il 1β  (PeproTech)

 
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    Name:
    Recombinant Murine IL 1β
    Description:
    IL 1β is a proinflammatory cytokine produced in a variety of cells including monocytes tissue macrophages keratinocytes and other epithelial cells Both IL 1α and IL 1β binds to the same receptor and has similar if not identical biological properties These cytokines have a broad range of activities including stimulation of thymocyte proliferation by inducing IL 2 release B cell maturation and proliferation mitogenic FGF like activity and the ability to stimulate the release of prostaglandin and collagenase from synovial cells However whereas IL 1β is a secreted cytokine IL 1α is predominantly a cell associated cytokine Recombinant murine IL 1β is a 17 5 kDa protein containing 153 amino acid residues
    Catalog Number:
    211-11B-100UG
    Price:
    1,040.00
    Category:
    Recombinant Proteins
    Source:
    E.coli
    Reactivity:
    Human Monkey Pig Rat Mouse Chicken
    Purity:
    98.0
    Quantity:
    100UG
    Buy from Supplier


    Structured Review

    PeproTech il 1β
    The effects of differential expression of CatC on M1 polarization of microglia in vitro. Primary cultured microglia from WT, CatCOE, and CatCKD mice were stimulated with LPS (50 ng/ml) for 24 h. A The mRNA expressions of TNF-α, <t>IL-1β,</t> IL-6, CD86, CD32, and CD16 were measured by qRT-PCR. B The levels of TNF-α and IL-1β in cellular lysate and culture supernatant were measured by ELISA. * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001
    IL 1β is a proinflammatory cytokine produced in a variety of cells including monocytes tissue macrophages keratinocytes and other epithelial cells Both IL 1α and IL 1β binds to the same receptor and has similar if not identical biological properties These cytokines have a broad range of activities including stimulation of thymocyte proliferation by inducing IL 2 release B cell maturation and proliferation mitogenic FGF like activity and the ability to stimulate the release of prostaglandin and collagenase from synovial cells However whereas IL 1β is a secreted cytokine IL 1α is predominantly a cell associated cytokine Recombinant murine IL 1β is a 17 5 kDa protein containing 153 amino acid residues
    https://www.bioz.com/result/il 1β/product/PeproTech
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    il 1β - by Bioz Stars, 2021-05
    86/100 stars

    Images

    1) Product Images from "Cathepsin C promotes microglia M1 polarization and aggravates neuroinflammation via activation of Ca2+-dependent PKC/p38MAPK/NF-κB pathway"

    Article Title: Cathepsin C promotes microglia M1 polarization and aggravates neuroinflammation via activation of Ca2+-dependent PKC/p38MAPK/NF-κB pathway

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-019-1398-3

    The effects of differential expression of CatC on M1 polarization of microglia in vitro. Primary cultured microglia from WT, CatCOE, and CatCKD mice were stimulated with LPS (50 ng/ml) for 24 h. A The mRNA expressions of TNF-α, IL-1β, IL-6, CD86, CD32, and CD16 were measured by qRT-PCR. B The levels of TNF-α and IL-1β in cellular lysate and culture supernatant were measured by ELISA. * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001
    Figure Legend Snippet: The effects of differential expression of CatC on M1 polarization of microglia in vitro. Primary cultured microglia from WT, CatCOE, and CatCKD mice were stimulated with LPS (50 ng/ml) for 24 h. A The mRNA expressions of TNF-α, IL-1β, IL-6, CD86, CD32, and CD16 were measured by qRT-PCR. B The levels of TNF-α and IL-1β in cellular lysate and culture supernatant were measured by ELISA. * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001

    Techniques Used: Expressing, In Vitro, Cell Culture, Mouse Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    The effects of CatC stimulation on the expression of M1 markers of microglia in vitro. Primary cultured microglia from WT mice was stimulated with active CatC (10 ng/ml) for 18 h. A The mRNA expressions of TNF-α, IL-1β, CD86, CD16, CD32, and IL-6 were measured by qRT-PCR. B The percentages of CD86 + and CD206 + cells were measured by flow cytometry. C CD86 and CatC co-expression was detected by immunofluorescent staining after LPS (50 ng/ml) treatment for 18 h. D The mRNA expressions of TNF-α, IL-1β, CD86, CD16, CD32, and IL-6 were measured by qRT-PCR in mice with injection of CatC (0.43 mg/μl, i.c.v.) for 6 h. Scale bars, 25 μm. The data represent mean ± SEM, n = 3. * P
    Figure Legend Snippet: The effects of CatC stimulation on the expression of M1 markers of microglia in vitro. Primary cultured microglia from WT mice was stimulated with active CatC (10 ng/ml) for 18 h. A The mRNA expressions of TNF-α, IL-1β, CD86, CD16, CD32, and IL-6 were measured by qRT-PCR. B The percentages of CD86 + and CD206 + cells were measured by flow cytometry. C CD86 and CatC co-expression was detected by immunofluorescent staining after LPS (50 ng/ml) treatment for 18 h. D The mRNA expressions of TNF-α, IL-1β, CD86, CD16, CD32, and IL-6 were measured by qRT-PCR in mice with injection of CatC (0.43 mg/μl, i.c.v.) for 6 h. Scale bars, 25 μm. The data represent mean ± SEM, n = 3. * P

    Techniques Used: Expressing, In Vitro, Cell Culture, Mouse Assay, Quantitative RT-PCR, Flow Cytometry, Cytometry, Staining, Injection

    The effects of differential expression of CatC on M1 polarization of microglia in the brain. TNF-α ISH staining in cortex and hippocampus was performed 24 h after LPS (1 mg/kg, i.c.v.) injection ( A ). The levels of TNF-α and IL-1β were measured by qRT-PCR ( B ) and ELISA ( D ), respectively. The activation of microglia in cortex and hippocampus was detected by Iba1 IHC staining ( C ). Scale bars, 200 μm (main panels, first and second rows), 500 μm (main panels, third row), 100 μm (main panels, fourth row), 50 μm (insets). * P ≤ 0.05, ** P ≤ 0.01. n = 3( A , B ), n = 3–5 ( C , D )
    Figure Legend Snippet: The effects of differential expression of CatC on M1 polarization of microglia in the brain. TNF-α ISH staining in cortex and hippocampus was performed 24 h after LPS (1 mg/kg, i.c.v.) injection ( A ). The levels of TNF-α and IL-1β were measured by qRT-PCR ( B ) and ELISA ( D ), respectively. The activation of microglia in cortex and hippocampus was detected by Iba1 IHC staining ( C ). Scale bars, 200 μm (main panels, first and second rows), 500 μm (main panels, third row), 100 μm (main panels, fourth row), 50 μm (insets). * P ≤ 0.05, ** P ≤ 0.01. n = 3( A , B ), n = 3–5 ( C , D )

    Techniques Used: Expressing, In Situ Hybridization, Staining, Injection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Activation Assay, Immunohistochemistry

    The effects of differential expression of CatC on microglia and proinflammatory cytokines in the brain. After spatial probe test, the activation of microglia was detected by Iba1 IHC staining in cortex and hippocampus of untreated mice ( A ) and LPS (i.p.)-injected mice ( D ). The levels of TNF-α and IL-1β were measured in untreated mice ( B , C ) and LPS-injected mice ( E , F ). The levels of TNF-α and IL-1β were determined by qRT-PCR and ELISA. Scale bars, 100 μm (main panels), 25 μm (insets). * P ≤ 0.05, ** P ≤ 0.01. n = 3 ( A – D ), n = 3–5 ( E , F )
    Figure Legend Snippet: The effects of differential expression of CatC on microglia and proinflammatory cytokines in the brain. After spatial probe test, the activation of microglia was detected by Iba1 IHC staining in cortex and hippocampus of untreated mice ( A ) and LPS (i.p.)-injected mice ( D ). The levels of TNF-α and IL-1β were measured in untreated mice ( B , C ) and LPS-injected mice ( E , F ). The levels of TNF-α and IL-1β were determined by qRT-PCR and ELISA. Scale bars, 100 μm (main panels), 25 μm (insets). * P ≤ 0.05, ** P ≤ 0.01. n = 3 ( A – D ), n = 3–5 ( E , F )

    Techniques Used: Expressing, Activation Assay, Immunohistochemistry, Staining, Mouse Assay, Injection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    2) Product Images from "P2X7 receptor signaling is not required but can enhance Th17 differentiation"

    Article Title: P2X7 receptor signaling is not required but can enhance Th17 differentiation

    Journal: bioRxiv

    doi: 10.1101/2021.03.19.435855

    Stimulation of P2X7R on DCs can promote IL-17 production in vitro . (A) Mouse BMDCs derived from C57BL/6J mice were stimulated with the indicated doses of BzATP and supernatants were collected at 3h and 6h to determine IL-6 and IL-1β production by ELISA. (B) Mixed leucocyte reaction (MLR) was induced by cocultering BMDCs derived from C57BL/6J, pretreated with 300μM BzATP or left untreated (controls), with naive CD4 + T cells isolated from BALB/cJ for 4 days. Supernatants were collected and L-17A levels assessed by ELISA. (C) BMDCs derived from WT or P2rx7 -/- mice, pretreated with BzATP or left untreated (controls), were cocultured with naive CD4 + T cells from BALB/cJ for 4 days. Supernatants were collected to determine IL-17A by ELISA. Results shown as mean ± SEM. Results are one representative of three independent experiments. *p
    Figure Legend Snippet: Stimulation of P2X7R on DCs can promote IL-17 production in vitro . (A) Mouse BMDCs derived from C57BL/6J mice were stimulated with the indicated doses of BzATP and supernatants were collected at 3h and 6h to determine IL-6 and IL-1β production by ELISA. (B) Mixed leucocyte reaction (MLR) was induced by cocultering BMDCs derived from C57BL/6J, pretreated with 300μM BzATP or left untreated (controls), with naive CD4 + T cells isolated from BALB/cJ for 4 days. Supernatants were collected and L-17A levels assessed by ELISA. (C) BMDCs derived from WT or P2rx7 -/- mice, pretreated with BzATP or left untreated (controls), were cocultured with naive CD4 + T cells from BALB/cJ for 4 days. Supernatants were collected to determine IL-17A by ELISA. Results shown as mean ± SEM. Results are one representative of three independent experiments. *p

    Techniques Used: In Vitro, Derivative Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Isolation

    3) Product Images from "Occupational exposure to carbon black nanoparticles increases inflammatory vascular disease risk: an implication of an ex vivo biosensor assay"

    Article Title: Occupational exposure to carbon black nanoparticles increases inflammatory vascular disease risk: an implication of an ex vivo biosensor assay

    Journal: Particle and Fibre Toxicology

    doi: 10.1186/s12989-020-00378-8

    The effect of IL-1β on expression (delta Ct) of biosensor genes in vitro using hUVECs. Serum samples obtained from three workers with lowest levels of IL-1β were spiked with exogenous IL-1β at 0, 125, 250 and 500 pg/ml as final concentrations. The highest concentration of added IL-1β is about 2-fold higher than the highest level seen in study subjects. Cultures of biosensor assay were conducted in duplicates. The slopes were listed as estimates in supplemental Table 3 . Among the seven biosensor genes studied, ICAM, VCAM, CCL2, and CXCL8 expressions were identified to be dramatically induced by IL-1β treatment. Relative quantification ranged from 2.43 for CCL2 to 349.71 for CXCL8 per 500 pg/ml addition of IL-1β in the culture medium
    Figure Legend Snippet: The effect of IL-1β on expression (delta Ct) of biosensor genes in vitro using hUVECs. Serum samples obtained from three workers with lowest levels of IL-1β were spiked with exogenous IL-1β at 0, 125, 250 and 500 pg/ml as final concentrations. The highest concentration of added IL-1β is about 2-fold higher than the highest level seen in study subjects. Cultures of biosensor assay were conducted in duplicates. The slopes were listed as estimates in supplemental Table 3 . Among the seven biosensor genes studied, ICAM, VCAM, CCL2, and CXCL8 expressions were identified to be dramatically induced by IL-1β treatment. Relative quantification ranged from 2.43 for CCL2 to 349.71 for CXCL8 per 500 pg/ml addition of IL-1β in the culture medium

    Techniques Used: Expressing, In Vitro, Concentration Assay, Biosensor Assay

    4) Product Images from "Interleukin 1β and tumor necrosis factor α promote hFOB1.19 cell viability via activating AP1"

    Article Title: Interleukin 1β and tumor necrosis factor α promote hFOB1.19 cell viability via activating AP1

    Journal: American Journal of Translational Research

    doi:

    ELISA analysis shows the concentration change of IL-6, IL-1β and TNF-α in peripheral blood serum samples of bone trauma patients after surgery. Samples were taken at 0, 1, 3, 5 and 7 days after surgery. * P
    Figure Legend Snippet: ELISA analysis shows the concentration change of IL-6, IL-1β and TNF-α in peripheral blood serum samples of bone trauma patients after surgery. Samples were taken at 0, 1, 3, 5 and 7 days after surgery. * P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay

    Relative luciferase activity of AP1 in cells treated with IL-1β, TNF-α, IL-6, PMA or SP600125. When cells are pretreated with SP600125, the effect of IL-1β and TNF-α is inhibited ( P
    Figure Legend Snippet: Relative luciferase activity of AP1 in cells treated with IL-1β, TNF-α, IL-6, PMA or SP600125. When cells are pretreated with SP600125, the effect of IL-1β and TNF-α is inhibited ( P

    Techniques Used: Luciferase, Activity Assay

    MTT assay shows the effects of IL-6, IL-1β and TNF-α on hFOB1.19 cell viability during an experimental period of 5 days. No significant change is observed in the first two days. Up-regulation of cell viability is observed after three day
    Figure Legend Snippet: MTT assay shows the effects of IL-6, IL-1β and TNF-α on hFOB1.19 cell viability during an experimental period of 5 days. No significant change is observed in the first two days. Up-regulation of cell viability is observed after three day

    Techniques Used: MTT Assay

    AP1 is necessary for the function of IL-1β and TNF-α in promoting hFOB1.19 cell viability. Cells are transfected with FOS-specific siRNA (si-FOS) or the control (si-Control). When FOS is inhibited, IL-1β or TNF-α treatment
    Figure Legend Snippet: AP1 is necessary for the function of IL-1β and TNF-α in promoting hFOB1.19 cell viability. Cells are transfected with FOS-specific siRNA (si-FOS) or the control (si-Control). When FOS is inhibited, IL-1β or TNF-α treatment

    Techniques Used: Transfection

    5) Product Images from "Quantification of Citrullinated Histone H3 Bound DNA for Detection of Neutrophil Extracellular Traps"

    Article Title: Quantification of Citrullinated Histone H3 Bound DNA for Detection of Neutrophil Extracellular Traps

    Journal: Cancers

    doi: 10.3390/cancers12113424

    Quantification of citrullinated histone H3 for detection of IL-1β-induced NETosis. Neutrophils were isolated from the BM of tumor-free mice ( n = 3) and stimulated in vitro with 200 ng/mL of murine IL-1β for 6 h. ( a ) Formation of NETs was evaluated by microscopy and quantified. ( b ) NET fractions were collected and used in the CitH3DNA binding assay. Mean ± SEM values obtained from neutrophils isolated from 3 individual mice are shown.
    Figure Legend Snippet: Quantification of citrullinated histone H3 for detection of IL-1β-induced NETosis. Neutrophils were isolated from the BM of tumor-free mice ( n = 3) and stimulated in vitro with 200 ng/mL of murine IL-1β for 6 h. ( a ) Formation of NETs was evaluated by microscopy and quantified. ( b ) NET fractions were collected and used in the CitH3DNA binding assay. Mean ± SEM values obtained from neutrophils isolated from 3 individual mice are shown.

    Techniques Used: Isolation, Mouse Assay, In Vitro, Microscopy, Binding Assay

    6) Product Images from "An additive interaction between the NF?B and estrogen receptor signalling pathways in human endometrial epithelial cells"

    Article Title: An additive interaction between the NF?B and estrogen receptor signalling pathways in human endometrial epithelial cells

    Journal: Human Reproduction (Oxford, England)

    doi: 10.1093/humrep/dep421

    The positive interaction between E2 and IL-1β in TERT -EECs is NFκB dependent and can be mediated by ERα and ERβ.
    Figure Legend Snippet: The positive interaction between E2 and IL-1β in TERT -EECs is NFκB dependent and can be mediated by ERα and ERβ.

    Techniques Used:

    Regulation of endogenous gene expression by E2 and IL-1β in TERT -EECs.
    Figure Legend Snippet: Regulation of endogenous gene expression by E2 and IL-1β in TERT -EECs.

    Techniques Used: Expressing

    E2 and IL-1β positively interact to enhance estrogen response element (ERE) activity in an estrogen receptor (ER) dependent manner in TERT -EECs.
    Figure Legend Snippet: E2 and IL-1β positively interact to enhance estrogen response element (ERE) activity in an estrogen receptor (ER) dependent manner in TERT -EECs.

    Techniques Used: Activity Assay

    7) Product Images from "IL-1R8 is a checkpoint in NK cells regulating anti-tumor and anti-viral activity"

    Article Title: IL-1R8 is a checkpoint in NK cells regulating anti-tumor and anti-viral activity

    Journal: Nature

    doi: 10.1038/nature24293

    Mechanism of IL-1R8-dependent regulation of NK cells (a) Splenic CD27 low NK cell frequency in wild type, Il1r8 -/- , Il18 -/- , and Il18 -/- /Il1r8 -/- mice. (b) Peripheral CD27 low NK cell frequency in wild-type , Il1r8 -/- , Il1r1 -/- and Il1r8 -/- Il1r1 -/- mice (left) and IFNγ production by splenic NK cells after IL-12 and IL-1β or IL-18 stimulation (right). (c, d) Splenic CD27 low NK cell frequency in Il1r8 +/+ and Il1r8 -/- mice upon commensal flora depletion (c) and breeding in co-housing conditions (d). (e) STED microscopy of human NK cells stimulated with IL-18. Magnification bar: 2μm. (a-d) *p
    Figure Legend Snippet: Mechanism of IL-1R8-dependent regulation of NK cells (a) Splenic CD27 low NK cell frequency in wild type, Il1r8 -/- , Il18 -/- , and Il18 -/- /Il1r8 -/- mice. (b) Peripheral CD27 low NK cell frequency in wild-type , Il1r8 -/- , Il1r1 -/- and Il1r8 -/- Il1r1 -/- mice (left) and IFNγ production by splenic NK cells after IL-12 and IL-1β or IL-18 stimulation (right). (c, d) Splenic CD27 low NK cell frequency in Il1r8 +/+ and Il1r8 -/- mice upon commensal flora depletion (c) and breeding in co-housing conditions (d). (e) STED microscopy of human NK cells stimulated with IL-18. Magnification bar: 2μm. (a-d) *p

    Techniques Used: Mouse Assay, Microscopy

    8) Product Images from "An anti-inflammatory role for C/EBPδ in human brain pericytes"

    Article Title: An anti-inflammatory role for C/EBPδ in human brain pericytes

    Journal: Scientific Reports

    doi: 10.1038/srep12132

    C/EBPδ is differentially induced by IFNγ, IL-1β and LPS. Human brain pericytes were treated with vehicle or 10 ng/mL IL-1β, IFNγ, or LPS for 24 hours. Representative immunocytochemistry images of C/EBPδ with treatments are shown ( a ). The percentage of cells positive for nuclear C/EBPδ was determined by immunocytochemistry ( b ) and the intensity of C/EBPδ expression was analysed by western blotting ( c,d ). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. Data is displayed as mean ± SEM of three independent experiments. * = p
    Figure Legend Snippet: C/EBPδ is differentially induced by IFNγ, IL-1β and LPS. Human brain pericytes were treated with vehicle or 10 ng/mL IL-1β, IFNγ, or LPS for 24 hours. Representative immunocytochemistry images of C/EBPδ with treatments are shown ( a ). The percentage of cells positive for nuclear C/EBPδ was determined by immunocytochemistry ( b ) and the intensity of C/EBPδ expression was analysed by western blotting ( c,d ). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. Data is displayed as mean ± SEM of three independent experiments. * = p

    Techniques Used: Immunocytochemistry, Expressing, Western Blot

    C/EBPδ knockdown modifies pericyte secretion of inflammatory mediators. Human brain pericytes were transfected with 50 nM of control or C/EBPδ siRNA for 48 hours. Following transfection cells were treated with vehicle or 10 ng/mL IL-1β for 24 hours and conditioned media collected. Concentration of sICAM-1 ( a ), MCP-1 ( b ), IL-8 ( c ) and IL-6 ( d ) in media was determined using a multiplex cytometric bead array. Data represent mean ± SEM (n = 4). *** = p
    Figure Legend Snippet: C/EBPδ knockdown modifies pericyte secretion of inflammatory mediators. Human brain pericytes were transfected with 50 nM of control or C/EBPδ siRNA for 48 hours. Following transfection cells were treated with vehicle or 10 ng/mL IL-1β for 24 hours and conditioned media collected. Concentration of sICAM-1 ( a ), MCP-1 ( b ), IL-8 ( c ) and IL-6 ( d ) in media was determined using a multiplex cytometric bead array. Data represent mean ± SEM (n = 4). *** = p

    Techniques Used: Transfection, Concentration Assay, Multiplex Assay

    C/EBPδ is induced in human brain pericytes by IL-1β/IFNγ. Human brain pericytes were treated with vehicle or 10 ng/mL IL-1β + IFNγ for 24 hours and RNA was extracted. Expression of C/EBPα, C/EBPβ and C/EBPδ was determined by microarray 10 ( a ) and qRT-PCR ( b ). Data is displayed as average fold change of five independent cases (a) or mean ± SEM of a separate three independent cases ( b ). *** = p
    Figure Legend Snippet: C/EBPδ is induced in human brain pericytes by IL-1β/IFNγ. Human brain pericytes were treated with vehicle or 10 ng/mL IL-1β + IFNγ for 24 hours and RNA was extracted. Expression of C/EBPα, C/EBPβ and C/EBPδ was determined by microarray 10 ( a ) and qRT-PCR ( b ). Data is displayed as average fold change of five independent cases (a) or mean ± SEM of a separate three independent cases ( b ). *** = p

    Techniques Used: Expressing, Microarray, Quantitative RT-PCR

    Knockdown of C/EBPδ using siRNA. Human brain pericytes were transfected with 50 nM of control or C/EBPδ siRNA for 48 hours. Following transfection, cells were treated with vehicle or 10 ng/mL IL-1β for 4–48 hours. Representative immunocytochemistry images of C/EBPδ with treatments are shown ( a ) and the percentage of cells positive for nuclear C/EBPδ was quantified ( b ). RNA was extracted following a six hour treatment with IL-1β and C/EBPδ transcript expression was determined via RT-qPCR ( c ). Protein was extracted following a 24 hour treatment with IL-1β and C/EBPδ expression determined by western blotting ( d,e ). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. *** = p
    Figure Legend Snippet: Knockdown of C/EBPδ using siRNA. Human brain pericytes were transfected with 50 nM of control or C/EBPδ siRNA for 48 hours. Following transfection, cells were treated with vehicle or 10 ng/mL IL-1β for 4–48 hours. Representative immunocytochemistry images of C/EBPδ with treatments are shown ( a ) and the percentage of cells positive for nuclear C/EBPδ was quantified ( b ). RNA was extracted following a six hour treatment with IL-1β and C/EBPδ transcript expression was determined via RT-qPCR ( c ). Protein was extracted following a 24 hour treatment with IL-1β and C/EBPδ expression determined by western blotting ( d,e ). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. *** = p

    Techniques Used: Transfection, Immunocytochemistry, Expressing, Quantitative RT-PCR, Western Blot

    Time-course and concentration-dependant induction of C/EBPδ expression. Human brain pericytes were treated with vehicle or 0.01–10 ng/mL IL-1β for 24 hours. Representative immunocytochemistry images of C/EBPδ are shown ( a ). The percentage of cells positive for nuclear C/EBPδ was determined by immunocytochemistry ( b ) and C/EBPδ intensity was analysed by western blotting ( c,d ). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. Human brain pericytes were treated with 10 ng/mL IL-1β for 0–48 hours. The percentage of C/EBPδ positive cells was determined by immunocytochemistry ( e ) and C/EBPδ intensity was analysed by western blotting ( f,g ). Data is displayed as mean ± SEM from three independent experiments. * = p
    Figure Legend Snippet: Time-course and concentration-dependant induction of C/EBPδ expression. Human brain pericytes were treated with vehicle or 0.01–10 ng/mL IL-1β for 24 hours. Representative immunocytochemistry images of C/EBPδ are shown ( a ). The percentage of cells positive for nuclear C/EBPδ was determined by immunocytochemistry ( b ) and C/EBPδ intensity was analysed by western blotting ( c,d ). Blots are cropped to improve clarity. Full-length blots are presented in Supplementary Figure S2. Human brain pericytes were treated with 10 ng/mL IL-1β for 0–48 hours. The percentage of C/EBPδ positive cells was determined by immunocytochemistry ( e ) and C/EBPδ intensity was analysed by western blotting ( f,g ). Data is displayed as mean ± SEM from three independent experiments. * = p

    Techniques Used: Concentration Assay, Expressing, Immunocytochemistry, Western Blot

    C/EBPδ knockdown modifies IL-1β induced inflammatory gene expression. Human brain pericytes were transfected with 50 nM of control or C/EBPδ siRNA for 48 hours. Following transfection, cells were treated with vehicle or 10 ng/mL IL-1β for six hours and RNA was extracted. The effect of C/EBPδ knockdown on IL-1β (a), ICAM-1 ( b ), MCP-1 ( c ), IL-8( d ), SOD2 ( e ), COX-2 ( f ) and IL-6 ( g ) was assayed by qRT-PCR. Data is displayed as mean ± SEM from four independent cases ** = p
    Figure Legend Snippet: C/EBPδ knockdown modifies IL-1β induced inflammatory gene expression. Human brain pericytes were transfected with 50 nM of control or C/EBPδ siRNA for 48 hours. Following transfection, cells were treated with vehicle or 10 ng/mL IL-1β for six hours and RNA was extracted. The effect of C/EBPδ knockdown on IL-1β (a), ICAM-1 ( b ), MCP-1 ( c ), IL-8( d ), SOD2 ( e ), COX-2 ( f ) and IL-6 ( g ) was assayed by qRT-PCR. Data is displayed as mean ± SEM from four independent cases ** = p

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR

    C/EBPδ knockdown enhances IL-1β induced MCP-1 and ICAM-1 protein expression. Human brain pericytes were transfected with 50 nM of control or C/EBPδ siRNA for 48 hours. Following transfection, cells were treated with vehicle or 10 ng/mL IL-1β for 2–24 hours and cells fixed and immunostained for ICAM-1 and MCP-1. Representative images of ICAM-1 and MCP-1 immunostaining respectively with vehicle and control siRNA ( a,f ), vehicle with C/EBPδ siRNA ( b,g ), 24 hours IL-1β with control siRNA ( c,h ) and 24 hours IL-1β with C/EBPδ siRNA are shown ( d,i ). Intensity of ICAM-1 staining ( e ) and the percentage of MCP-1 positive cells (j) were determined. Data is displayed as mean ± SEM from three independent experiments. ** = p
    Figure Legend Snippet: C/EBPδ knockdown enhances IL-1β induced MCP-1 and ICAM-1 protein expression. Human brain pericytes were transfected with 50 nM of control or C/EBPδ siRNA for 48 hours. Following transfection, cells were treated with vehicle or 10 ng/mL IL-1β for 2–24 hours and cells fixed and immunostained for ICAM-1 and MCP-1. Representative images of ICAM-1 and MCP-1 immunostaining respectively with vehicle and control siRNA ( a,f ), vehicle with C/EBPδ siRNA ( b,g ), 24 hours IL-1β with control siRNA ( c,h ) and 24 hours IL-1β with C/EBPδ siRNA are shown ( d,i ). Intensity of ICAM-1 staining ( e ) and the percentage of MCP-1 positive cells (j) were determined. Data is displayed as mean ± SEM from three independent experiments. ** = p

    Techniques Used: Expressing, Transfection, Immunostaining, Staining

    9) Product Images from "Erythromycin acts through the ghrelin receptor to attenuate inflammatory responses in chondrocytes and maintain joint integrity"

    Article Title: Erythromycin acts through the ghrelin receptor to attenuate inflammatory responses in chondrocytes and maintain joint integrity

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2019.03.014

    EM and ghrelin enhance cartilage matrix levels in human OA cartilage. (A) RT-qPCR analysis of Agg, Col-II, Col-IX, MMP13, iNOS, IL-1β and ghrelin in human normal cartilage from 4 healthy donors and OA cartilage from 5 OA donors. TBP served as a reference gene. (B) Safranin O staining analysis on sections of OA articular cartilage explants after 3 weeks of culturing with 10μM EM. Images of explants from donor 54/F (age/sex) are shown. (C) Safranin O staining analysis on sections of OA articular cartilage explants after 3 weeks of culturing with 10μM EM900. Images of explants from donor 54/F (age/sex) are shown. (D) Safranin O staining analysis on sections of OA articular cartilage explants after 3 weeks of culturing with 100nM ghrelin. Images of explants from donor 53/F (age/sex) are shown. For each experiment, duplicate technical repeats were analyzed. Experiments were repeated three times using samples from three different OA donors. As the extent of cartilage matrix loss and OA severity of the donor samples differed among experiments, cellularity and matrix levels varied, which is evident from the differences in control samples used for EM, EM900 and ghrelin experiments. Scale bar = 100μm. SZ = superficial zone. DZ = deep zone.
    Figure Legend Snippet: EM and ghrelin enhance cartilage matrix levels in human OA cartilage. (A) RT-qPCR analysis of Agg, Col-II, Col-IX, MMP13, iNOS, IL-1β and ghrelin in human normal cartilage from 4 healthy donors and OA cartilage from 5 OA donors. TBP served as a reference gene. (B) Safranin O staining analysis on sections of OA articular cartilage explants after 3 weeks of culturing with 10μM EM. Images of explants from donor 54/F (age/sex) are shown. (C) Safranin O staining analysis on sections of OA articular cartilage explants after 3 weeks of culturing with 10μM EM900. Images of explants from donor 54/F (age/sex) are shown. (D) Safranin O staining analysis on sections of OA articular cartilage explants after 3 weeks of culturing with 100nM ghrelin. Images of explants from donor 53/F (age/sex) are shown. For each experiment, duplicate technical repeats were analyzed. Experiments were repeated three times using samples from three different OA donors. As the extent of cartilage matrix loss and OA severity of the donor samples differed among experiments, cellularity and matrix levels varied, which is evident from the differences in control samples used for EM, EM900 and ghrelin experiments. Scale bar = 100μm. SZ = superficial zone. DZ = deep zone.

    Techniques Used: Quantitative RT-PCR, Staining

    EM900, a non-antibiotic EM derivative, inhibits IL-1β-induced MMP13 expression and NF-κB activation, and maintains joint health in MIA-injected knees. (A) Live-dead assay on chondrocytes treated with EM900 (0, 1, 10 and 100μM) for 4 days. Percentage of dead cells/total cells was quantified. (B) RT-qPCR analysis of MMP13 mRNA on nHACs treated with EM900 (1, 10 and 25μM) overnight followed by IL-1β (1ng/mL) treatment for 3 days. TBP served as a reference gene. (C) NF-κB transactivation assay. nHACs were transiently transfected with the NF-κB reporter construct for 24 hours and then treated with EM900 (1μM) and IL-1β (1ng/mL). A Renilla luciferase construct was co-transfected as an internal control for normalization. (D) Serum Response Element (SRE) luciferase reporter assay as a readout of ghrelin receptor signaling. nHACs were transiently transfected with the SRE reporter construct for 24 hours and then treated with EM900 (10μM) and IL-1β (1ng/mL). A Renilla luciferase construct was co-transfected as an internal control for normalization. Data were presented as “fold activation” compared to untreated samples. (E) Safranin O staining images for cartilage matrix analysis of WT mouse knee joints. MIA was intra-articularly injected to knee joints at day 0. EM900 was IP-delivered daily from the day of MIA injection. Samples were collected for analysis 7 days later. The degree of cartilage matrix loss was scored according to the OARSI scoring system. Scale bar = 200μm. M = Meniscus. T = Tibia. F = Femur. (F) Synovitis analysis on sections from the medial tibial plateau of the WT mice knees injected with EM900. MIA was intra-articularly injected into knee joints at day 0. EM900 (PBS as control) was IP-injected into the mice daily until sample harvest at day 7. Arrowheads indicate thickening of the synovial layers and increased cellularity of resident cells. Synovitis was scored according to established synovitis scoring protocols. For A to D, data were reported as mean ± SD and analyzed by Dunnett’s test (B) and an unpaired t -test (C, D). For E and F, data were reported in a box plot, where the median, as well as the maximum and the minimum data points were indicated. Data from E and F were analyzed by Kruskal-Wallis statistical test followed by Mann-Whitney U test. * p
    Figure Legend Snippet: EM900, a non-antibiotic EM derivative, inhibits IL-1β-induced MMP13 expression and NF-κB activation, and maintains joint health in MIA-injected knees. (A) Live-dead assay on chondrocytes treated with EM900 (0, 1, 10 and 100μM) for 4 days. Percentage of dead cells/total cells was quantified. (B) RT-qPCR analysis of MMP13 mRNA on nHACs treated with EM900 (1, 10 and 25μM) overnight followed by IL-1β (1ng/mL) treatment for 3 days. TBP served as a reference gene. (C) NF-κB transactivation assay. nHACs were transiently transfected with the NF-κB reporter construct for 24 hours and then treated with EM900 (1μM) and IL-1β (1ng/mL). A Renilla luciferase construct was co-transfected as an internal control for normalization. (D) Serum Response Element (SRE) luciferase reporter assay as a readout of ghrelin receptor signaling. nHACs were transiently transfected with the SRE reporter construct for 24 hours and then treated with EM900 (10μM) and IL-1β (1ng/mL). A Renilla luciferase construct was co-transfected as an internal control for normalization. Data were presented as “fold activation” compared to untreated samples. (E) Safranin O staining images for cartilage matrix analysis of WT mouse knee joints. MIA was intra-articularly injected to knee joints at day 0. EM900 was IP-delivered daily from the day of MIA injection. Samples were collected for analysis 7 days later. The degree of cartilage matrix loss was scored according to the OARSI scoring system. Scale bar = 200μm. M = Meniscus. T = Tibia. F = Femur. (F) Synovitis analysis on sections from the medial tibial plateau of the WT mice knees injected with EM900. MIA was intra-articularly injected into knee joints at day 0. EM900 (PBS as control) was IP-injected into the mice daily until sample harvest at day 7. Arrowheads indicate thickening of the synovial layers and increased cellularity of resident cells. Synovitis was scored according to established synovitis scoring protocols. For A to D, data were reported as mean ± SD and analyzed by Dunnett’s test (B) and an unpaired t -test (C, D). For E and F, data were reported in a box plot, where the median, as well as the maximum and the minimum data points were indicated. Data from E and F were analyzed by Kruskal-Wallis statistical test followed by Mann-Whitney U test. * p

    Techniques Used: Expressing, Activation Assay, Injection, Live Dead Assay, Quantitative RT-PCR, Transactivation Assay, Transfection, Construct, Luciferase, Reporter Assay, Staining, Mouse Assay, MANN-WHITNEY

    The anti-inflammatory activity of EM in chondrocytes is mediated by the ghrelin receptor GHSR. (A) Immunohistochemical (IHC) analysis using an anti-GHSR antibody (anti-GHS-R1a) on WT and GHSR null mouse knee joints. Rectangles denote areas magnified. Arrows and arrowhead indicate GHSR positive and negative cells, respectively. Scale bar = 200μm. (B) Relative MMP13 mRNA analysis on WT and GHSR null chondrocytes treated with EM (0, 10, and 25μM) and IL-1β (1ng/ml). TBP served as a loading control. (C) Immunocytochemistry (ICC) analysis with anti-EM antibody (red) on WT chondrocytes treated with different doses of EM (0, 1, 10, and 25μM) for 1hr. Arrows indicate cells with EM staining. Percentage of EM-positive cells was calculated. (D) ICC analysis with EM presence on WT and GHSR null chondrocytes treated with EM (10μM) for 1hr. Percentage of EM-positive cells was calculated. Data were reported as mean ± SD and analyzed by Dunnett’s test (B and C) and an unpaired t -test (D). At least three independent experiments were performed. * p
    Figure Legend Snippet: The anti-inflammatory activity of EM in chondrocytes is mediated by the ghrelin receptor GHSR. (A) Immunohistochemical (IHC) analysis using an anti-GHSR antibody (anti-GHS-R1a) on WT and GHSR null mouse knee joints. Rectangles denote areas magnified. Arrows and arrowhead indicate GHSR positive and negative cells, respectively. Scale bar = 200μm. (B) Relative MMP13 mRNA analysis on WT and GHSR null chondrocytes treated with EM (0, 10, and 25μM) and IL-1β (1ng/ml). TBP served as a loading control. (C) Immunocytochemistry (ICC) analysis with anti-EM antibody (red) on WT chondrocytes treated with different doses of EM (0, 1, 10, and 25μM) for 1hr. Arrows indicate cells with EM staining. Percentage of EM-positive cells was calculated. (D) ICC analysis with EM presence on WT and GHSR null chondrocytes treated with EM (10μM) for 1hr. Percentage of EM-positive cells was calculated. Data were reported as mean ± SD and analyzed by Dunnett’s test (B and C) and an unpaired t -test (D). At least three independent experiments were performed. * p

    Techniques Used: Activity Assay, Immunohistochemistry, Immunocytochemistry, Staining

    Erythromycin (EM) inhibits the catabolic activity of IL-1β on primary human articular chondrocytes (nHACs). (A) Live-dead assay on chondrocytes treated with EM (1, 10 and 100μM) for 4 days. Percentage of dead cells/total cells was quantified. (B) RT-qPCR analysis of Col-II, Col-X, iNOS, MMP1, MMP9 and MMP13 on nHACs treated with EM (0, 1, and 10μM) and IL-1β (1ng/mL). TBP served as a reference gene for normalization. (C) NF-κB transactivation assay. nHACs were transiently transfected with an NF-κB reporter and treated with EM (10μM) and IL-1β (1ng/ml). A Renilla luciferase construct was co-transfected as an internal control for normalization. Data were reported as mean ± SD and analyzed by Dunnett’s test (B) and an unpaired t -test (C). At least three independent experiments were performed. * p
    Figure Legend Snippet: Erythromycin (EM) inhibits the catabolic activity of IL-1β on primary human articular chondrocytes (nHACs). (A) Live-dead assay on chondrocytes treated with EM (1, 10 and 100μM) for 4 days. Percentage of dead cells/total cells was quantified. (B) RT-qPCR analysis of Col-II, Col-X, iNOS, MMP1, MMP9 and MMP13 on nHACs treated with EM (0, 1, and 10μM) and IL-1β (1ng/mL). TBP served as a reference gene for normalization. (C) NF-κB transactivation assay. nHACs were transiently transfected with an NF-κB reporter and treated with EM (10μM) and IL-1β (1ng/ml). A Renilla luciferase construct was co-transfected as an internal control for normalization. Data were reported as mean ± SD and analyzed by Dunnett’s test (B) and an unpaired t -test (C). At least three independent experiments were performed. * p

    Techniques Used: Activity Assay, Live Dead Assay, Quantitative RT-PCR, Transactivation Assay, Transfection, Luciferase, Construct

    Ghrelin signaling is necessary and sufficient to inhibit IL-1β-induced catabolic gene expression and NF-κB activation in chondrocytes. (A) RT-qPCR analysis of Col-II, Col-X, iNOS, MMP1, MMP9 and MMP13 on nHACs treated with ghrelin (0, 10 and 100nM) and IL-1β (1ng/mL). TBP served as a reference gene. (B) NF-κB transactivation assay. nHACs were transiently transfected with NF-κB reporter construct for 24 hours and then treated with ghrelin (10nM) and IL-1β (1ng/mL). A Renilla luciferase construct was co-transfected as an internal control for normalization. (C) Serum Response Element (SRE) luciferase reporter assay. nHACs were transiently transfected with the SRE reporter construct for 24 hours and then treated with ghrelin (10nM) or EM (10μM) for 16 hours. A Renilla luciferase construct was co-transfected as an internal control for normalization. Data are presented as fold activation relative to untreated samples. (D) RT-qPCR analysis of MMP13 on WT and GHSR null mouse chondrocytes treated with ghrelin (0, 10, 100nM) and IL-1β (1ng/mL). TBP served as a reference gene. (E) ICC analysis with an anti-EM antibody on WT chondrocytes co-treated with EM (10μM) and different concentrations of ghrelin (1, 10 and 100nM). Arrows indicate cells with EM staining. Percentage of EM-positive cells was calculated. Data were reported as mean ± SD and analyzed by Dunnett’s test (A, D and E) and an unpaired t -test (B and C). At least three independent experiments were performed. * p
    Figure Legend Snippet: Ghrelin signaling is necessary and sufficient to inhibit IL-1β-induced catabolic gene expression and NF-κB activation in chondrocytes. (A) RT-qPCR analysis of Col-II, Col-X, iNOS, MMP1, MMP9 and MMP13 on nHACs treated with ghrelin (0, 10 and 100nM) and IL-1β (1ng/mL). TBP served as a reference gene. (B) NF-κB transactivation assay. nHACs were transiently transfected with NF-κB reporter construct for 24 hours and then treated with ghrelin (10nM) and IL-1β (1ng/mL). A Renilla luciferase construct was co-transfected as an internal control for normalization. (C) Serum Response Element (SRE) luciferase reporter assay. nHACs were transiently transfected with the SRE reporter construct for 24 hours and then treated with ghrelin (10nM) or EM (10μM) for 16 hours. A Renilla luciferase construct was co-transfected as an internal control for normalization. Data are presented as fold activation relative to untreated samples. (D) RT-qPCR analysis of MMP13 on WT and GHSR null mouse chondrocytes treated with ghrelin (0, 10, 100nM) and IL-1β (1ng/mL). TBP served as a reference gene. (E) ICC analysis with an anti-EM antibody on WT chondrocytes co-treated with EM (10μM) and different concentrations of ghrelin (1, 10 and 100nM). Arrows indicate cells with EM staining. Percentage of EM-positive cells was calculated. Data were reported as mean ± SD and analyzed by Dunnett’s test (A, D and E) and an unpaired t -test (B and C). At least three independent experiments were performed. * p

    Techniques Used: Expressing, Activation Assay, Quantitative RT-PCR, Transactivation Assay, Transfection, Construct, Luciferase, Reporter Assay, Immunocytochemistry, Staining

    The Ghrelin receptor is required for EM to inhibit cartilage matrix loss and synovitis in vivo . (A) Analysis of total glucose consumption, intracellular ATP levels and reactive oxygen species (ROS) levels at day 2 of culture in healthy and OA human articular chondrocytes. (B) Analysis of total glucose consumption, intracellular ATP levels and reactive oxygen species (ROS) levels at day 2 of culture in nHACs after MIA treatment. (C) RT-qPCR analysis of aggrecan (agg), Col-II, Col-X, IL-1β, TNF⍺ and MMP13 on nHACs treated with MIA (0, 0.1 and 1μM) for 2 days. TBP served as a reference gene for normalization. (D) Safranin O staining analysis of WT and GHSR null mouse knee joints. MIA (50μg/joint) was intra-articularly injected to knee joints at day 0, and samples were harvested at day 7. The degree of cartilage matrix loss was examined according to the OARSI scoring system. (E) Safranin O staining analysis of WT and GHSR null mouse knee joints injected with MIA. MIA was intra-articularly injected to knee joints at day 0. EM (PBS as control) was IP-injected at 50μg/g into the mice daily until sample harvest at day 7. The degree of cartilage matrix loss was examined according to the OARSI scoring system. (F) Synovitis analysis on sections from the medial tibial plateau of WT and GHSR null mice knees injected with EM. MIA was intra-articularly injected to knee joints at day 0. EM (PBS as control) was IP-injected into the mice daily until sample harvest at day 7. Arrowheads indicate thickening of the synovial layers and increased cellularity of resident cells. M = Meniscus. T = Tibia. F=Femur. Synovitis was scored according to established synovitis scoring protocols. Scale bar = 200μm. For A, data were reported as mean ± SD and analyzed by an unpaired t -test. For B and C, data were reported as mean ± SD and analyzed by Dunnett’s test. Comparisons were made between MIA treatments over untreated controls for each gene. For D-F, data were reported as a box plot, where the median, as well as the maximum and the minimum data points were indicated. Data from D-F were analyzed by Kruskal-Wallis statistical test followed by Mann-Whitney U test. * p
    Figure Legend Snippet: The Ghrelin receptor is required for EM to inhibit cartilage matrix loss and synovitis in vivo . (A) Analysis of total glucose consumption, intracellular ATP levels and reactive oxygen species (ROS) levels at day 2 of culture in healthy and OA human articular chondrocytes. (B) Analysis of total glucose consumption, intracellular ATP levels and reactive oxygen species (ROS) levels at day 2 of culture in nHACs after MIA treatment. (C) RT-qPCR analysis of aggrecan (agg), Col-II, Col-X, IL-1β, TNF⍺ and MMP13 on nHACs treated with MIA (0, 0.1 and 1μM) for 2 days. TBP served as a reference gene for normalization. (D) Safranin O staining analysis of WT and GHSR null mouse knee joints. MIA (50μg/joint) was intra-articularly injected to knee joints at day 0, and samples were harvested at day 7. The degree of cartilage matrix loss was examined according to the OARSI scoring system. (E) Safranin O staining analysis of WT and GHSR null mouse knee joints injected with MIA. MIA was intra-articularly injected to knee joints at day 0. EM (PBS as control) was IP-injected at 50μg/g into the mice daily until sample harvest at day 7. The degree of cartilage matrix loss was examined according to the OARSI scoring system. (F) Synovitis analysis on sections from the medial tibial plateau of WT and GHSR null mice knees injected with EM. MIA was intra-articularly injected to knee joints at day 0. EM (PBS as control) was IP-injected into the mice daily until sample harvest at day 7. Arrowheads indicate thickening of the synovial layers and increased cellularity of resident cells. M = Meniscus. T = Tibia. F=Femur. Synovitis was scored according to established synovitis scoring protocols. Scale bar = 200μm. For A, data were reported as mean ± SD and analyzed by an unpaired t -test. For B and C, data were reported as mean ± SD and analyzed by Dunnett’s test. Comparisons were made between MIA treatments over untreated controls for each gene. For D-F, data were reported as a box plot, where the median, as well as the maximum and the minimum data points were indicated. Data from D-F were analyzed by Kruskal-Wallis statistical test followed by Mann-Whitney U test. * p

    Techniques Used: In Vivo, Quantitative RT-PCR, Staining, Injection, Mouse Assay, MANN-WHITNEY

    10) Product Images from "Altered levels of soluble CD18 may associate immune mechanisms with outcome in sepsis"

    Article Title: Altered levels of soluble CD18 may associate immune mechanisms with outcome in sepsis

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/cei.13016

    In‐vitro shedding of CD18 and function of sCD18. (a) Inflammatory induction of CD18 shedding. The levels of sCD18 were increased in the supernatants from peripheral blood mononuclear cells (PBMCs) incubated with tumour necrosis factor (TNF)‐α, phorbol‐12‐myristate‐13‐acetate (PMA), lipopolysaccharide (LPS), interleukin (IL)‐1β and prostaglandin E 2 (PGE 2 ). Lines indicate median and whiskers indicate interquartile range (IQR). Data were analysed using the Wilcoxon signed‐rank test. (b) Antagonistic function of sCD18. Depletion of sCD18 in healthy volunteer serum resulted in increased Mac‐1‐mediated cell adhesion. * P
    Figure Legend Snippet: In‐vitro shedding of CD18 and function of sCD18. (a) Inflammatory induction of CD18 shedding. The levels of sCD18 were increased in the supernatants from peripheral blood mononuclear cells (PBMCs) incubated with tumour necrosis factor (TNF)‐α, phorbol‐12‐myristate‐13‐acetate (PMA), lipopolysaccharide (LPS), interleukin (IL)‐1β and prostaglandin E 2 (PGE 2 ). Lines indicate median and whiskers indicate interquartile range (IQR). Data were analysed using the Wilcoxon signed‐rank test. (b) Antagonistic function of sCD18. Depletion of sCD18 in healthy volunteer serum resulted in increased Mac‐1‐mediated cell adhesion. * P

    Techniques Used: In Vitro, Incubation

    11) Product Images from "RAPAMYCIN ACTIVATES TGF RECEPTOR INDEPENDENTLY OF ITS LIGAND: IMPLICATIONS FOR ENDOTHELIAL DYSFUNCTION."

    Article Title: RAPAMYCIN ACTIVATES TGF RECEPTOR INDEPENDENTLY OF ITS LIGAND: IMPLICATIONS FOR ENDOTHELIAL DYSFUNCTION.

    Journal: Clinical science (London, England : 1979)

    doi: 10.1042/CS20171457

    Rapamycin induces Endothelial-to-Mesenchymal Transition, which is potentiated in the presence of IL-1β. (A – C) Immunocytochemistry of EndMT markers after rapamycin treatment for 96 hours in the presence or absence of TGFRI inhibitor. Endothelial markers in green, mesenchymal markers in red, nuclei in blue (DAPI). (A) Double staining for VE-cadherin and Phalloidin; (B) double staining for PECAM-1 and Calponin; (C) SM22. Each image is representative of 3 independent experiments. (D-G) Western blot for EndMT markers: (D) VE-Cadherin, N=7; (E) PECAM-1, N=4; (F) Calponin, N=8; (G) SM22, N=5. Each bar represents mean ± SEM. * indicates p
    Figure Legend Snippet: Rapamycin induces Endothelial-to-Mesenchymal Transition, which is potentiated in the presence of IL-1β. (A – C) Immunocytochemistry of EndMT markers after rapamycin treatment for 96 hours in the presence or absence of TGFRI inhibitor. Endothelial markers in green, mesenchymal markers in red, nuclei in blue (DAPI). (A) Double staining for VE-cadherin and Phalloidin; (B) double staining for PECAM-1 and Calponin; (C) SM22. Each image is representative of 3 independent experiments. (D-G) Western blot for EndMT markers: (D) VE-Cadherin, N=7; (E) PECAM-1, N=4; (F) Calponin, N=8; (G) SM22, N=5. Each bar represents mean ± SEM. * indicates p

    Techniques Used: Immunocytochemistry, Double Staining, Western Blot

    12) Product Images from "Muscle cells enhance resistance to pro-inflammatory cytokine-induced cartilage destruction"

    Article Title: Muscle cells enhance resistance to pro-inflammatory cytokine-induced cartilage destruction

    Journal:

    doi: 10.1016/j.bbrc.2009.12.138

    Cartilage-specific collagen expression in muscle and cartilage cell cocultures upon IL-1β (1ng/ml) and TNFα (2ng/ml) treatment
    Figure Legend Snippet: Cartilage-specific collagen expression in muscle and cartilage cell cocultures upon IL-1β (1ng/ml) and TNFα (2ng/ml) treatment

    Techniques Used: Expressing

    13) Product Images from "A dual role for microglia in promoting tissue inhibitor of metalloproteinase (TIMP) expression in glial cells in response to neuroinflammatory stimuli"

    Article Title: A dual role for microglia in promoting tissue inhibitor of metalloproteinase (TIMP) expression in glial cells in response to neuroinflammatory stimuli

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-8-61

    Analysis of glial cell TIMP-1 expression in response to cytokines and LPS . TIMP-1 expression was examined by western blot of cell culture supernatants, as described in Materials and Methods. A. The influence of LPS on TIMP-1 expression in the three different types of glial culture. MGC, astrocytes, and microglial cultures were treated with LPS for 2 days, then cell culture supernatants examined for TIMP-1 expression. Note that cultures not treated with LPS did not express TIMP-1, and TIMP-1 protein (~28 kD band) was induced only in MGC following LPS stimulation. B. TIMP-1 induction in astrocytes in the presence of activated microglia or MG-CM. Pure astrocyte cultures received either: LPS, microglia + LPS, or LPS-activated MG-CM. Note that the presence of LPS-activated microglia or LPS-activated MG-CM strongly induced TIMP-1 expression in astrocytes. C. The influence of cytokines on TIMP-1 expression by astrocytes. Pure astrocyte cultures were treated with a panel of different cytokines for 2 days, and the supernatants analyzed for TIMP-1 expression. Note that of the cytokines tested, only IL-1β stimulated TIMP-1 expression in astrocytes.
    Figure Legend Snippet: Analysis of glial cell TIMP-1 expression in response to cytokines and LPS . TIMP-1 expression was examined by western blot of cell culture supernatants, as described in Materials and Methods. A. The influence of LPS on TIMP-1 expression in the three different types of glial culture. MGC, astrocytes, and microglial cultures were treated with LPS for 2 days, then cell culture supernatants examined for TIMP-1 expression. Note that cultures not treated with LPS did not express TIMP-1, and TIMP-1 protein (~28 kD band) was induced only in MGC following LPS stimulation. B. TIMP-1 induction in astrocytes in the presence of activated microglia or MG-CM. Pure astrocyte cultures received either: LPS, microglia + LPS, or LPS-activated MG-CM. Note that the presence of LPS-activated microglia or LPS-activated MG-CM strongly induced TIMP-1 expression in astrocytes. C. The influence of cytokines on TIMP-1 expression by astrocytes. Pure astrocyte cultures were treated with a panel of different cytokines for 2 days, and the supernatants analyzed for TIMP-1 expression. Note that of the cytokines tested, only IL-1β stimulated TIMP-1 expression in astrocytes.

    Techniques Used: Expressing, Western Blot, Cell Culture

    Examining the role of microglia-derived IL-1β in promoting astrocyte TIMP-1 expression . A. Determination of IL-1β in LPS-activated MG-CM. Microglial cultures were treated with LPS for 2 days, then subject to either six rounds of IL-1β immunodepletion, or mock depletion, as described in Materials and Methods, and samples analyzed for the presence of IL-1β. Note that IL-1β was detected in the LPS-activated MG-CM before, but not after the IL-1β immunodepletion, demonstrating the effectiveness of the immunodepletion strategy. B and C. The effect of removing IL-1β from LPS-activated MG-CM on astrocyte TIMP-1 production. Pure astrocyte cultures received either: LPS, microglia + LPS, LPS-activated MG-CM (mock depleted) or LPS-activated MG-CM depleted of IL-1β. Note that the presence of LPS-activated microglia or LPS-activated MG-CM strongly induced TIMP-1 expression in astrocytes, but this effect was markedly attenuated after IL-1β removal from the MG-CM. * p
    Figure Legend Snippet: Examining the role of microglia-derived IL-1β in promoting astrocyte TIMP-1 expression . A. Determination of IL-1β in LPS-activated MG-CM. Microglial cultures were treated with LPS for 2 days, then subject to either six rounds of IL-1β immunodepletion, or mock depletion, as described in Materials and Methods, and samples analyzed for the presence of IL-1β. Note that IL-1β was detected in the LPS-activated MG-CM before, but not after the IL-1β immunodepletion, demonstrating the effectiveness of the immunodepletion strategy. B and C. The effect of removing IL-1β from LPS-activated MG-CM on astrocyte TIMP-1 production. Pure astrocyte cultures received either: LPS, microglia + LPS, LPS-activated MG-CM (mock depleted) or LPS-activated MG-CM depleted of IL-1β. Note that the presence of LPS-activated microglia or LPS-activated MG-CM strongly induced TIMP-1 expression in astrocytes, but this effect was markedly attenuated after IL-1β removal from the MG-CM. * p

    Techniques Used: Derivative Assay, Expressing

    14) Product Images from "Salidroside Regulates Inflammatory Response in Raw 264.7 Macrophages via TLR4/TAK1 and Ameliorates Inflammation in Alcohol Binge Drinking-Induced Liver Injury"

    Article Title: Salidroside Regulates Inflammatory Response in Raw 264.7 Macrophages via TLR4/TAK1 and Ameliorates Inflammation in Alcohol Binge Drinking-Induced Liver Injury

    Journal: Molecules

    doi: 10.3390/molecules21111490

    Effect of SDS on binge drinking induced liver injury. C57BL/6 mice were orally administrated SDS (100 mg/kg) for 5 days, followed by three gavages of dextrin maltose or ethanol every 30 min. ( A ) Representative H E staining and Oil Red O staining of liver tissues; ( B ) serum ALT and AST; ( C ) serum IL-1β protein. Black arrows represent damaged hepatocytes. Values represent means ± SD. * p
    Figure Legend Snippet: Effect of SDS on binge drinking induced liver injury. C57BL/6 mice were orally administrated SDS (100 mg/kg) for 5 days, followed by three gavages of dextrin maltose or ethanol every 30 min. ( A ) Representative H E staining and Oil Red O staining of liver tissues; ( B ) serum ALT and AST; ( C ) serum IL-1β protein. Black arrows represent damaged hepatocytes. Values represent means ± SD. * p

    Techniques Used: Mouse Assay, Staining, AST Assay

    Effect of SDS on the synthesis and release of proinflammatory cytokines. RAW 264.7 cells were pretreated with SDS (100 μM) for 1 h, followed by stimulated with LPS (1 μg/mL) for 1, 3 or 6 h. mRNA expression of IL-1β ( A ) and TNF-α ( C ) was analyzed by real time PCR. Release of IL-1β ( B ) and TNF-α ( D ) in culture media was measured using ELISA. Data are expressed as means ± SD of three independent experiments. ## p
    Figure Legend Snippet: Effect of SDS on the synthesis and release of proinflammatory cytokines. RAW 264.7 cells were pretreated with SDS (100 μM) for 1 h, followed by stimulated with LPS (1 μg/mL) for 1, 3 or 6 h. mRNA expression of IL-1β ( A ) and TNF-α ( C ) was analyzed by real time PCR. Release of IL-1β ( B ) and TNF-α ( D ) in culture media was measured using ELISA. Data are expressed as means ± SD of three independent experiments. ## p

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

    15) Product Images from "The novel NADPH oxidase 4 selective inhibitor GLX7013114 counteracts human islet cell death in vitro"

    Article Title: The novel NADPH oxidase 4 selective inhibitor GLX7013114 counteracts human islet cell death in vitro

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0204271

    Immunoblot analysis of Nox2 and Nox4 in human islets (A-C) and EndoC- β H1 cells (D). Nox2 (A) and Nox4 (B) expression was assessed after 4h and 24h of IL-1β (20 ng/ml) + IFN-γ (20 ng/ml) (cyt) and palmitate (1.5 mM in 2% BSA) + high glucose (20 mM) (PH) exposure. The expression of Nox2 and Nox4 was normalized to total ERK, which was used as a loading control. Results are means ± S.E.M for 5 independent experiments. * denotes P
    Figure Legend Snippet: Immunoblot analysis of Nox2 and Nox4 in human islets (A-C) and EndoC- β H1 cells (D). Nox2 (A) and Nox4 (B) expression was assessed after 4h and 24h of IL-1β (20 ng/ml) + IFN-γ (20 ng/ml) (cyt) and palmitate (1.5 mM in 2% BSA) + high glucose (20 mM) (PH) exposure. The expression of Nox2 and Nox4 was normalized to total ERK, which was used as a loading control. Results are means ± S.E.M for 5 independent experiments. * denotes P

    Techniques Used: Expressing

    16) Product Images from "Colchicine inhibits cationic dye uptake induced by ATP in P2X2 and P2X7 receptor-expressing cells: implications for its therapeutic action"

    Article Title: Colchicine inhibits cationic dye uptake induced by ATP in P2X2 and P2X7 receptor-expressing cells: implications for its therapeutic action

    Journal: British Journal of Pharmacology

    doi: 10.1111/j.1476-5381.2011.01254.x

    ROS, NO and IL-1β production induced by ATP are inhibited by colchicine. Macrophages obtained by peritoneal wash were pretreated with or without 50 µM colchicine for 30 min, followed by incubation with H 2 CFDA for additional 30 min. ATP
    Figure Legend Snippet: ROS, NO and IL-1β production induced by ATP are inhibited by colchicine. Macrophages obtained by peritoneal wash were pretreated with or without 50 µM colchicine for 30 min, followed by incubation with H 2 CFDA for additional 30 min. ATP

    Techniques Used: Incubation

    17) Product Images from "Rescue of IL-1β-induced reduction of human neurogenesis by omega-3 fatty acids and antidepressants"

    Article Title: Rescue of IL-1β-induced reduction of human neurogenesis by omega-3 fatty acids and antidepressants

    Journal: Brain, Behavior, and Immunity

    doi: 10.1016/j.bbi.2017.05.006

    IL-1β modulates the production of metabolites and enzymes of the kynurenine pathway. Treatment with IL-1β affected the kynurenine pathway by (a) increasing levels of kynurenine and quinolinic acid and decreasing levels of anthranilic acid released into the supernatant, and (b) up-regulating the expression of IDO, KMO, KYNU and ACMSD; * p
    Figure Legend Snippet: IL-1β modulates the production of metabolites and enzymes of the kynurenine pathway. Treatment with IL-1β affected the kynurenine pathway by (a) increasing levels of kynurenine and quinolinic acid and decreasing levels of anthranilic acid released into the supernatant, and (b) up-regulating the expression of IDO, KMO, KYNU and ACMSD; * p

    Techniques Used: Expressing

    Monoaminergic antidepressants and ω-3 fatty acids reverse the IL-β-induced reduction of human hippocampal cells neurogenesis. Treatment with IL-1β decrease DCX - positive cells (immature neuronal phenotype), and MAP2 positive cells (mature neuronal phenotype) (a). Treatment with EPA, DHA, sertraline or venlafaxine reverse the IL-1β-induced decrease in DCX positive cells (b), and MAP2-positive cells (c); **** p
    Figure Legend Snippet: Monoaminergic antidepressants and ω-3 fatty acids reverse the IL-β-induced reduction of human hippocampal cells neurogenesis. Treatment with IL-1β decrease DCX - positive cells (immature neuronal phenotype), and MAP2 positive cells (mature neuronal phenotype) (a). Treatment with EPA, DHA, sertraline or venlafaxine reverse the IL-1β-induced decrease in DCX positive cells (b), and MAP2-positive cells (c); **** p

    Techniques Used:

    Antidepressants and ω-3 fatty acids modulate changes of kynurenine pathway metabolites caused by IL-1β treatment. Co-treatment of cells with IL-1β and ω-3 fatty acids partially abrogated the increase in kynurenine caused by the cytokine (a). Co-treatment with sertraline fully reverted the decrease in levels of ANA caused by IL-1β (b). Co-incubation with DHA and sertraline partially reverted the increase in QUIN levels caused by IL-1β (c). No significant changes in PIC were observed by any co-incubation (d); * p
    Figure Legend Snippet: Antidepressants and ω-3 fatty acids modulate changes of kynurenine pathway metabolites caused by IL-1β treatment. Co-treatment of cells with IL-1β and ω-3 fatty acids partially abrogated the increase in kynurenine caused by the cytokine (a). Co-treatment with sertraline fully reverted the decrease in levels of ANA caused by IL-1β (b). Co-incubation with DHA and sertraline partially reverted the increase in QUIN levels caused by IL-1β (c). No significant changes in PIC were observed by any co-incubation (d); * p

    Techniques Used: Incubation

    Antidepressants and ω-3 fatty acids modulate IL-1β-induced changes to the expression of enzymes of the kynurenine pathway. Co-treatment of cells with IL-1β and ω-3 fatty acids or antidepressants decreased the expression of IDO (a). Co-treatment with DHA, sertraline or venlafaxine decreased expression of KMO (b). Co-treatment with DHA upregulated KMO production (c). Co-incubation with sertraline or venlafaxine decreased the upregulation of ACMSD (d); * p
    Figure Legend Snippet: Antidepressants and ω-3 fatty acids modulate IL-1β-induced changes to the expression of enzymes of the kynurenine pathway. Co-treatment of cells with IL-1β and ω-3 fatty acids or antidepressants decreased the expression of IDO (a). Co-treatment with DHA, sertraline or venlafaxine decreased expression of KMO (b). Co-treatment with DHA upregulated KMO production (c). Co-incubation with sertraline or venlafaxine decreased the upregulation of ACMSD (d); * p

    Techniques Used: Expressing, Incubation

    18) Product Images from "Benzylpenicillin differentially conjugates to IFN-?, TNF-?, IL-1?, IL-4 and IL-13 but selectively reduces IFN-? activity"

    Article Title: Benzylpenicillin differentially conjugates to IFN-?, TNF-?, IL-1?, IL-4 and IL-13 but selectively reduces IFN-? activity

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.2003.02069.x

    Effect of BP on the ability of(a)IL-1β,(b)TNF-α or(c)IFN-γ to induce CD54 expression on A549 cells. Each cytokine (100 ng/ml) was incubated in RT2 with (○) or without (•) BP (2 mg/ml) for 4 days at 37°C, then each preparation was assayed at 0·02, 0·2, 2 or 10 ng/ml. Confluent layers of A549 cells in 96-well plates were incubated overnight with cytokine preparations. After fixing, CD54 expression was measured by ELISA. ▴ Cytokine incubated 4 days alone, 2 mg/ml BP added immediately before assay. Results represent mean ± SEM of 3 (IL-1β and TNF-α) or 4 (IFN-γ) experiments, each performed in triplictate. * P
    Figure Legend Snippet: Effect of BP on the ability of(a)IL-1β,(b)TNF-α or(c)IFN-γ to induce CD54 expression on A549 cells. Each cytokine (100 ng/ml) was incubated in RT2 with (○) or without (•) BP (2 mg/ml) for 4 days at 37°C, then each preparation was assayed at 0·02, 0·2, 2 or 10 ng/ml. Confluent layers of A549 cells in 96-well plates were incubated overnight with cytokine preparations. After fixing, CD54 expression was measured by ELISA. ▴ Cytokine incubated 4 days alone, 2 mg/ml BP added immediately before assay. Results represent mean ± SEM of 3 (IL-1β and TNF-α) or 4 (IFN-γ) experiments, each performed in triplictate. * P

    Techniques Used: Expressing, Incubation, Enzyme-linked Immunosorbent Assay

    19) Product Images from "Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells"

    Article Title: Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003100

    Activation of gp120-primed DC reduced the expression of Bcl2 and activated Akt, and the induction of cell apoptosis is ASK1-dependent. ( A ) moDC treated with immune-complex gp120 ADA were exposed to CL40L Tf or mock Tf, or LPS, TNFα, IL-1β for 3 d, and cellular proteins of moDC (recovered from coculture) were extracted for Western blotting analysis. MoDC treated by anti-His Ab (used to cross-link recombinant gp120 ADA ) were used as a control (control DC). *p-ASK1 represent results from untreated moDC (lane 1) and DC treated as indicated but no gp120 pulsing (lanes 2–6), whereas #p-ASK-1 represents results from pre-treatment of anti-DC-SIGN mAbs. Data are representative of 3 experiments. ( B ) moDC were transfected with siRNA against human ASK1 or control siRNA (scrambled) before pulse with immune-complex gp120 and subsequent exposure to CD40L Tf, LPS, TNFα or IL-1β. Data are expressed as mean ± SD from 3 experiments. **p
    Figure Legend Snippet: Activation of gp120-primed DC reduced the expression of Bcl2 and activated Akt, and the induction of cell apoptosis is ASK1-dependent. ( A ) moDC treated with immune-complex gp120 ADA were exposed to CL40L Tf or mock Tf, or LPS, TNFα, IL-1β for 3 d, and cellular proteins of moDC (recovered from coculture) were extracted for Western blotting analysis. MoDC treated by anti-His Ab (used to cross-link recombinant gp120 ADA ) were used as a control (control DC). *p-ASK1 represent results from untreated moDC (lane 1) and DC treated as indicated but no gp120 pulsing (lanes 2–6), whereas #p-ASK-1 represents results from pre-treatment of anti-DC-SIGN mAbs. Data are representative of 3 experiments. ( B ) moDC were transfected with siRNA against human ASK1 or control siRNA (scrambled) before pulse with immune-complex gp120 and subsequent exposure to CD40L Tf, LPS, TNFα or IL-1β. Data are expressed as mean ± SD from 3 experiments. **p

    Techniques Used: Activation Assay, Expressing, Western Blot, Recombinant, Transfection

    Cross-linked recombinant gp120 or HIV(+) serum sensitizes moDC for apoptosis after activation by LPS, TNF-α or IL-1β, and DC-SIGN(+) cells in HIV(+) blood are pre-sensitized for LPS/TNFα/IL-1β-induced apoptosis. ( A,B ) moDC were treated with gp120 ADA in the presence of isotype control or anti-DC-SIGN Abs and subsequently cultured in the absence or presence of 100 ng/ml LPS for 3 d. Data are representative of 5 experiments in A and are expressed as mean ± SD in B ; **p
    Figure Legend Snippet: Cross-linked recombinant gp120 or HIV(+) serum sensitizes moDC for apoptosis after activation by LPS, TNF-α or IL-1β, and DC-SIGN(+) cells in HIV(+) blood are pre-sensitized for LPS/TNFα/IL-1β-induced apoptosis. ( A,B ) moDC were treated with gp120 ADA in the presence of isotype control or anti-DC-SIGN Abs and subsequently cultured in the absence or presence of 100 ng/ml LPS for 3 d. Data are representative of 5 experiments in A and are expressed as mean ± SD in B ; **p

    Techniques Used: Recombinant, Activation Assay, Cell Culture

    20) Product Images from "Transendothelial Migration Enables Subsequent Transmigration of Neutrophils through Underlying Pericytes"

    Article Title: Transendothelial Migration Enables Subsequent Transmigration of Neutrophils through Underlying Pericytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0060025

    CD18-dependent post-transendothelial migration. (A) CD18 (β 2 integrin) expression on naïve and transendothelial migrated (TEM) neutrophils (across 4 hr IL-1β activated EC monolayers) and (B) naive 0.7 ng/ml IL-8 and 25 ng/ml IL-8 stimulated neutrophils assessed by flow cytometry. Table 1 . Corrected mean fluorescent intensity (MFI) reflect CD18 expression from flow cytometry. (C) Neutrophil adhesion on EC, PC, post-TEM PC, or post-TEM ICAM-1+PC monolayers following 4 hr IL-1β activation. Freshly isolated neutrophils were pre-incubated with anti-CD18 antibodies and seeded onto the monolayers in Sykes-Moore chambers and allowed to adhere prior to counting. Bars represent average neutrophil adhesion ± SEM. *P
    Figure Legend Snippet: CD18-dependent post-transendothelial migration. (A) CD18 (β 2 integrin) expression on naïve and transendothelial migrated (TEM) neutrophils (across 4 hr IL-1β activated EC monolayers) and (B) naive 0.7 ng/ml IL-8 and 25 ng/ml IL-8 stimulated neutrophils assessed by flow cytometry. Table 1 . Corrected mean fluorescent intensity (MFI) reflect CD18 expression from flow cytometry. (C) Neutrophil adhesion on EC, PC, post-TEM PC, or post-TEM ICAM-1+PC monolayers following 4 hr IL-1β activation. Freshly isolated neutrophils were pre-incubated with anti-CD18 antibodies and seeded onto the monolayers in Sykes-Moore chambers and allowed to adhere prior to counting. Bars represent average neutrophil adhesion ± SEM. *P

    Techniques Used: Migration, Expressing, Transmission Electron Microscopy, Flow Cytometry, Cytometry, Activation Assay, Isolation, Incubation

    IL-8 expression and presentation. (A) EC or PC monolayers and EC/PC bilayers were grown until confluence on Transwells and incubated for 4 hr with IL-1β. Following incubation, media were removed and analyzed for IL-8 by ELISA. Bars represent average IL-8 expression ± SEM. IL-1β activated (B) EC and (C) PC monolayers were stained and imaged for cell surface bound IL-8. Bar in B and C 100 µm.
    Figure Legend Snippet: IL-8 expression and presentation. (A) EC or PC monolayers and EC/PC bilayers were grown until confluence on Transwells and incubated for 4 hr with IL-1β. Following incubation, media were removed and analyzed for IL-8 by ELISA. Bars represent average IL-8 expression ± SEM. IL-1β activated (B) EC and (C) PC monolayers were stained and imaged for cell surface bound IL-8. Bar in B and C 100 µm.

    Techniques Used: Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Staining

    Neutrophil adhesion to IL-1β-activated EC or PC monolayers. Freshly isolated (naïve) neutrophils were seeded onto EC or PC monolayers previously activated with IL-1β for 4 or 24 hr in Sykes-Moore chambers and allowed to adhere prior to counting. Data are represented (A) as a line plot of neutrophil adhesion to EC or PC or as a scatterplot of neutrophil adhesion to (B) EC or (C) PC. Line data represents average neutrophil adhesion ± SEM. *P
    Figure Legend Snippet: Neutrophil adhesion to IL-1β-activated EC or PC monolayers. Freshly isolated (naïve) neutrophils were seeded onto EC or PC monolayers previously activated with IL-1β for 4 or 24 hr in Sykes-Moore chambers and allowed to adhere prior to counting. Data are represented (A) as a line plot of neutrophil adhesion to EC or PC or as a scatterplot of neutrophil adhesion to (B) EC or (C) PC. Line data represents average neutrophil adhesion ± SEM. *P

    Techniques Used: Isolation

    EC and PC ICAM-1 expression and effects of CD18 blocking on neutrophil adhesion. ICAM-1 expression on (A) EC or (D) PC monolayers before and after 4 hr IL-1β activation was assessed by flow cytometry (IgG labeled control white). ICAM-1 expression on (B,C) EC and (E,F) PC following 4 hr IL-1β activation was imaged using confocal microscopy. Scale bars are 20 µm. (G) Neutrophil adhesion inhibition to IL-1β activated EC or PC monolayers. Freshly isolated neutrophils were pre-incubated with anti-Mac-1 or anti-LFA-1 antibodies and seeded onto EC or PC monolayers (activated for 4 with IL-1β) in Sykes-Moore chambers and allowed to adhere prior to counting. Bars represent average neutrophil adhesion ± SEM. *P
    Figure Legend Snippet: EC and PC ICAM-1 expression and effects of CD18 blocking on neutrophil adhesion. ICAM-1 expression on (A) EC or (D) PC monolayers before and after 4 hr IL-1β activation was assessed by flow cytometry (IgG labeled control white). ICAM-1 expression on (B,C) EC and (E,F) PC following 4 hr IL-1β activation was imaged using confocal microscopy. Scale bars are 20 µm. (G) Neutrophil adhesion inhibition to IL-1β activated EC or PC monolayers. Freshly isolated neutrophils were pre-incubated with anti-Mac-1 or anti-LFA-1 antibodies and seeded onto EC or PC monolayers (activated for 4 with IL-1β) in Sykes-Moore chambers and allowed to adhere prior to counting. Bars represent average neutrophil adhesion ± SEM. *P

    Techniques Used: Expressing, Blocking Assay, Activation Assay, Flow Cytometry, Cytometry, Labeling, Confocal Microscopy, Inhibition, Isolation, Incubation

    ICAM-1 transduced PC. ICAM-1 expression on (A) non-transduced PC and (B) ICAM-1-transduced PC (ICAM-1+PC) monolayers following 4 hr of activation by IL-1β. ICAM-1 assessed using flow cytometry. ICAM-1 expression on (C,D) ICAM-1 transduced PC following 4 hr of IL-1β activation imaged using confocal microscopy. Bar in (C,D), 20 µm.(E) Neutrophil adhesion on EC, PC or ICAM-1 transduced PC monolayers following 4 hr of IL-1β activation. Freshly isolated neutrophils were pre-incubated with anti-CD18 antibodies and seeded onto the monolayers in Sykes-Moore chambers and allowed to adhere prior to counting. Bars represent average neutrophil adhesion ± SEM. # P
    Figure Legend Snippet: ICAM-1 transduced PC. ICAM-1 expression on (A) non-transduced PC and (B) ICAM-1-transduced PC (ICAM-1+PC) monolayers following 4 hr of activation by IL-1β. ICAM-1 assessed using flow cytometry. ICAM-1 expression on (C,D) ICAM-1 transduced PC following 4 hr of IL-1β activation imaged using confocal microscopy. Bar in (C,D), 20 µm.(E) Neutrophil adhesion on EC, PC or ICAM-1 transduced PC monolayers following 4 hr of IL-1β activation. Freshly isolated neutrophils were pre-incubated with anti-CD18 antibodies and seeded onto the monolayers in Sykes-Moore chambers and allowed to adhere prior to counting. Bars represent average neutrophil adhesion ± SEM. # P

    Techniques Used: Expressing, Activation Assay, Flow Cytometry, Cytometry, Confocal Microscopy, Isolation, Incubation

    Neutrophil transmigration across IL-1β activated EC or PC monolayers. Freshly isolated neutrophils were seeded onto monolayers (activated with IL-1β for 4 or 24 hr) and allowed to transmigrate for 1 hr. Data are represented as a (A) line plot of naive neutrophil transmigration across EC or PC, and transendothelial migrated neutrophils (Post-TEM) transmigration across PC. Figure 3B is demonstrative of (B) EC and PC co-cultured on a single membrane to form an EC/PC bilayer. (C) Freshly isolated naive neutrophils were seeded onto EC/PC bilayers (activated with IL-1β for 4 or 24 hr) and allowed to transmigrate for 1 hr. Line data represent average neutrophil transmigration ± SEM. # P
    Figure Legend Snippet: Neutrophil transmigration across IL-1β activated EC or PC monolayers. Freshly isolated neutrophils were seeded onto monolayers (activated with IL-1β for 4 or 24 hr) and allowed to transmigrate for 1 hr. Data are represented as a (A) line plot of naive neutrophil transmigration across EC or PC, and transendothelial migrated neutrophils (Post-TEM) transmigration across PC. Figure 3B is demonstrative of (B) EC and PC co-cultured on a single membrane to form an EC/PC bilayer. (C) Freshly isolated naive neutrophils were seeded onto EC/PC bilayers (activated with IL-1β for 4 or 24 hr) and allowed to transmigrate for 1 hr. Line data represent average neutrophil transmigration ± SEM. # P

    Techniques Used: Transmigration Assay, Isolation, Transmission Electron Microscopy, Cell Culture

    21) Product Images from "Inflammatory Cytokines Associated with Degenerative Disc Disease Control Aggrecanase-1 (ADAMTS-4) Expression in Nucleus Pulposus Cells through MAPK and NF-?B"

    Article Title: Inflammatory Cytokines Associated with Degenerative Disc Disease Control Aggrecanase-1 (ADAMTS-4) Expression in Nucleus Pulposus Cells through MAPK and NF-?B

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2013.02.037

    NF-κB regulation of ADAMTS-4 expression. A: Rat NP cells were transfected with RelA/p65, and ADAMTS4 promoter activity was measured. There was a dose-dependent increase in promoter activity up to 100 ng of p65. B: Cotransfection with RelB and c-Rel had no effect on ADAMTS4 promoter activity in rat NP cells. C: Rat NP cells were cotransfected with RelA/p65 and/or p50, and promoter activity was measured. When p65 and p50 were added together, p50 significantly blocked p65-mediated induction in promoter activity. D: Rat NP cells were cotransfected with p65 alone and with increasing doses of p50. Even at 50 ng, p50 completely blocked p65-mediated induction of promoter activity. E and F: Rat NP cells cotransfected with p50, and ADAMTS4 promoter activity was measured after TNF-α ( E ) and IL-1β ( F ) treatment. Cytokine-mediated induction in promoter activity was completely blocked by p50. G: TNF-α– and IL-1β–mediated induction in promoter activity was completely blocked by the NF-κB inhibitor SM7368. H: Cotransfection of cells with DN-NF-κB/IκBαM resulted in a significant inhibition of IL-1β–dependent ADAMTS4 promoter activity. Data are expressed as means ± SEM from three independent experiments. ∗ P
    Figure Legend Snippet: NF-κB regulation of ADAMTS-4 expression. A: Rat NP cells were transfected with RelA/p65, and ADAMTS4 promoter activity was measured. There was a dose-dependent increase in promoter activity up to 100 ng of p65. B: Cotransfection with RelB and c-Rel had no effect on ADAMTS4 promoter activity in rat NP cells. C: Rat NP cells were cotransfected with RelA/p65 and/or p50, and promoter activity was measured. When p65 and p50 were added together, p50 significantly blocked p65-mediated induction in promoter activity. D: Rat NP cells were cotransfected with p65 alone and with increasing doses of p50. Even at 50 ng, p50 completely blocked p65-mediated induction of promoter activity. E and F: Rat NP cells cotransfected with p50, and ADAMTS4 promoter activity was measured after TNF-α ( E ) and IL-1β ( F ) treatment. Cytokine-mediated induction in promoter activity was completely blocked by p50. G: TNF-α– and IL-1β–mediated induction in promoter activity was completely blocked by the NF-κB inhibitor SM7368. H: Cotransfection of cells with DN-NF-κB/IκBαM resulted in a significant inhibition of IL-1β–dependent ADAMTS4 promoter activity. Data are expressed as means ± SEM from three independent experiments. ∗ P

    Techniques Used: Expressing, Transfection, Activity Assay, Cotransfection, Inhibition

    Modulation of cytokine-dependent expression of ADAMTS-4 and -5 expression by NF-κB and MAPK signaling in rat NP cells. A and B: Western blot analysis of NF-κB and MAPK signaling proteins after treatment of NP cells with TNF-α ( A ) and IL-1β ( B ). Cytokine treatment induced phosphorylation of p65, p38, ERK, JNK within the first 15 minutes. No appreciable change was observed in expression of p65, p38, ERK, and JNK. C–F: RT-qPCR analysis of ADAMTS4 and ADAMTS5 expression by NP cells after treatment with TNF-α ( C and E ) or IL-1β ( D and F ) for 24 hours with or without inhibitors for NF-κB (SM7368, 10 μmol/L), p38 (SB203580, 10 μmol/L), ERK (PD98059, 10 μmol/L), and JNK (SP600125, 10 μmol/L). Inhibition of NF-κB signaling and MAPK signaling resulted in a significant blocking of cytokine-dependent induction in ADAMTS4 and ADAMTS5 mRNA expression. G and H: Western blot analysis indicates that treatment with NF-κB and MAPK inhibitors completely abolished ADAMTS-4 protein induction (the bands are indicated by asterisk ) by TNF-α ( G ) and IL-1β ( H ). Data are expressed as means ± SEM from three independent experiments. ∗ P
    Figure Legend Snippet: Modulation of cytokine-dependent expression of ADAMTS-4 and -5 expression by NF-κB and MAPK signaling in rat NP cells. A and B: Western blot analysis of NF-κB and MAPK signaling proteins after treatment of NP cells with TNF-α ( A ) and IL-1β ( B ). Cytokine treatment induced phosphorylation of p65, p38, ERK, JNK within the first 15 minutes. No appreciable change was observed in expression of p65, p38, ERK, and JNK. C–F: RT-qPCR analysis of ADAMTS4 and ADAMTS5 expression by NP cells after treatment with TNF-α ( C and E ) or IL-1β ( D and F ) for 24 hours with or without inhibitors for NF-κB (SM7368, 10 μmol/L), p38 (SB203580, 10 μmol/L), ERK (PD98059, 10 μmol/L), and JNK (SP600125, 10 μmol/L). Inhibition of NF-κB signaling and MAPK signaling resulted in a significant blocking of cytokine-dependent induction in ADAMTS4 and ADAMTS5 mRNA expression. G and H: Western blot analysis indicates that treatment with NF-κB and MAPK inhibitors completely abolished ADAMTS-4 protein induction (the bands are indicated by asterisk ) by TNF-α ( G ) and IL-1β ( H ). Data are expressed as means ± SEM from three independent experiments. ∗ P

    Techniques Used: Expressing, Western Blot, Quantitative RT-PCR, Inhibition, Blocking Assay

    MAPK signaling controls ADAMTS4 promoter activity in rat NP cells. A and B: Cotransfection of rat NP cells with DN-p38α abolished TNF-α–dependent ( A ) and IL-1β–dependent ( B ) induction in ADAMTS4 promoter activity. C and D: IL-1β–dependent increase in ADAMTS4 promoter activity was blocked by DN-p38β2 ( C ) and DN-p38γ ( D ). E and F: DN-ERK1 suppressed induction in ADAMTS4 promoter activity by TNF-α ( C ) and IL-1β ( D ) treatment. For IL-1β treatment, inhibition was seen only at the highest dose (150 ng). G: NP cells were cotransfected with LIP and ADAMTS4 promoter, and luciferase activity was measured after TNF-α treatment. Addition of LIP further increased TNF-α–dependent ADAMTS-4 reporter activity. H: ADAMTS4 promoter activity was measured after cotransfection with LAP2. LAP2 suppressed ADAMTS4 promoter activity. Data are expressed as means ± SEM from three independent experiments. ∗ P
    Figure Legend Snippet: MAPK signaling controls ADAMTS4 promoter activity in rat NP cells. A and B: Cotransfection of rat NP cells with DN-p38α abolished TNF-α–dependent ( A ) and IL-1β–dependent ( B ) induction in ADAMTS4 promoter activity. C and D: IL-1β–dependent increase in ADAMTS4 promoter activity was blocked by DN-p38β2 ( C ) and DN-p38γ ( D ). E and F: DN-ERK1 suppressed induction in ADAMTS4 promoter activity by TNF-α ( C ) and IL-1β ( D ) treatment. For IL-1β treatment, inhibition was seen only at the highest dose (150 ng). G: NP cells were cotransfected with LIP and ADAMTS4 promoter, and luciferase activity was measured after TNF-α treatment. Addition of LIP further increased TNF-α–dependent ADAMTS-4 reporter activity. H: ADAMTS4 promoter activity was measured after cotransfection with LAP2. LAP2 suppressed ADAMTS4 promoter activity. Data are expressed as means ± SEM from three independent experiments. ∗ P

    Techniques Used: Activity Assay, Cotransfection, Inhibition, Luciferase

    Expression and cytokine dependency of ADAMTS-4 in rat NP cells. A: RT-qPCR analysis shows ADAMTS-4 was expressed at a very low level in adult rat NP and annulus fibrosus (AF) tissues. B: Western blot analysis of ADAMTS4 expression in adult disk tissues reveals bands at 58 and 73 kDa, representing mature/processed protein. C and D: RT-qPCR analysis of ADAMTS4 expression by rat NP cells treated with the cytokines TNF-α ( C ) and IL-1β ( D ) for 24 hours. There was a dose-dependent increase in ADAMTS4 mRNA expression by the cytokine treatment. E and F: Western blot analysis of NP cells indicates increased expression of ADAMTS-4 after TNF-α and IL-1β treatment. G: Schematic of ADAMTS4 promoter constructs, showing important transcription factor and regulatory elements. H: Treatment of NP cells with TNF-α and IL-1β resulted in significant induction of ADAMTS4 promoter activity. Data are expressed as means ± SEM from three independent experiments. ∗ P
    Figure Legend Snippet: Expression and cytokine dependency of ADAMTS-4 in rat NP cells. A: RT-qPCR analysis shows ADAMTS-4 was expressed at a very low level in adult rat NP and annulus fibrosus (AF) tissues. B: Western blot analysis of ADAMTS4 expression in adult disk tissues reveals bands at 58 and 73 kDa, representing mature/processed protein. C and D: RT-qPCR analysis of ADAMTS4 expression by rat NP cells treated with the cytokines TNF-α ( C ) and IL-1β ( D ) for 24 hours. There was a dose-dependent increase in ADAMTS4 mRNA expression by the cytokine treatment. E and F: Western blot analysis of NP cells indicates increased expression of ADAMTS-4 after TNF-α and IL-1β treatment. G: Schematic of ADAMTS4 promoter constructs, showing important transcription factor and regulatory elements. H: Treatment of NP cells with TNF-α and IL-1β resulted in significant induction of ADAMTS4 promoter activity. Data are expressed as means ± SEM from three independent experiments. ∗ P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Construct, Activity Assay

    22) Product Images from "S-allylmercaptocysteine reduces carbon tetrachloride-induced hepatic oxidative stress and necroinflammation via nuclear factor kappa B-dependent pathways in mice"

    Article Title: S-allylmercaptocysteine reduces carbon tetrachloride-induced hepatic oxidative stress and necroinflammation via nuclear factor kappa B-dependent pathways in mice

    Journal: European Journal of Nutrition

    doi: 10.1007/s00394-011-0217-0

    Anti-inflammatory properties of SAMC. Expression levels of a TNF-α mRNA, b iNOS mRNA, c IL-1β mRNA, d IL-1β protein, e COX-2 mRNA, and f COX-2 protein were measured in mice liver after CCl 4 with or without pre-treatment of SAMC. GAPDH was used as internal controls for target gene’s mRNA expression. Data presented are expressed as Mean ± SEM ( n = 8–10) and experimental groups marked by different letters represented significant differences between groups at p
    Figure Legend Snippet: Anti-inflammatory properties of SAMC. Expression levels of a TNF-α mRNA, b iNOS mRNA, c IL-1β mRNA, d IL-1β protein, e COX-2 mRNA, and f COX-2 protein were measured in mice liver after CCl 4 with or without pre-treatment of SAMC. GAPDH was used as internal controls for target gene’s mRNA expression. Data presented are expressed as Mean ± SEM ( n = 8–10) and experimental groups marked by different letters represented significant differences between groups at p

    Techniques Used: Expressing, Mouse Assay

    23) Product Images from "Single cell analysis of kynurenine and System L amino acid transport in T cells"

    Article Title: Single cell analysis of kynurenine and System L amino acid transport in T cells

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04366-7

    System L-mediated transport of kynurenine drives AHR signalling. a CD4 + T cells were activated and maintained in TH17 conditions (CD3/CD28; IL-6, IL-1β and TGFb) (TH17 cond ). Uptake of 3 H-kynurenine in the presence or absence of BCH (5 mM) or non-radiolabelled kynurenine (5 mM) at times D2, D3 and D5 post activation is shown. b Histograms showing 450 nm fluorescence of D0, D3 and D5 TH17 cond CD4 + T cells treated with kynurenine (200 μM, 4 min) in the presence or absence of BCH (5 mM), or with no kynurenine. c Ratio of the 450 nm emission of D0, D3 and D5 TH17 cond CD4 + T cells treated with kynurenine ± BCH 5 mM (4 mins, 37 °C). d The graph shows the mean fluorescence intensity (MFI) of D3 TH17 cond CD4 + T cells treated with kynurenine (100 μM, 4 min, emission 450 nm) or FICZ (300 nM, 4 min, emission 530 nm) in the presence or absence of BCH (10 mM). e , f Representative flow cytometry histograms of ( d ); kynurenine uptake ( e ) and FICZ uptake ( f ). g The graph shows Cyp1a1 mRNA from D3 or D5 TH17 cond CD4 + T cells stimulated with kynurenine (100 μM, 2 h) or FICZ (300 nM, 2 hr), relative to non-treated control. h Induction of Cyp1a1 mRNA in D3 TH17 cond CD4 + T cells stimulated with kynurenine (100 μM, 2 h) or FICZ (300 nM, 2 h) in the presence or absence of BCH (25 mM). The data are shown relative to non-treated control. i Dose–response induction of Cyp1a1 mRNA in D3 TH17 cond CD4 + T cells stimulated with kynurenine at indicated doses for 2 h. The data are shown relative to non-treated control. j Induction of Cyp1a1 mRNA in D3 TH17 cond CD4 + T cells stimulated with kynurenine (10 μM, 2 h) in the presence or absence of AHR antagonist (CH223191, 30 μM). The data are shown relative to non-treated control. Error bars are ±s.d. Individual points indicate biological replicates. MFIs are indicated in the histograms, data are from three biological replicates ( a – g , i – j ) and six (Kyn) or three biological replicates (FICZ) ( h ). P values * =
    Figure Legend Snippet: System L-mediated transport of kynurenine drives AHR signalling. a CD4 + T cells were activated and maintained in TH17 conditions (CD3/CD28; IL-6, IL-1β and TGFb) (TH17 cond ). Uptake of 3 H-kynurenine in the presence or absence of BCH (5 mM) or non-radiolabelled kynurenine (5 mM) at times D2, D3 and D5 post activation is shown. b Histograms showing 450 nm fluorescence of D0, D3 and D5 TH17 cond CD4 + T cells treated with kynurenine (200 μM, 4 min) in the presence or absence of BCH (5 mM), or with no kynurenine. c Ratio of the 450 nm emission of D0, D3 and D5 TH17 cond CD4 + T cells treated with kynurenine ± BCH 5 mM (4 mins, 37 °C). d The graph shows the mean fluorescence intensity (MFI) of D3 TH17 cond CD4 + T cells treated with kynurenine (100 μM, 4 min, emission 450 nm) or FICZ (300 nM, 4 min, emission 530 nm) in the presence or absence of BCH (10 mM). e , f Representative flow cytometry histograms of ( d ); kynurenine uptake ( e ) and FICZ uptake ( f ). g The graph shows Cyp1a1 mRNA from D3 or D5 TH17 cond CD4 + T cells stimulated with kynurenine (100 μM, 2 h) or FICZ (300 nM, 2 hr), relative to non-treated control. h Induction of Cyp1a1 mRNA in D3 TH17 cond CD4 + T cells stimulated with kynurenine (100 μM, 2 h) or FICZ (300 nM, 2 h) in the presence or absence of BCH (25 mM). The data are shown relative to non-treated control. i Dose–response induction of Cyp1a1 mRNA in D3 TH17 cond CD4 + T cells stimulated with kynurenine at indicated doses for 2 h. The data are shown relative to non-treated control. j Induction of Cyp1a1 mRNA in D3 TH17 cond CD4 + T cells stimulated with kynurenine (10 μM, 2 h) in the presence or absence of AHR antagonist (CH223191, 30 μM). The data are shown relative to non-treated control. Error bars are ±s.d. Individual points indicate biological replicates. MFIs are indicated in the histograms, data are from three biological replicates ( a – g , i – j ) and six (Kyn) or three biological replicates (FICZ) ( h ). P values * =

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

    24) Product Images from "IL-1 receptor antagonist-deficient mice develop autoimmune arthritis due to intrinsic activation of IL-17-producing CCR2+Vγ6+γδ T cells"

    Article Title: IL-1 receptor antagonist-deficient mice develop autoimmune arthritis due to intrinsic activation of IL-17-producing CCR2+Vγ6+γδ T cells

    Journal: Nature Communications

    doi: 10.1038/ncomms8464

    IL-23 induces expression of IL-1R on the surface of γδ T cells, whereas IL-1Ra suppresses its expression. ( a , b ) Concentrations of IL-17 in culture supernatants of magnetic-activated cell sorting (MACS)-purified (about 80%) ( a ) or FACS-purified ( > 99% pure) ( b ) splenic γδ T cells from pools of 16 WT mice ( a , b ) or 16 Il1a −/− b −/− mice ( b ) stimulated for 3 days with medium only, IL-1β, IL-23 or IL-23 plus IL-1β, without γδ TCR stimulation. IL-17 was detected by enzyme-linked immunosorbent assay (ELISA). *** P
    Figure Legend Snippet: IL-23 induces expression of IL-1R on the surface of γδ T cells, whereas IL-1Ra suppresses its expression. ( a , b ) Concentrations of IL-17 in culture supernatants of magnetic-activated cell sorting (MACS)-purified (about 80%) ( a ) or FACS-purified ( > 99% pure) ( b ) splenic γδ T cells from pools of 16 WT mice ( a , b ) or 16 Il1a −/− b −/− mice ( b ) stimulated for 3 days with medium only, IL-1β, IL-23 or IL-23 plus IL-1β, without γδ TCR stimulation. IL-17 was detected by enzyme-linked immunosorbent assay (ELISA). *** P

    Techniques Used: Expressing, FACS, Magnetic Cell Separation, Purification, Mouse Assay, Enzyme-linked Immunosorbent Assay

    25) Product Images from "Altered environmental perception by parental stress and depression vulnerability: impact on mothers and offspring"

    Article Title: Altered environmental perception by parental stress and depression vulnerability: impact on mothers and offspring

    Journal: bioRxiv

    doi: 10.1101/2021.02.23.432580

    Graphical summary of main findings. Our results showed that mothers with different depression-like vulnerabilities facing parental stress (induced by MS) adopted different strategies. Although both strains increased the maternal behaviour in response to MS, non-depressive-like mothers (Wistar) exhibited a higher quality maternal behaviour (affiliative), showing a more adaptive response and more pro-social behaviour (more active strategy). On the other hand, depressive-like mothers (Kyoto) displayed a more passive/defensive strategy (non-adaptive response) in response to MS. The higher levels of oxytocin observed in Kyoto mothers that experienced MS seem to be part of the stress-response mechanism, which amplifies the negative perception of the environment (as insecure), leading to a more defensive behaviour by keeping the pups very close to them and away from outsiders (out-group anti-social behaviour). This increase in oxytocin expression seems to decrease the anxiety-like state in Kyoto dams in response to MS. Furthermore, the quality of maternal behaviour observed in non-depressive-like mothers (Wistar) after MS seems to protect the cognitive performance of adolescent rats from the negative effects of MS, leading to an increase of BDNF expression in the offspring hippocampus. On the other hand, depressive-like adolescents showed lower resilience to MS effects, exhibiting worst performance in the cognitive tests and alteration in the IL-1β levels.
    Figure Legend Snippet: Graphical summary of main findings. Our results showed that mothers with different depression-like vulnerabilities facing parental stress (induced by MS) adopted different strategies. Although both strains increased the maternal behaviour in response to MS, non-depressive-like mothers (Wistar) exhibited a higher quality maternal behaviour (affiliative), showing a more adaptive response and more pro-social behaviour (more active strategy). On the other hand, depressive-like mothers (Kyoto) displayed a more passive/defensive strategy (non-adaptive response) in response to MS. The higher levels of oxytocin observed in Kyoto mothers that experienced MS seem to be part of the stress-response mechanism, which amplifies the negative perception of the environment (as insecure), leading to a more defensive behaviour by keeping the pups very close to them and away from outsiders (out-group anti-social behaviour). This increase in oxytocin expression seems to decrease the anxiety-like state in Kyoto dams in response to MS. Furthermore, the quality of maternal behaviour observed in non-depressive-like mothers (Wistar) after MS seems to protect the cognitive performance of adolescent rats from the negative effects of MS, leading to an increase of BDNF expression in the offspring hippocampus. On the other hand, depressive-like adolescents showed lower resilience to MS effects, exhibiting worst performance in the cognitive tests and alteration in the IL-1β levels.

    Techniques Used: Expressing

    MS disturbed mothers’ neurobehaviour. After weaning, MS increased Kyoto (A) oxytocin levels, in agreement with the decreased of anxiety observed. (E) Kyoto showed high levels of TNF than Wistar strain. Quantification of (A) oxytocin (pg/ml) levels in hypothalamus by ELISA; OXTR in (B) PFC, (C) hippocampus and (D) amygdala by Western blotting; (E) TNF (pg/ml), (F) IL-6 (pg/ml) and (G) IL-1β (pg/ml) levels in hippocampus by ELISA. Data were analysed by two-way ANOVA (2 x 2 factorial design) with Bonferroni comparisons. (A), (B), (C), (F), (G) data were log-transformed. n = 5, except for (B) MS Kyoto, and (C) (D) for control Wistar: n = 4. Results are expressed as mean ±SEM. * p
    Figure Legend Snippet: MS disturbed mothers’ neurobehaviour. After weaning, MS increased Kyoto (A) oxytocin levels, in agreement with the decreased of anxiety observed. (E) Kyoto showed high levels of TNF than Wistar strain. Quantification of (A) oxytocin (pg/ml) levels in hypothalamus by ELISA; OXTR in (B) PFC, (C) hippocampus and (D) amygdala by Western blotting; (E) TNF (pg/ml), (F) IL-6 (pg/ml) and (G) IL-1β (pg/ml) levels in hippocampus by ELISA. Data were analysed by two-way ANOVA (2 x 2 factorial design) with Bonferroni comparisons. (A), (B), (C), (F), (G) data were log-transformed. n = 5, except for (B) MS Kyoto, and (C) (D) for control Wistar: n = 4. Results are expressed as mean ±SEM. * p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Western Blot, Transformation Assay

    MS worsened Kyoto adolescent offspring cognitive performance. MS increased Kyoto (A) latency to reach the platform, showing learning impairment. Regarding probe day (C), (D), (E), (F) and (G) MS slightly affected both strains. (H) MS did not impact working memory. (J) VGlut expression was higher in Kyoto strain. (K) MS led to an increase of BDNF level on Wistar, which may be related to the increase of affiliative behaviour received by their mothers. (M) MS altered IL1-β levels on Kyoto that can be associated with cognitive impairments presented. Quantification of (I) PSD95 and (J) VGlut in hippocampus by Western blotting; (K) BDNF (pg/ml), (L) IL-6 (pg/ml) and (M) IL-1β (pg/ml) levels in hippocampus by ELISA. (A), (H) data were analysed by linear regression. (C), (G), (I), (J), (K), (L) data were analysed by two-way ANOVA (2 x 2 factorial design) with Bonferroni comparations. (D), (E), (F) data were analysed by logistic regression. (I), (J), (M) data were log-transformed. (A), (C), (D), (E), (F) and (G) n = 22. (I), (J), (K), (L), (M) n = 16, except for (I) n = 13 for control Wistar and Kyoto, n = 11 for MS Wistar and Kyoto; (K) n = 12 for control Wistar and MS Kyoto, (L) n = 15 for control Wistar and n = 14 for MS Kyoto, (M) n = 15 for MS Kyoto. Results are expressed as mean ±SEM. * p
    Figure Legend Snippet: MS worsened Kyoto adolescent offspring cognitive performance. MS increased Kyoto (A) latency to reach the platform, showing learning impairment. Regarding probe day (C), (D), (E), (F) and (G) MS slightly affected both strains. (H) MS did not impact working memory. (J) VGlut expression was higher in Kyoto strain. (K) MS led to an increase of BDNF level on Wistar, which may be related to the increase of affiliative behaviour received by their mothers. (M) MS altered IL1-β levels on Kyoto that can be associated with cognitive impairments presented. Quantification of (I) PSD95 and (J) VGlut in hippocampus by Western blotting; (K) BDNF (pg/ml), (L) IL-6 (pg/ml) and (M) IL-1β (pg/ml) levels in hippocampus by ELISA. (A), (H) data were analysed by linear regression. (C), (G), (I), (J), (K), (L) data were analysed by two-way ANOVA (2 x 2 factorial design) with Bonferroni comparations. (D), (E), (F) data were analysed by logistic regression. (I), (J), (M) data were log-transformed. (A), (C), (D), (E), (F) and (G) n = 22. (I), (J), (K), (L), (M) n = 16, except for (I) n = 13 for control Wistar and Kyoto, n = 11 for MS Wistar and Kyoto; (K) n = 12 for control Wistar and MS Kyoto, (L) n = 15 for control Wistar and n = 14 for MS Kyoto, (M) n = 15 for MS Kyoto. Results are expressed as mean ±SEM. * p

    Techniques Used: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Transformation Assay

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    PeproTech recombinant murine il 1β protein
    Secretome profile of <t>IL-1β</t> treated spinal astrocytes reveals the expression of 24 cytokines and chemokines. (A) Coordinates of cytokines on array membranes and developed membranes. The Proteome Profiler array used in the experiments allowed the detection of 40 cytokines, chemokines and other soluble factors. Upper panel shows array coordinates. Middle panel shows expression of cytokines/chemokines in the supernatant of unstimulated spinal astrocytes, and lower panel shows expression of cytokines/chemokines by IL-1β stimulated astrocytes. (B) Heat map analysis of spinal astrocytic cytokine profile after 24 h of IL-1β stimulus. Left column shows array coordinates of cytokines/chemokines in the middle column and right column shows the fold change of their amount. (C) List of cytokines/chemokines which are significantly overexpressed in the supernatant of spinal astrocyte cultures upon IL-1β stimulation.
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    Secretome profile of IL-1β treated spinal astrocytes reveals the expression of 24 cytokines and chemokines. (A) Coordinates of cytokines on array membranes and developed membranes. The Proteome Profiler array used in the experiments allowed the detection of 40 cytokines, chemokines and other soluble factors. Upper panel shows array coordinates. Middle panel shows expression of cytokines/chemokines in the supernatant of unstimulated spinal astrocytes, and lower panel shows expression of cytokines/chemokines by IL-1β stimulated astrocytes. (B) Heat map analysis of spinal astrocytic cytokine profile after 24 h of IL-1β stimulus. Left column shows array coordinates of cytokines/chemokines in the middle column and right column shows the fold change of their amount. (C) List of cytokines/chemokines which are significantly overexpressed in the supernatant of spinal astrocyte cultures upon IL-1β stimulation.

    Journal: Frontiers in Physiology

    Article Title: IL-1β Induced Cytokine Expression by Spinal Astrocytes Can Play a Role in the Maintenance of Chronic Inflammatory Pain

    doi: 10.3389/fphys.2020.543331

    Figure Lengend Snippet: Secretome profile of IL-1β treated spinal astrocytes reveals the expression of 24 cytokines and chemokines. (A) Coordinates of cytokines on array membranes and developed membranes. The Proteome Profiler array used in the experiments allowed the detection of 40 cytokines, chemokines and other soluble factors. Upper panel shows array coordinates. Middle panel shows expression of cytokines/chemokines in the supernatant of unstimulated spinal astrocytes, and lower panel shows expression of cytokines/chemokines by IL-1β stimulated astrocytes. (B) Heat map analysis of spinal astrocytic cytokine profile after 24 h of IL-1β stimulus. Left column shows array coordinates of cytokines/chemokines in the middle column and right column shows the fold change of their amount. (C) List of cytokines/chemokines which are significantly overexpressed in the supernatant of spinal astrocyte cultures upon IL-1β stimulation.

    Article Snippet: Prior to the assay, cells were stimulated by 10 pg/ml recombinant murine IL-1β protein (PeproTech) for 24 h. Then the supernatants were pooled from 12 well non-treated, control and 12 well IL-1β-stimulated cultures.

    Techniques: Expressing

    IL-1β activates the NF-κB signaling pathway in spinal astrocyte cultures. (A) Representative immunoblots show time-dependent changes of the NF-κB signaling pathway upon IL-1β stimulation of spinal astrocyte cultures. NF-κB p65 expression increase after 2 h of stimulation in the cytosol and the p65 protein appears in the nuclear fraction after 2 h of treatment. The inhibitory iκB unit is downregulated in the cytosol of the cultures upon 4 h of stimulation. (B) Micrographs of single 1 μm thick laser scanning confocal optical sections illustrating cytoplasmic expression [GFAP astrocytic marker (green)] of p65 protein in control cultures (panels a–d ). The nuclear translocation of the p65 protein was observed after 2 (panels e–h ) and 4 h (panels i–l ) of IL-1β treatment. On all images DAPI was used to label cell nuclei (blue). Mixed color (purple) on the superimposed images ( d,h,l ) indicate double labeled structures. Scale bars: 10 μm.

    Journal: Frontiers in Physiology

    Article Title: IL-1β Induced Cytokine Expression by Spinal Astrocytes Can Play a Role in the Maintenance of Chronic Inflammatory Pain

    doi: 10.3389/fphys.2020.543331

    Figure Lengend Snippet: IL-1β activates the NF-κB signaling pathway in spinal astrocyte cultures. (A) Representative immunoblots show time-dependent changes of the NF-κB signaling pathway upon IL-1β stimulation of spinal astrocyte cultures. NF-κB p65 expression increase after 2 h of stimulation in the cytosol and the p65 protein appears in the nuclear fraction after 2 h of treatment. The inhibitory iκB unit is downregulated in the cytosol of the cultures upon 4 h of stimulation. (B) Micrographs of single 1 μm thick laser scanning confocal optical sections illustrating cytoplasmic expression [GFAP astrocytic marker (green)] of p65 protein in control cultures (panels a–d ). The nuclear translocation of the p65 protein was observed after 2 (panels e–h ) and 4 h (panels i–l ) of IL-1β treatment. On all images DAPI was used to label cell nuclei (blue). Mixed color (purple) on the superimposed images ( d,h,l ) indicate double labeled structures. Scale bars: 10 μm.

    Article Snippet: Prior to the assay, cells were stimulated by 10 pg/ml recombinant murine IL-1β protein (PeproTech) for 24 h. Then the supernatants were pooled from 12 well non-treated, control and 12 well IL-1β-stimulated cultures.

    Techniques: Western Blot, Expressing, Marker, Translocation Assay, Labeling

    Cultured spinal astrocytes express IL-1R1 and their activity is significantly increased upon IL-1β stimulation. (A) Cultured spinal astrocytes express the ligand binding unit or IL-1 receptor (IL-1R1). Micrographs of fluorescent images illustrating co-localization between GFAP astrocytic marker (a,c; green) and IL-1R1 (b,c; red). Panel a–c represent control cultures. Mixed colors (yellow) on the superimposed image (c) indicate double labeled structures. On all images DAPI was used to label cell nuclei (blue). (B) Dose-dependent (1–100 ng/ml) enhancement of astrocytic activity by IL-1β was determined by MTT assay after 24 h of treatment. Quantification of MTT activity is presented as fold change over the control cells in percentage. Data are shown as mean ± SEM of three independent experiments in duplicate assay (ANOVA Repeated Measures, followed by Tukey’s pairwise comparison ∗ p

    Journal: Frontiers in Physiology

    Article Title: IL-1β Induced Cytokine Expression by Spinal Astrocytes Can Play a Role in the Maintenance of Chronic Inflammatory Pain

    doi: 10.3389/fphys.2020.543331

    Figure Lengend Snippet: Cultured spinal astrocytes express IL-1R1 and their activity is significantly increased upon IL-1β stimulation. (A) Cultured spinal astrocytes express the ligand binding unit or IL-1 receptor (IL-1R1). Micrographs of fluorescent images illustrating co-localization between GFAP astrocytic marker (a,c; green) and IL-1R1 (b,c; red). Panel a–c represent control cultures. Mixed colors (yellow) on the superimposed image (c) indicate double labeled structures. On all images DAPI was used to label cell nuclei (blue). (B) Dose-dependent (1–100 ng/ml) enhancement of astrocytic activity by IL-1β was determined by MTT assay after 24 h of treatment. Quantification of MTT activity is presented as fold change over the control cells in percentage. Data are shown as mean ± SEM of three independent experiments in duplicate assay (ANOVA Repeated Measures, followed by Tukey’s pairwise comparison ∗ p

    Article Snippet: Prior to the assay, cells were stimulated by 10 pg/ml recombinant murine IL-1β protein (PeproTech) for 24 h. Then the supernatants were pooled from 12 well non-treated, control and 12 well IL-1β-stimulated cultures.

    Techniques: Cell Culture, Activity Assay, Ligand Binding Assay, Marker, Labeling, MTT Assay

    Localization of cytokines in cytoplasmic compartment of GFAP + spinal astrocyte cultures: enhanced cytokine expression was observed in IL-1β stimulated spinal astrocytes. Micrographs of single 1 μm thick laser scanning confocal optical sections illustrating co-localization between GFAP astrocytic marker (green) and IL-6, GM-CSF, CCL5 cytokines (red). Panels (a–c , g,h , l–n) represent control, while (d–f,i–k,o–q) show cultures which received 24 h of IL-1β treatment. Mixed colors (yellow) on the superimposed images (c,f,h,k,n,r) indicate double labeled structures. On all images DAPI was used to label cell nuclei (blue). Scale bar 10 μm.

    Journal: Frontiers in Physiology

    Article Title: IL-1β Induced Cytokine Expression by Spinal Astrocytes Can Play a Role in the Maintenance of Chronic Inflammatory Pain

    doi: 10.3389/fphys.2020.543331

    Figure Lengend Snippet: Localization of cytokines in cytoplasmic compartment of GFAP + spinal astrocyte cultures: enhanced cytokine expression was observed in IL-1β stimulated spinal astrocytes. Micrographs of single 1 μm thick laser scanning confocal optical sections illustrating co-localization between GFAP astrocytic marker (green) and IL-6, GM-CSF, CCL5 cytokines (red). Panels (a–c , g,h , l–n) represent control, while (d–f,i–k,o–q) show cultures which received 24 h of IL-1β treatment. Mixed colors (yellow) on the superimposed images (c,f,h,k,n,r) indicate double labeled structures. On all images DAPI was used to label cell nuclei (blue). Scale bar 10 μm.

    Article Snippet: Prior to the assay, cells were stimulated by 10 pg/ml recombinant murine IL-1β protein (PeproTech) for 24 h. Then the supernatants were pooled from 12 well non-treated, control and 12 well IL-1β-stimulated cultures.

    Techniques: Expressing, Marker, Labeling

    Time course experiments show different kinetics of cytokine secretion due to IL-1β stimulation. (A) Secretion of pro inflammatory cytokines was significantly increased in the supernatant of IL-1β stimulated astrocytes by ELISA experiments. Data are shown as mean ± SEM of three independent experiments in duplicate assay. ANOVA Repeated Measures, followed by Tukey’s pairwise comparison, ∗ p

    Journal: Frontiers in Physiology

    Article Title: IL-1β Induced Cytokine Expression by Spinal Astrocytes Can Play a Role in the Maintenance of Chronic Inflammatory Pain

    doi: 10.3389/fphys.2020.543331

    Figure Lengend Snippet: Time course experiments show different kinetics of cytokine secretion due to IL-1β stimulation. (A) Secretion of pro inflammatory cytokines was significantly increased in the supernatant of IL-1β stimulated astrocytes by ELISA experiments. Data are shown as mean ± SEM of three independent experiments in duplicate assay. ANOVA Repeated Measures, followed by Tukey’s pairwise comparison, ∗ p

    Article Snippet: Prior to the assay, cells were stimulated by 10 pg/ml recombinant murine IL-1β protein (PeproTech) for 24 h. Then the supernatants were pooled from 12 well non-treated, control and 12 well IL-1β-stimulated cultures.

    Techniques: Enzyme-linked Immunosorbent Assay

    During the course of CFA-induced inflammatory pain IL-1β production correlates with enhanced mechanical pain sensitivity. (A) Line chart shows the mean mechanical withdrawal threshold (MWT) values on both hind limbs of control animals and animals receiving CFA injection into the right (ipsilateral) hind paw. Note that MWT values appeared to be remarkably stable throughout the entire length of the experimental period in control animals and in the untreated left (contralateral) hind paw of animals receiving CFA injection. However, CFA injection resulted in a significant drop in MWT values in the ipsilateral hind paw of animals on experimental days 1–4. Mechanical pain sensitivity peaked on post-injection day 3 and 4. Data are presented as mean ± SEM. (One way ANOVA, ∗∗∗ p

    Journal: Frontiers in Physiology

    Article Title: IL-1β Induced Cytokine Expression by Spinal Astrocytes Can Play a Role in the Maintenance of Chronic Inflammatory Pain

    doi: 10.3389/fphys.2020.543331

    Figure Lengend Snippet: During the course of CFA-induced inflammatory pain IL-1β production correlates with enhanced mechanical pain sensitivity. (A) Line chart shows the mean mechanical withdrawal threshold (MWT) values on both hind limbs of control animals and animals receiving CFA injection into the right (ipsilateral) hind paw. Note that MWT values appeared to be remarkably stable throughout the entire length of the experimental period in control animals and in the untreated left (contralateral) hind paw of animals receiving CFA injection. However, CFA injection resulted in a significant drop in MWT values in the ipsilateral hind paw of animals on experimental days 1–4. Mechanical pain sensitivity peaked on post-injection day 3 and 4. Data are presented as mean ± SEM. (One way ANOVA, ∗∗∗ p

    Article Snippet: Prior to the assay, cells were stimulated by 10 pg/ml recombinant murine IL-1β protein (PeproTech) for 24 h. Then the supernatants were pooled from 12 well non-treated, control and 12 well IL-1β-stimulated cultures.

    Techniques: Injection

    Astrocytic cytokine expression was significantly reduced by SN50, inhibitor of NF-κB nuclear translocation. (A) Histogram shows relative GM-CSF levels determined by ELISA method after 1 h of pre-treatment by SN50 and 8 h of IL-1β stimulation. The GM-CSF secretion of the astrocyte cultures is significantly down regulated by 5 μM SN50 if compared with the IL-1β-treated cultures. (B) Histogram shows relative IL-6 and CCL5 levels in the supernatant of the astrocyte cultures measured by ELISA method after 1 h of SN50 pre-treatment and 16 h of IL-1β stimulation. Both cytokine levels show significant reduction due to 10 μM SN50 treatment if compared with the IL-1β-treated cultures. Data are shown as mean ± SEM of two independent experiments in duplicate. One-way ANOVA, followed by Student-Newman-Keuls pairwise comparison, ∗ p

    Journal: Frontiers in Physiology

    Article Title: IL-1β Induced Cytokine Expression by Spinal Astrocytes Can Play a Role in the Maintenance of Chronic Inflammatory Pain

    doi: 10.3389/fphys.2020.543331

    Figure Lengend Snippet: Astrocytic cytokine expression was significantly reduced by SN50, inhibitor of NF-κB nuclear translocation. (A) Histogram shows relative GM-CSF levels determined by ELISA method after 1 h of pre-treatment by SN50 and 8 h of IL-1β stimulation. The GM-CSF secretion of the astrocyte cultures is significantly down regulated by 5 μM SN50 if compared with the IL-1β-treated cultures. (B) Histogram shows relative IL-6 and CCL5 levels in the supernatant of the astrocyte cultures measured by ELISA method after 1 h of SN50 pre-treatment and 16 h of IL-1β stimulation. Both cytokine levels show significant reduction due to 10 μM SN50 treatment if compared with the IL-1β-treated cultures. Data are shown as mean ± SEM of two independent experiments in duplicate. One-way ANOVA, followed by Student-Newman-Keuls pairwise comparison, ∗ p

    Article Snippet: Prior to the assay, cells were stimulated by 10 pg/ml recombinant murine IL-1β protein (PeproTech) for 24 h. Then the supernatants were pooled from 12 well non-treated, control and 12 well IL-1β-stimulated cultures.

    Techniques: Expressing, Translocation Assay, Enzyme-linked Immunosorbent Assay

    Effect of GSK3 inhibitor CHIR99021 on LPS-induced pro-inflammatory cytokines in adipose tissue. ( A ) Human omental adipose tissue and skeletal muscle were incubated in the absence or presence of 10 µM CHIR99021 (CHIR) for 20 h. Representative Western blot demonstrating GSK3β protein expression. ( B–G ) Human omental adipose tissue was incubated with 10 µg/ml LPS in the absence or presence of 10 µM CHIR99021 (CHIR) for 20 h (n = 6 patients). ( B–D ) Gene expression for TNF-α, IL-1β and IL-6 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to LPS. Data displayed as mean ±SEM. * P

    Journal: PLoS ONE

    Article Title: GSK3β Is Increased in Adipose Tissue and Skeletal Muscle from Women with Gestational Diabetes Where It Regulates the Inflammatory Response

    doi: 10.1371/journal.pone.0115854

    Figure Lengend Snippet: Effect of GSK3 inhibitor CHIR99021 on LPS-induced pro-inflammatory cytokines in adipose tissue. ( A ) Human omental adipose tissue and skeletal muscle were incubated in the absence or presence of 10 µM CHIR99021 (CHIR) for 20 h. Representative Western blot demonstrating GSK3β protein expression. ( B–G ) Human omental adipose tissue was incubated with 10 µg/ml LPS in the absence or presence of 10 µM CHIR99021 (CHIR) for 20 h (n = 6 patients). ( B–D ) Gene expression for TNF-α, IL-1β and IL-6 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to LPS. Data displayed as mean ±SEM. * P

    Article Snippet: Tissues were incubated in the absence or presence of 10 µM CHIR99021 (Selleck Chemicals; Houston, TX, USA) for 60 min before the addition of 10 µg/ml LPS (derived from Escherichia coli 026:B6; Sigma-Aldrich, St. Louis, MO, USA) or 1 ng/ml IL-1β (PeproTech; Rocky Hill, NJ, USA) for 20 h. For adipose tissue, additional explants were performed using another GSK3 inhibitor SB216763 (Selleck Chemicals; Houston, TX, USA).

    Techniques: Incubation, Western Blot, Expressing, Quantitative RT-PCR

    Effect of GSK3 inhibitor CHIR99021 on IL-1β-induced adhesion molecules in adipose tissue and skeletal muscle. Human ( A–D ) omental adipose tissue and ( E–H ) skeletal muscle were incubated with 1 ng/ml IL-1β in the absence or presence of 10 µM CHIR99021 (CHIR) for 20 h (n = 6 patients). ( A,B,E,F ) Gene expression for VCAM-1 and ICAM-1 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to IL-1β. Data displayed as mean ±SEM. * P

    Journal: PLoS ONE

    Article Title: GSK3β Is Increased in Adipose Tissue and Skeletal Muscle from Women with Gestational Diabetes Where It Regulates the Inflammatory Response

    doi: 10.1371/journal.pone.0115854

    Figure Lengend Snippet: Effect of GSK3 inhibitor CHIR99021 on IL-1β-induced adhesion molecules in adipose tissue and skeletal muscle. Human ( A–D ) omental adipose tissue and ( E–H ) skeletal muscle were incubated with 1 ng/ml IL-1β in the absence or presence of 10 µM CHIR99021 (CHIR) for 20 h (n = 6 patients). ( A,B,E,F ) Gene expression for VCAM-1 and ICAM-1 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to IL-1β. Data displayed as mean ±SEM. * P

    Article Snippet: Tissues were incubated in the absence or presence of 10 µM CHIR99021 (Selleck Chemicals; Houston, TX, USA) for 60 min before the addition of 10 µg/ml LPS (derived from Escherichia coli 026:B6; Sigma-Aldrich, St. Louis, MO, USA) or 1 ng/ml IL-1β (PeproTech; Rocky Hill, NJ, USA) for 20 h. For adipose tissue, additional explants were performed using another GSK3 inhibitor SB216763 (Selleck Chemicals; Houston, TX, USA).

    Techniques: Incubation, Expressing, Quantitative RT-PCR

    Effect of GSK3 inhibitor CHIR99021 on IL-1β induced pro-inflammatory chemokines in adipose tissue and skeletal muscle. Human ( A–D ) omental adipose tissue and ( E–H ) skeletal muscle were incubated with 1 ng/ml IL-1β in the absence or presence of 10 µM CHIR99021 (CHIR) for 20 h (n = 6 patients). ( A,B,E,F ) Gene expression for IL-8 and MCP-1 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to IL-1β. Data displayed as mean ±SEM. * P

    Journal: PLoS ONE

    Article Title: GSK3β Is Increased in Adipose Tissue and Skeletal Muscle from Women with Gestational Diabetes Where It Regulates the Inflammatory Response

    doi: 10.1371/journal.pone.0115854

    Figure Lengend Snippet: Effect of GSK3 inhibitor CHIR99021 on IL-1β induced pro-inflammatory chemokines in adipose tissue and skeletal muscle. Human ( A–D ) omental adipose tissue and ( E–H ) skeletal muscle were incubated with 1 ng/ml IL-1β in the absence or presence of 10 µM CHIR99021 (CHIR) for 20 h (n = 6 patients). ( A,B,E,F ) Gene expression for IL-8 and MCP-1 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to IL-1β. Data displayed as mean ±SEM. * P

    Article Snippet: Tissues were incubated in the absence or presence of 10 µM CHIR99021 (Selleck Chemicals; Houston, TX, USA) for 60 min before the addition of 10 µg/ml LPS (derived from Escherichia coli 026:B6; Sigma-Aldrich, St. Louis, MO, USA) or 1 ng/ml IL-1β (PeproTech; Rocky Hill, NJ, USA) for 20 h. For adipose tissue, additional explants were performed using another GSK3 inhibitor SB216763 (Selleck Chemicals; Houston, TX, USA).

    Techniques: Incubation, Expressing, Quantitative RT-PCR

    Effect of GSK3 inhibitor CHIR99021 on LPS-induced pro-inflammatory cytokines in skeletal muscle. Human skeletal muscle was incubated with 10 µg/ml LPS in the absence or presence of 10 µM CHIR99021 (CHIR) for 20 h (n = 6 patients). ( A–C ) Gene expression for TNF-α, IL-1β and IL-6 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to LPS. Data displayed as mean ±SEM. * P

    Journal: PLoS ONE

    Article Title: GSK3β Is Increased in Adipose Tissue and Skeletal Muscle from Women with Gestational Diabetes Where It Regulates the Inflammatory Response

    doi: 10.1371/journal.pone.0115854

    Figure Lengend Snippet: Effect of GSK3 inhibitor CHIR99021 on LPS-induced pro-inflammatory cytokines in skeletal muscle. Human skeletal muscle was incubated with 10 µg/ml LPS in the absence or presence of 10 µM CHIR99021 (CHIR) for 20 h (n = 6 patients). ( A–C ) Gene expression for TNF-α, IL-1β and IL-6 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to LPS. Data displayed as mean ±SEM. * P

    Article Snippet: Tissues were incubated in the absence or presence of 10 µM CHIR99021 (Selleck Chemicals; Houston, TX, USA) for 60 min before the addition of 10 µg/ml LPS (derived from Escherichia coli 026:B6; Sigma-Aldrich, St. Louis, MO, USA) or 1 ng/ml IL-1β (PeproTech; Rocky Hill, NJ, USA) for 20 h. For adipose tissue, additional explants were performed using another GSK3 inhibitor SB216763 (Selleck Chemicals; Houston, TX, USA).

    Techniques: Incubation, Expressing, Quantitative RT-PCR

    Effect of GSK3 inhibitor SB216763 on LPS-induced pro-inflammatory cytokines and chemokines in adipose tissue. Human omental adipose tissue was incubated with 10 µg/ml LPS in the absence or presence of 20 µM SB216763 (SB) for 20 h (n = 5 patients). ( A–E ) Gene expression for TNF-α, IL-1β, IL-6, IL-8 and MCP-1 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to LPS. Data displayed as mean ±SEM. * P

    Journal: PLoS ONE

    Article Title: GSK3β Is Increased in Adipose Tissue and Skeletal Muscle from Women with Gestational Diabetes Where It Regulates the Inflammatory Response

    doi: 10.1371/journal.pone.0115854

    Figure Lengend Snippet: Effect of GSK3 inhibitor SB216763 on LPS-induced pro-inflammatory cytokines and chemokines in adipose tissue. Human omental adipose tissue was incubated with 10 µg/ml LPS in the absence or presence of 20 µM SB216763 (SB) for 20 h (n = 5 patients). ( A–E ) Gene expression for TNF-α, IL-1β, IL-6, IL-8 and MCP-1 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to LPS. Data displayed as mean ±SEM. * P

    Article Snippet: Tissues were incubated in the absence or presence of 10 µM CHIR99021 (Selleck Chemicals; Houston, TX, USA) for 60 min before the addition of 10 µg/ml LPS (derived from Escherichia coli 026:B6; Sigma-Aldrich, St. Louis, MO, USA) or 1 ng/ml IL-1β (PeproTech; Rocky Hill, NJ, USA) for 20 h. For adipose tissue, additional explants were performed using another GSK3 inhibitor SB216763 (Selleck Chemicals; Houston, TX, USA).

    Techniques: Incubation, Expressing, Quantitative RT-PCR

    Effect of GSK3 inhibitor CHIR99021 on IL-1β-induced pro-inflammatory cytokine IL-6 in adipose tissue and skeletal muscle. Human ( A,B ) omental adipose tissue and ( C,D ) skeletal muscle were incubated with 1 ng/ml IL-1β in the absence or presence of 10 µM CHIR99021 (CHIR) for 20 h (n = 6 patients). ( A,C ) Gene expression for IL-6 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to IL-1β. Data displayed as mean ±SEM. * P

    Journal: PLoS ONE

    Article Title: GSK3β Is Increased in Adipose Tissue and Skeletal Muscle from Women with Gestational Diabetes Where It Regulates the Inflammatory Response

    doi: 10.1371/journal.pone.0115854

    Figure Lengend Snippet: Effect of GSK3 inhibitor CHIR99021 on IL-1β-induced pro-inflammatory cytokine IL-6 in adipose tissue and skeletal muscle. Human ( A,B ) omental adipose tissue and ( C,D ) skeletal muscle were incubated with 1 ng/ml IL-1β in the absence or presence of 10 µM CHIR99021 (CHIR) for 20 h (n = 6 patients). ( A,C ) Gene expression for IL-6 was analysed by qRT-PCR. Gene expression was normalised to GAPDH mRNA expression and the fold change was calculated relative to IL-1β. Data displayed as mean ±SEM. * P

    Article Snippet: Tissues were incubated in the absence or presence of 10 µM CHIR99021 (Selleck Chemicals; Houston, TX, USA) for 60 min before the addition of 10 µg/ml LPS (derived from Escherichia coli 026:B6; Sigma-Aldrich, St. Louis, MO, USA) or 1 ng/ml IL-1β (PeproTech; Rocky Hill, NJ, USA) for 20 h. For adipose tissue, additional explants were performed using another GSK3 inhibitor SB216763 (Selleck Chemicals; Houston, TX, USA).

    Techniques: Incubation, Expressing, Quantitative RT-PCR

    ESE-1 upregulated GP73 expression in HCC cells. (A) pCR3.1-ESE-1 plasmid was transfected into HEK293T cells and ESE-1 expression was tested by Western blot 36 h after transfection. (B) pCR3.1-ESE-1 plasmid was transfected into Hep3B cells for 36 h, and GP73 mRNA and protein levels were analyzed with qPCR and Western blot, respectively. (C) GP73 mRNA and protein levels were analyzed by adopting qPCR and Western blot in Huh7 cells transfected with pCR3.1-ESE-1 plasmid. (D) pCR3.1-ΔESE-1 plasmid with deletion ETS domain of ESE-1 was constructed and transfected into HEK293T cells. ΔESE-1 expression was confirmed by Western blot. (E) The empty vector (Mock) or ESE-1 or ΔESE-1 was transfected into Hep3B and Huh7 cells, and GP73 expression was tested by Western blot. (F) ESE-1 was downregulated in Hep3B and (G) Huh7 cells by infecting them with virus carrying the shRNA expression cassette against ESE-1 (shESE-1) or a non-target control (shGFP) with or without IL-1β stimulation. After 96 h, the mRNA and protein levels of ESE-1 and GP73 were analyzed with qPCR and Western blot, respectively. (H) Hep3B and (I) Huh7 cells infected with shESE-1 virus particles were transfected with pCR3.1-ESE-1 plasmid at 48 h post-infection. At 48 h after transfection, cells were harvested, and ESE-1 and GP73 proteins were analyzed with Western blot.

    Journal: Cell & Bioscience

    Article Title: Epithelium-Specific ETS (ESE)-1 upregulated GP73 expression in hepatocellular carcinoma cells

    doi: 10.1186/2045-3701-4-76

    Figure Lengend Snippet: ESE-1 upregulated GP73 expression in HCC cells. (A) pCR3.1-ESE-1 plasmid was transfected into HEK293T cells and ESE-1 expression was tested by Western blot 36 h after transfection. (B) pCR3.1-ESE-1 plasmid was transfected into Hep3B cells for 36 h, and GP73 mRNA and protein levels were analyzed with qPCR and Western blot, respectively. (C) GP73 mRNA and protein levels were analyzed by adopting qPCR and Western blot in Huh7 cells transfected with pCR3.1-ESE-1 plasmid. (D) pCR3.1-ΔESE-1 plasmid with deletion ETS domain of ESE-1 was constructed and transfected into HEK293T cells. ΔESE-1 expression was confirmed by Western blot. (E) The empty vector (Mock) or ESE-1 or ΔESE-1 was transfected into Hep3B and Huh7 cells, and GP73 expression was tested by Western blot. (F) ESE-1 was downregulated in Hep3B and (G) Huh7 cells by infecting them with virus carrying the shRNA expression cassette against ESE-1 (shESE-1) or a non-target control (shGFP) with or without IL-1β stimulation. After 96 h, the mRNA and protein levels of ESE-1 and GP73 were analyzed with qPCR and Western blot, respectively. (H) Hep3B and (I) Huh7 cells infected with shESE-1 virus particles were transfected with pCR3.1-ESE-1 plasmid at 48 h post-infection. At 48 h after transfection, cells were harvested, and ESE-1 and GP73 proteins were analyzed with Western blot.

    Article Snippet: For cells treated with cytokines, 10% FBS-cultured medium was removed and washed twice with PBS before adding 2% FBS-cultured medium with IL-1β (Peprotech, USA).

    Techniques: Expressing, Plasmid Preparation, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Construct, shRNA, Infection

    ESE-1 and GP73 expressions were induced by IL-1β stimulation in vitro . (A) The basal levels of ESE-1 and GP73 expressions in HepG2, Hep3B, and Huh7 cells were analyzed by conducting Western blot. (B) Hep3B cells were stimulated with different doses of IL-1β (2.5, 10, and 20 ng/mL) for 48 h. IL-1β-induced ESE-1 and GP73 mRNA and protein levels were analyzed by performing qPCR and Western blot. (C) Huh7 cells were stimulated with different doses of IL-1β (10 and 20 ng/mL) for 48 h. ESE-1 and GP73 mRNA and protein levels were analyzed with qPCR and Western blot. The values were normalized to GAPDH . Western blot was reprobed for GAPDH as a loading reference control.

    Journal: Cell & Bioscience

    Article Title: Epithelium-Specific ETS (ESE)-1 upregulated GP73 expression in hepatocellular carcinoma cells

    doi: 10.1186/2045-3701-4-76

    Figure Lengend Snippet: ESE-1 and GP73 expressions were induced by IL-1β stimulation in vitro . (A) The basal levels of ESE-1 and GP73 expressions in HepG2, Hep3B, and Huh7 cells were analyzed by conducting Western blot. (B) Hep3B cells were stimulated with different doses of IL-1β (2.5, 10, and 20 ng/mL) for 48 h. IL-1β-induced ESE-1 and GP73 mRNA and protein levels were analyzed by performing qPCR and Western blot. (C) Huh7 cells were stimulated with different doses of IL-1β (10 and 20 ng/mL) for 48 h. ESE-1 and GP73 mRNA and protein levels were analyzed with qPCR and Western blot. The values were normalized to GAPDH . Western blot was reprobed for GAPDH as a loading reference control.

    Article Snippet: For cells treated with cytokines, 10% FBS-cultured medium was removed and washed twice with PBS before adding 2% FBS-cultured medium with IL-1β (Peprotech, USA).

    Techniques: In Vitro, Western Blot, Real-time Polymerase Chain Reaction

    Inter-cellular gaps in vascular endothelial monolayers treated with RSPO3 or IL-1β. a Immunofluorescence staining of VE-cadherin (red) and DAPI staining of nuclei (blue) in HCAEC either untreated, or treated with RSPO3, IL-1β and combined IL-1β and RSPO3 for 6 h. Microphotographs were taken using a Zeiss Axioskope equipped with Axio-CamMRm and AxioVision Rel.4.6 software and are representative of three independent experiments run in triplicates. Original magnification, 630×. b Quantification of inter-endothelial gap index and gap size index in HCAEC treated with RSPO3. Data are mean ± S.E.M of three independent experiments run in triplicates, * P

    Journal: Molecular Medicine

    Article Title: RSPO3 impairs barrier function of human vascular endothelial monolayers and synergizes with pro-inflammatory IL-1

    doi: 10.1186/s10020-018-0048-z

    Figure Lengend Snippet: Inter-cellular gaps in vascular endothelial monolayers treated with RSPO3 or IL-1β. a Immunofluorescence staining of VE-cadherin (red) and DAPI staining of nuclei (blue) in HCAEC either untreated, or treated with RSPO3, IL-1β and combined IL-1β and RSPO3 for 6 h. Microphotographs were taken using a Zeiss Axioskope equipped with Axio-CamMRm and AxioVision Rel.4.6 software and are representative of three independent experiments run in triplicates. Original magnification, 630×. b Quantification of inter-endothelial gap index and gap size index in HCAEC treated with RSPO3. Data are mean ± S.E.M of three independent experiments run in triplicates, * P

    Article Snippet: Human coronary artery endothelial cells (HCAEC), human pulmonary artery endothelial cells (HPAEC), human cardiac microvascular endothelial cells (HCMVEC), human dermal microvascular endothelial cells (HDMVEC, all from Clonetics, Lonza), and human brain microvascular endothelial cells (HBMVEC, Cell Systems) were propagated as described (Skaria et al., ) and treated with recombinant human RSPO3 (doses ranging from 250 to 1000 ng/mL, PeproTech) and recombinant human IL-1β (20 U/mL, PeproTech) as given in the supplementary methods (Additional file : Supplementary methods ).

    Techniques: Immunofluorescence, Staining, Software

    Automated real-time assessment of endothelial monolayer barrier function using the ECIS system. Uniform tight monolayers of HCAEC, HPAEC, HCMVEC and HBMVEC cultured in stabilized and collagen coated 8W10E+ ECIS culture chambers were treated with RSPO3 (500 ng/mL), IL-1β (20 U/mL) or a combination of RSPO3 and IL-1β. The line graphs are the original ECIS measurements obtained using ECIS Z Theta system equipped with v.1.2.126 PC software and from one representative of three independent experiments run in duplicates at 4000 Hz (indicative of cell-cell adhesion tightness). Bar graphs show the data of barrier function measurements continuously recorded for and at 12 h from three independent experiments run in duplicates. Black, vehicle; Green, IL-1β; Yellow, RSPO3; Purple, RSPO3 + IL-1β. Error bars are mean ± S.D. *** P

    Journal: Molecular Medicine

    Article Title: RSPO3 impairs barrier function of human vascular endothelial monolayers and synergizes with pro-inflammatory IL-1

    doi: 10.1186/s10020-018-0048-z

    Figure Lengend Snippet: Automated real-time assessment of endothelial monolayer barrier function using the ECIS system. Uniform tight monolayers of HCAEC, HPAEC, HCMVEC and HBMVEC cultured in stabilized and collagen coated 8W10E+ ECIS culture chambers were treated with RSPO3 (500 ng/mL), IL-1β (20 U/mL) or a combination of RSPO3 and IL-1β. The line graphs are the original ECIS measurements obtained using ECIS Z Theta system equipped with v.1.2.126 PC software and from one representative of three independent experiments run in duplicates at 4000 Hz (indicative of cell-cell adhesion tightness). Bar graphs show the data of barrier function measurements continuously recorded for and at 12 h from three independent experiments run in duplicates. Black, vehicle; Green, IL-1β; Yellow, RSPO3; Purple, RSPO3 + IL-1β. Error bars are mean ± S.D. *** P

    Article Snippet: Human coronary artery endothelial cells (HCAEC), human pulmonary artery endothelial cells (HPAEC), human cardiac microvascular endothelial cells (HCMVEC), human dermal microvascular endothelial cells (HDMVEC, all from Clonetics, Lonza), and human brain microvascular endothelial cells (HBMVEC, Cell Systems) were propagated as described (Skaria et al., ) and treated with recombinant human RSPO3 (doses ranging from 250 to 1000 ng/mL, PeproTech) and recombinant human IL-1β (20 U/mL, PeproTech) as given in the supplementary methods (Additional file : Supplementary methods ).

    Techniques: Electric Cell-substrate Impedance Sensing, Cell Culture, Software