incubated with dopamine (DA) in the presence or absence of interleukin (IL)-6 for 15 min. Afterwards, the extent of phosphorylated STAT3 (pSTAT3) was evaluated by intracellular immunostaining in the CD14 + population and analysed by flow cytometry using the gating strategy indicated in Figure S7 in Supplementary Material. (A) Representative histograms for pSTAT3 in peripheral blood CD14 + cells unstimulated (black lines) or treated with 1 µM DA (green lines) in the absence (left panel) or in the presence of IL-6 (right panel). The extent of pSTAT3 was also determined in peripheral blood CD14 + cells stimulated with IL-6 alone (red line). (B) The extent of pSTAT3 in unstimulated (white symbols) or IL-6 treated (black symbols) CD14 + in the presence of increasing DA concentrations was quantified as the mean fluorescence intensity (MFI) associated with pSTAT3-immunostaining (left panel) or as the ratio of the pSTAT3-associated MFI of stimulated cells to the pSTAT3-associated MFI of unstimulated cells (in-fold, right panel). Values represent mean ± SEM, n = 5. * p

    Techniques Used: Incubation, Immunostaining, Flow Cytometry, Cytometry, Fluorescence

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    Western Blot:

    Article Title: Bone Morphogenetic Proteins Stimulate Mammary Fibroblasts to Promote Mammary Carcinoma Cell Invasion
    Article Snippet: Paragraph title: Protein Isolation, Western Blot and ELISA ... ELISA for human IL-6 (bioLegend) Cat # 430504 Human IL-6 ELISA MAX™ Deluxe.

    Recombinant:

    Article Title: Varicella zoster virus differentially alters morphology and suppresses proinflammatory cytokines in primary human spinal cord and hippocampal astrocytes
    Article Snippet: To determine reactivity of HA-sps and HA-hps, quiescent cells were treated with 10 ng/mL of recombinant human IL-1β (R & D Systems, Minneapolis, MN, USA) or vehicle PBS containing 0.01% bovine serum albumin then IL-6 was quantified as previously described [ ]. .. After 24 h, supernatant was collected, flash-frozen, and analyzed for IL-6 concentrations using the Human IL-6 ELISA MAX Deluxe set as per the manufacturer’s instructions (Biolegend, San Diego, CA, USA).

    Immunofluorescence:

    Article Title: Varicella zoster virus differentially alters morphology and suppresses proinflammatory cytokines in primary human spinal cord and hippocampal astrocytes
    Article Snippet: After 24 h, supernatant was collected, flash-frozen, and analyzed for IL-6 concentrations using the Human IL-6 ELISA MAX Deluxe set as per the manufacturer’s instructions (Biolegend, San Diego, CA, USA). .. In addition, cells were fixed and analyzed at 24 h post-treatment by IFA for GFAP expression and morphological changes.

    Blocking Assay:

    Article Title: The induced RNA‐binding protein, HuR, targets 3′‐UTR region of IL‐6 mRNA and enhances its stabilization in periodontitis
    Article Snippet: The supernatant of the culture medium of OBA‐9 was collected and IL‐6 was measured in the supernatant by human IL‐6 ELISA MAX™ Deluxe (Cat. no. 9430504; Biolegend, San Diego, CA, USA), as described previously . .. After blocking each well with 1% BSA in PBST, the supernatant or standard (diluted in PBST from 1 ng/ml to zero) was applied to each well.

    HMT Assay:

    Article Title: Increased ROS production in non-polarized mammary epithelial cells induces monocyte infiltration in 3D culture
    Article Snippet: After HMT-3522 mammary epithelial cells were cultured in 3D lrECM gel for 3 days, culture medium was replaced with fresh T4 medium and incubated for an additional 48 h. Conditioned medium was collected, centrifuged at 4058 g for 5 min to pellet floating cells and debris. .. The supernatant was analyzed for secreted IL-6 (Biolegend, 430504) and CXCL1 (Abcam, ab100530) by ELISA assay according to the manufacturer's instructions.

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    BioLegend human il 6 elisa max deluxe
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