il-1α Search Results


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  • 94
    Millipore il 1α
    <t>IL-1α</t> localization and knockdown in THP-1 macrophages. A ) THP-1 macrophages were transfected with control (ctr) or IL-1α siRNA for 48 h, followed by 24 h stimulation with MSU (100 µg/ml). Cell lysates were used to examine the expression of pro-IL-1α, pro-IL-1β, cleaved capase-1, and NLRP3 in IL-1α knockdown cells. B ) THP-1 macrophages were stimulated with MSU (100 μg/ml) for 30 min. Nuclear and cyotsolic fractions were prepared to evaluate the expression of pro–IL-1α and –IL-1β. C ) Conditioned medium was collected and used for the quantification of IL-1β, TNF-α, IL-8, and MCP-1 production with without MSU stimulation. NS, nonstimulated. * P
    Il 1α, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 537 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson il 1α
    CD11a hi FcγRIII hi B cells produce a high level of IFN-γ in response to CD40 ligation. C57BL/6 mice were infected with 2 × 10 6 LM. (A) The intracellular expression of <t>IL-1α,</t> IL-2, IL-6, IL-10 and IFN-γ in CD11a hi FcγRIII hi and conventional B cells was assayed on day 3 post-infection. (B - G) Splenic CD11a hi FcγRIII hi and conventional B cells were sorted on day 3 post-infection. IL-1β, IL-2, IL-6, IL-12p70, IFN-γ, or TNF-α secretion by CD11a hi FcγRIII hi and conventional B cells was detected by ELISA. Data shown represent the mean ± SD of triplicate experiments. * P
    Il 1α, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 470 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad il 1α
    Tissue cytokine changes in hippocampal organotypic cultures after SD. Tissue cytokine measurements from sham-treated (light-gray bars) and SD-treated (dark-gray bars) cultures were made after recovery of 6 hours, 1 day, and 3 days (left, middle, and right of each cytokine panel, respectively). Units of gray bars in each panel are log picograms per milligram. Right axis units are linear × 1,000 pg/mg. Colored bars in each panel emphasize the difference between SD- and sham-treated cytokine levels for each time point. For example, in the top left panel, the cytokine levels of <t>IL-1α</t> at 6 hours of recovery are 1,081 ± 411 and 2,488 ± 1,003 for sham-and SD-treated cultures, respectively. Subtracting the sham value from the SD value and dividing by 1,000 equals 1.41, the value represented by the blue bar. Each measurement represents tissue samples from a total of five to nine cultures. Significant differences ( P
    Il 1α, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 337 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeproTech il 1α
    Kinetics of cytokine secretion in Max Planck Institute (MPI) cells. Production of interleukin 6 (IL-6), TNF-α, <t>IL-1α,</t> IL-1β, and IL-10 by MPI cells (P32) in response to (A) heat-killed (HK) Mycobacterium tuberculosis ( Mtb ) or 10 ng/mL lipopolysaccharide (LPS) or (B) live Mtb infection challenge. Mtb was used at different multiplicity of infection (MOI) and cytokines were quantified at 24 h post-treatment for LPS, 4, 8, 24, and 48 h post-infection with HK bacteria and 4, 8, 24, and 48 h (MOI 5) or 4, 8, and 24 h (MOI 10 and 20) post-infection with live bacteria. Data are representative of three independent experiments and expressed as mean ± SD for three biological replicates. Statistical significance between time points (black asterisks) or between stimulated and non-stimulated cells at corresponding time points (red asterisks) were determined by Prism software using the unpaired Student’s t -test. *, p -value
    Il 1α, supplied by PeproTech, used in various techniques. Bioz Stars score: 94/100, based on 345 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher il 1α
    IL-1R signaling instructs AECII to produce GM-CSF during pulmonary Legionella infection (A-C) GM-CSF levels in the BAL of C57BL/6 mice at 24, 48, and 72 hpi (A), WT, Il1a -/- , Il1b -/- and Il1r1 -/- mice at 24 hpi (B), and WT: Csf2 -/- BM chimeras at 24 hpi (C). (D) Csf2 transcript levels in the indicated cell types isolated from the lungs of naïve and L . p .-infected C57BL/6 mice at 24 hpi. (E) Representative flow cytometric plots showing intracellular staining for GM-CSF in AECII and AECI from the lungs of naïve and L . p .-infected C57BL/6 mice at 24 hpi. Frequency and total number of GM-CSF-producing AECII and AECI. (F-H) GM-CSF levels in the BAL of WT: Il1r1 -/- BM chimeras (F), Il1r1 fl/fl ;Spc-cre ERT2 and Il1r1 fl/fl mice (G), and Il1r1 r/r ;Spc-cre ERT2 and Il1r1 r/r mice (H) at 24 hpi. (I) GM-CSF levels in the supernatants of primary WT or Il1r1 -/- AECII treated with 10 ng/ml each of recombinant <t>IL-1α</t> and IL-1β (rIL-1α/β) or PBS vehicle control and infected with L . p . (MOI=5) at 12 hpi. Data shown are the pooled results of three (A, B, G, and H) or two (C-F) independent experiments with 3-4 mice per condition in each experiment. Data shown in I are the pooled results of two independent experiments with 3 mice and triplicate wells per condition in each experiment. NS, not significant; *p
    Il 1α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeproTech recombinant human il 1α
    CIK Cell Culture, Identification, and Adenovirus-Binding Ability Detection (A–C) CIK cells were isolated from human peripheral blood and differentiated by adding IFN-γ, anti-CD3 mAb, <t>IL-1α,</t> and IL-2 in different culture stages. CIK cells were observed under a phase contrast microscope at 200× after culturing for 5 (A), 10 (B), and 14 (C) days. Microscope observation showed that the cell number increased gradually and reached its peak and formed a cell colony on day 14, which is a defining characteristic of CIK cells. (D–I) CIK cells were harvested on day 14 and embedded in wax blocks. (D) CIK cells were sectioned and stained with H E, and they were observed under a microscope at 400× magnification. Microscope observation showed morphologically regular cells, with larger nuclei and less cytoplasm. (E and F) CIK cell markers (E, CD3 and F, CD56) were detected by immunohistochemistry and found to be expressed on CIK cell membranes at 400× magnification. (G) CD46 protein (the receptor for KGHV500 and KGHV400 adenoviruses) was also detected on CIK cell surfaces at 400× magnification. (H and I) KGHV500 (H) and KGHV400 (I) adenovirus hexon was detected on CIK cell membranes at 400× magnification.
    Recombinant Human Il 1α, supplied by PeproTech, used in various techniques. Bioz Stars score: 97/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam il 1α
    TRAIL expression and modulation in pIL6-TRAIL + -GFP + -UC-MSCs. a Left: transfection efficiency of UC-MSCs evaluated by flow cytometry for GFP fluorescence. Wildtype UC-MSCs were negative as compared to GFP + -UC-MSCs (red histogram: 83.5%) in a similar amount as for pIL6-TRAIL + -GFP + -UC-MSCs (blue histogram: 87.6%). Right: FACS analysis revealed constitutive TRAIL expression in transduced UC-MSCs (green histogram: 62.5%) that was significantly enhanced by the conditioned medium from U-266 cells (blue histogram: 80.3%) as well as in response to <t>IL-1α/IL-1β</t> (red histogram: 91.2%). b Confocal microscopy of pIL6-TRAIL + -GFP + -UC-MSCs confirmed the upregulation of TRAIL (FITC, green) induced by either U-266 supernatant or IL-1α/IL-1β treatment. Phalloidin staining was used to show actin (TRITC-red); nuclei were counterstained with Hoechst 33342 (blue). Magnification: 100×. c Evaluation of TRAIL expression in transduced cells by RT-qPCR, western blot analysis, and ELISA. (i) After either U-266 or IL-1α/IL-1β treatment, TRAIL mRNA levels in pIL6-TRAIL + -GFP + -UC-MSCs increased up to 2.2-fold and 2.5-fold compared to basal condition. Data are relative amount of mRNA expression normalized to GAPDH and are presented as mean ± SD of three independent experiments. * p
    Il 1α, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology il 1α
    Experimental evidence that regulation of parkin expression and phosphorylation is related to glial activation and excess IL-1β in Alzheimer brain and in AD Model Systems. a Parkin in neurons in brains of age-matched control patients (AMC) is diffusely distributed within the cytoplasm whereas parkin in neurons in brains of Alzheimer patients (AD) is present in rosette-like aggregates ( b ). c Parkin in Alzheimer brain is colocalized with tau aggregates. d Activated glia overexpressing <t>IL-1α</t> (brown) are immediately adjacent to parkin-immunoreactive neurons (blue), scale bar 20 μM. e Comparative levels of IL-1α, IL-1β, and parkin mRNAs in wild-type littermates or PD-APP mice. f Representative western immunoblot showing proteins from NT2 cells either treated, or not, overnight with IL-1β. g Phospho-parkin levels (P-parkin Ser 65 ) in two representative western immunoblots of proteins from NT2 cell and rat primary neuron cultures treated, or not, with IL-1β. h Quantification of parkin in untreated and treated cell cultures ( n = 6), data is mean ± SEM (** p
    Il 1α, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genzyme il 1α
    Flow cytometric analysis of P-selectin on HDMECs. A: Basal expression of P-selectin was minimal or undetectable. When HDMECs were incubated with histamine (10 −5 mol/L), transient expression was observed 5 and 10 minutes after histamine treatment. B: HDMECs were incubated with <t>IL-1α</t> (10 ng/ml), TNF-α (10 ng/ml), interferon-γ (10 ng/ml), IL-4 (10 ng/ml), IL-13 (10 ng/ml), or substance P (100 nmol/L) for 24 hours. Whereas IL-4, IL-13, and substance P increased P-selectin expression, TNF-α had no effect on P-selectin expression. The concentration of each cytokine that induced maximal response was determined in preliminary experiments. C and D: Time course changes in P-selectin expression by HDMECs ( C ) and HUVECs ( D ) after treatment with IL-4. Surface P-selectin expression in HDMECs peaked 24 hours after treatment with IL-4. Black line , control; green line , treated with stimulants. Results are from a single representative of three separate experiments.
    Il 1α, supplied by Genzyme, used in various techniques. Bioz Stars score: 92/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant human il 1α
    Priming by C5a induces expression of pro-IL-1β protein. Expression of pro-IL-1β protein (36 kDa) was assessed in ARPE-19 cells following priming with <t>IL-1α,</t> complement-competent normal human serum (NHS), recombinant human C5a,
    Recombinant Human Il 1α, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeproTech recombinant il 1α
    Neutrophil-recruiting chemokines in the oral mucosa are reduced in absence of IL-1R signaling. ( A ) Chemokine expression in the tongues of naïve (n) and infected (inf) WT mice was measured by qRT-PCR 24 hours post-infection. ( B ) Chemokine expression in the tongues of naïve and infected WT and infected Il1r1 -/- mice 24 hours post-infection. ( C ) CD45 + leukocytes, CD45 - EpCAM + CD31 - epithelial cells, and CD45 - EpCAM - CD31 + endothelial cells were isolated from the tongues of naïve and infected WT mice by FACS sorting 24 hours post-infection, and Cxcl1 mRNA was quantified by qRT-PCR. ( D ) The tongue-derived keratinocyte (TDK) cell line was treated with recombinant <t>IL-1α</t> (20 ng/ml), anakinra (250 μg/ml), or PBS, and CXCL1 levels in the culture supernatant were determined by ELISA. Bar graphs show the group mean + SD. Data are representative of two (A, C–D) or pooled from two (B) independent experiments, with the exception of the naïve group in B, which is from one experiment. Statistical analysis was performed using log 10 transformation and Student’s t-test with Welch’s correction (A) or one-way ANOVA with Dunnett’s test (B, D).
    Recombinant Il 1α, supplied by PeproTech, used in various techniques. Bioz Stars score: 94/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems mouse il 1 alpha il 1f1 antibody
    Neutrophil-recruiting chemokines in the oral mucosa are reduced in absence of IL-1R signaling. ( A ) Chemokine expression in the tongues of naïve (n) and infected (inf) WT mice was measured by qRT-PCR 24 hours post-infection. ( B ) Chemokine expression in the tongues of naïve and infected WT and infected Il1r1 -/- mice 24 hours post-infection. ( C ) CD45 + leukocytes, CD45 - EpCAM + CD31 - epithelial cells, and CD45 - EpCAM - CD31 + endothelial cells were isolated from the tongues of naïve and infected WT mice by FACS sorting 24 hours post-infection, and Cxcl1 mRNA was quantified by qRT-PCR. ( D ) The tongue-derived keratinocyte (TDK) cell line was treated with recombinant <t>IL-1α</t> (20 ng/ml), anakinra (250 μg/ml), or PBS, and CXCL1 levels in the culture supernatant were determined by ELISA. Bar graphs show the group mean + SD. Data are representative of two (A, C–D) or pooled from two (B) independent experiments, with the exception of the naïve group in B, which is from one experiment. Statistical analysis was performed using log 10 transformation and Student’s t-test with Welch’s correction (A) or one-way ANOVA with Dunnett’s test (B, D).
    Mouse Il 1 Alpha Il 1f1 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti il 1α
    Secreted cytokines induced by Helicobacter pylori (Hp) exosomes using a protein array. (a) Map of antibodies against cytokines on the RayBiotech human inflammation antibody array. (b) Cell culture supernatants from gastric epithelial cells (GES)‐1 cells treated with exosomes were used in the array. Bound cytokines were recognized by a pool of anti‐cytokine antibodies corresponding to the antibodies spotted on the array. The Hp exosome group revealed increased expression of <t>IL‐1α</t> and sIL‐6R. (c) Semi‐quantification of scanned antibody arrays. The levels were normalized to internal positive controls present in the membrane. Semiquantitative levels are represented in the heat map.
    Anti Il 1α, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    BioLegend il 1α
    IL-1β/α induction by 25-HC is highly specific. The levels of secreted IL-1β ( a ), <t>IL-1α</t> ( b ), or IL-6 ( c ) in the medium of primary microglia treated with LPS (10 ng/ml) in the presence of 25-HC (10 μg/ml), cholesterol (10μg/ml), or 7α-HC (10 μg/ml) for 24 h. d Ent-25-HC (10 μg/ml) has much weaker effects in augmenting IL-1β production in primary microglia treated with LPS (10 ng/ml) for 24 h. The levels of cytokines were determined by ELISA. Statistical significances were determined by ordinary two-way ANOVA with Tukey multiple comparisons test. * p
    Il 1α, supplied by BioLegend, used in various techniques. Bioz Stars score: 94/100, based on 249 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc il 1α
    PPARγ loss of function is associated with impaired IL-1 cytokine release in primary macrophages. ( A-C ) Peritoneal macrophages (pMACs) isolated from WT (open bars) or mPPARγKO mice (filled bars) were treated with control (BSA-LPS), palm (250 μM)-LPS (100 ng), or stearate (150 μM)-LPS for 20h and the release of IL-1β ( A ), ( B ) <t>IL-1α,</t> and ( C ) TNFα was determined by ELISA. ( D-F ) pMACs isolated from WT (open bars) or mPPARγKO mice (filled bars) were treated with LPS and ATP, alum or silica as described in the methods and IL-1β ( D ), ( E ) IL-1α, and ( F ) TNFα was determined by ELISA. WT pMACs were treated with veh (open bars) or T0070907 (T007; gray bars) for 24h after which they received the indicated stimuli. The release of IL-1β ( G ), ( H ) IL-1α, and ( I ) TNFα was determined by ELISA. Bar graphs report the mean ± standard error (SE) for a minimum of 3 experiments, each performed in triplicate. *, p
    Il 1α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Glaxo Smith il 1α
    PPARγ loss of function is associated with impaired IL-1 cytokine release in primary macrophages. ( A-C ) Peritoneal macrophages (pMACs) isolated from WT (open bars) or mPPARγKO mice (filled bars) were treated with control (BSA-LPS), palm (250 μM)-LPS (100 ng), or stearate (150 μM)-LPS for 20h and the release of IL-1β ( A ), ( B ) <t>IL-1α,</t> and ( C ) TNFα was determined by ELISA. ( D-F ) pMACs isolated from WT (open bars) or mPPARγKO mice (filled bars) were treated with LPS and ATP, alum or silica as described in the methods and IL-1β ( D ), ( E ) IL-1α, and ( F ) TNFα was determined by ELISA. WT pMACs were treated with veh (open bars) or T0070907 (T007; gray bars) for 24h after which they received the indicated stimuli. The release of IL-1β ( G ), ( H ) IL-1α, and ( I ) TNFα was determined by ELISA. Bar graphs report the mean ± standard error (SE) for a minimum of 3 experiments, each performed in triplicate. *, p
    Il 1α, supplied by Glaxo Smith, used in various techniques. Bioz Stars score: 91/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeproTech recombinant murine il 1α
    Immunological properties of 3-week mDC cultures in EGM . <t>IL-1α</t> ( A ) and IL-6 ( B ) were detected by ELISA on reseeded cultures after 48 h stimulation with TNFα and LPS. ( A ) ANOVA analysis: p
    Recombinant Murine Il 1α, supplied by PeproTech, used in various techniques. Bioz Stars score: 94/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti il 1α antibody
    Tissue expression of <t>IL-1α.</t> a Right lung samples demonstrated higher tissue IL-1α expression compared to the untreated control samples. b Left lung samples revealed upregulated tissue IL-1α expression. Immunohistochemical staining with anti-IL-1α antibody of c normal control and d right lung sample treated with helical tomotherapy with CCRT. * p
    Anti Il 1α Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 88/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems il 1α
    Synergism and antagonism in growth factor and cytokine-stimulated myoblast proliferation and differentiation. Primary mouse myoblasts were cultured in differentiation medium (DM) with 0.1% DMSO (control) and were stimulated with either FGF2 + EGF + IGF1 (FEI), TNF-α + <t>IL-1α</t> (TI), IL-6 + OSM + LIF (IOL), or their two-way combinations for 72 h. ( a , b ) Proliferative index was determined by nuclei counting from DAPI images. ( a ) Representative DAPI images. Scale bar, 200 μ m. ( c – e ) RT-qPCR analysis of myogenic differentiation genes Myod1, Myogenin , and Myh2 quantified relative to 36b4 (reference control). ( f – h ) Immunoblots for MHC, Myogenin, and Hsp90 (as a loading control). Myogenin ( g ) and MHC ( h ) densitometry, normalized to Hsp90. In ( b – e , g – h ), n = 3 replicates are plotted as log 2 of mean fold change relative to control ± SEM. For co-treatments, an additive model used to identify synergy or antagonism using a modified Bliss independence model with p
    Il 1α, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 1582 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher recombinant human il 1α
    Cytokine-induced p38 MAPK subtype activation in human CF. (A) Cells stimulated with 10 ng/ml TNFα, 10 ng/ml <t>IL-1α</t> or 25 μg/ml anisomycin for 5–60 min before preparing whole cell homogenates and immunoblotting with phospho-specific and expression antibodies for p38 MAPK, MAPKAPK2 and HSP27. Blots representative of n = 3. (B) CF stimulated with 10 ng/ml TNFα, 10 ng/ml IL-1α or 25 μg/ml anisomycin for 15 min before preparing cell extracts for analysis of p38 subtype phosphorylation by IP/IB method. Samples were immunoprecipitated with pan phospho-p38 antibody then immunoblotted with individual p38 subtype expression antibodies. Blots representative of n = 3. (C) CF stimulated as for (B) and p38 subtype phosphorylation analyzed by ELISA. Bar chart depicts concentration of phosphorylated p38 subtypes (pg/ml). ∗∗∗ P
    Recombinant Human Il 1α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad interleukin il 1 α
    Cytokine-induced p38 MAPK subtype activation in human CF. (A) Cells stimulated with 10 ng/ml TNFα, 10 ng/ml <t>IL-1α</t> or 25 μg/ml anisomycin for 5–60 min before preparing whole cell homogenates and immunoblotting with phospho-specific and expression antibodies for p38 MAPK, MAPKAPK2 and HSP27. Blots representative of n = 3. (B) CF stimulated with 10 ng/ml TNFα, 10 ng/ml IL-1α or 25 μg/ml anisomycin for 15 min before preparing cell extracts for analysis of p38 subtype phosphorylation by IP/IB method. Samples were immunoprecipitated with pan phospho-p38 antibody then immunoblotted with individual p38 subtype expression antibodies. Blots representative of n = 3. (C) CF stimulated as for (B) and p38 subtype phosphorylation analyzed by ELISA. Bar chart depicts concentration of phosphorylated p38 subtypes (pg/ml). ∗∗∗ P
    Interleukin Il 1 α, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    R&D Systems recombinant mouse il 1α
    Growth and MVD of DA/3 mammary cancer cells in WT BALB/c, <t>IL-1α,</t> or IL-1β KO mice. ( A ) Mice were injected i.f.p. with DA/3 cells (5 × 10 5 cells per mouse). The results represent one of three experiments. Mean ± SEM from each group ( n = 8 per group) are shown. ( B ) MVD ± SEM in DA/3-embedded Matrigel plugs (2 × 10 5 ) in WT, IL-1β, or IL-1α KO mice ( n = 5 in each group).
    Recombinant Mouse Il 1α, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of combined therapy of methotrexate and coenzyme Q 10 on plasmatic level of <t>IL-1α</t> assessed on day 28. Values are given as arithmetic mean ± S.E.M. Statistical significance was evaluated using unpaired Student′s t-test: ** p
    Il 1α, supplied by Bender MedSystems, used in various techniques. Bioz Stars score: 93/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    IL-1α localization and knockdown in THP-1 macrophages. A ) THP-1 macrophages were transfected with control (ctr) or IL-1α siRNA for 48 h, followed by 24 h stimulation with MSU (100 µg/ml). Cell lysates were used to examine the expression of pro-IL-1α, pro-IL-1β, cleaved capase-1, and NLRP3 in IL-1α knockdown cells. B ) THP-1 macrophages were stimulated with MSU (100 μg/ml) for 30 min. Nuclear and cyotsolic fractions were prepared to evaluate the expression of pro–IL-1α and –IL-1β. C ) Conditioned medium was collected and used for the quantification of IL-1β, TNF-α, IL-8, and MCP-1 production with without MSU stimulation. NS, nonstimulated. * P

    Journal: The FASEB Journal

    Article Title: Critical role of IL-1α in IL-1β–induced inflammatory responses: cooperation with NF-κBp65 in transcriptional regulation

    doi: 10.1096/fj.201801513R

    Figure Lengend Snippet: IL-1α localization and knockdown in THP-1 macrophages. A ) THP-1 macrophages were transfected with control (ctr) or IL-1α siRNA for 48 h, followed by 24 h stimulation with MSU (100 µg/ml). Cell lysates were used to examine the expression of pro-IL-1α, pro-IL-1β, cleaved capase-1, and NLRP3 in IL-1α knockdown cells. B ) THP-1 macrophages were stimulated with MSU (100 μg/ml) for 30 min. Nuclear and cyotsolic fractions were prepared to evaluate the expression of pro–IL-1α and –IL-1β. C ) Conditioned medium was collected and used for the quantification of IL-1β, TNF-α, IL-8, and MCP-1 production with without MSU stimulation. NS, nonstimulated. * P

    Article Snippet: Small interfering RNA (siRNA) sequences for IL-1α were purchased from MilliporeSigma (eMission predesigned siRNA sequences SASI_Hs01_00167953 and SASI_Hs02_00302829).

    Techniques: Transfection, Expressing

    NF-κB governs IL-1α and IL-1β expression. A ) RASFs were pretreated with SB202190 (SB, 10 µM), SP600125 (SP, 10 µM), PD98059 (PD, 10 µM), PDTC (100 µM), or diacerein (DIC, 10 µM) for 2 h prior to IL-1β stimulation for 24 h. Cell lysates were analyzed for IL-1α and IL-1β expression. B ) RASFs (8-well chamber slides) were pretreated with IL-1R antagonist (100 ng/ml) for 2 h followed by IL-1β stimulation for 8 h. C ) Similarly, RASFs were treated with PDTC (100 µM) for 2 h followed by IL-1β stimulation for 8 h. Cells were fixed and analyzed for IL-1α expression using immunofluorescence method. D , E ) Conditioned media from inhibitors study in A were used for determining IL-6 and IL-8 using ELISA. PDTC inhibits both IL-1α and IL-1β, as well as IL-6 and IL-8 production. Cells were lysed and analyzed for IL-1α and IL-1β. * P

    Journal: The FASEB Journal

    Article Title: Critical role of IL-1α in IL-1β–induced inflammatory responses: cooperation with NF-κBp65 in transcriptional regulation

    doi: 10.1096/fj.201801513R

    Figure Lengend Snippet: NF-κB governs IL-1α and IL-1β expression. A ) RASFs were pretreated with SB202190 (SB, 10 µM), SP600125 (SP, 10 µM), PD98059 (PD, 10 µM), PDTC (100 µM), or diacerein (DIC, 10 µM) for 2 h prior to IL-1β stimulation for 24 h. Cell lysates were analyzed for IL-1α and IL-1β expression. B ) RASFs (8-well chamber slides) were pretreated with IL-1R antagonist (100 ng/ml) for 2 h followed by IL-1β stimulation for 8 h. C ) Similarly, RASFs were treated with PDTC (100 µM) for 2 h followed by IL-1β stimulation for 8 h. Cells were fixed and analyzed for IL-1α expression using immunofluorescence method. D , E ) Conditioned media from inhibitors study in A were used for determining IL-6 and IL-8 using ELISA. PDTC inhibits both IL-1α and IL-1β, as well as IL-6 and IL-8 production. Cells were lysed and analyzed for IL-1α and IL-1β. * P

    Article Snippet: Small interfering RNA (siRNA) sequences for IL-1α were purchased from MilliporeSigma (eMission predesigned siRNA sequences SASI_Hs01_00167953 and SASI_Hs02_00302829).

    Techniques: Expressing, Immunofluorescence, Enzyme-linked Immunosorbent Assay

    A ) The interacting surface of the conformation of IL-1R (green) and IL-1β (gray) complex at 100 ns molecular dynamics simulation. The electrostatic potential map of interacting surface of receptor shown in red, blue, and white represent electronegative, electropositive, and hydrophobic surface area of the binding site, respectively. The H-bonds and π-π interaction are shown as black and cyan dotted line, respectively. B ) The interacting surface of the conformation of IL-1R (green) and IL-1α (gray) complex at 100 ns molecular dynamics simulation. The electrostatic potential map of interacting surface of receptor shown in red, blue, and white represent electronegative, electropositive, and hydrophobic surface area of the binding site, respectively. The H-bonds and cation-π interaction are shown as black and green dotted line, respectively.

    Journal: The FASEB Journal

    Article Title: Critical role of IL-1α in IL-1β–induced inflammatory responses: cooperation with NF-κBp65 in transcriptional regulation

    doi: 10.1096/fj.201801513R

    Figure Lengend Snippet: A ) The interacting surface of the conformation of IL-1R (green) and IL-1β (gray) complex at 100 ns molecular dynamics simulation. The electrostatic potential map of interacting surface of receptor shown in red, blue, and white represent electronegative, electropositive, and hydrophobic surface area of the binding site, respectively. The H-bonds and π-π interaction are shown as black and cyan dotted line, respectively. B ) The interacting surface of the conformation of IL-1R (green) and IL-1α (gray) complex at 100 ns molecular dynamics simulation. The electrostatic potential map of interacting surface of receptor shown in red, blue, and white represent electronegative, electropositive, and hydrophobic surface area of the binding site, respectively. The H-bonds and cation-π interaction are shown as black and green dotted line, respectively.

    Article Snippet: Small interfering RNA (siRNA) sequences for IL-1α were purchased from MilliporeSigma (eMission predesigned siRNA sequences SASI_Hs01_00167953 and SASI_Hs02_00302829).

    Techniques: Binding Assay

    Effects of IL-1α knockdown. A ) IL-1α expression was knocked down using IL-1α siRNA for 48 h prior to IL-1β stimulation for 24 h. We observed that knocking down IL-1α also reduced IL-1β expression. B ) We reconfirmed the knockdown of IL-1α using an IL-1α ELISA and stimulating RASFs with ATP (5 nM) and LPS (10 μg/ml) to trigger IL-1α release into conditioned medium. C ) Upon confirming IL-1α has been knocked down, we observed a statistically significant decrease of IL-6 production upon IL-1β stimulation. D ) We also observed a decrease in IL-8 production; however, this was not statistically significant. E ) In addition to released proteins, we analyzed expression of cyclooxygenases 1 and 2 (COX-1 and -2) and saw a statistically significant reduction in IL-1β–induced COX-2 and intercellular adhesion molecule 1 expression in IL-1α knockdown samples. * P

    Journal: The FASEB Journal

    Article Title: Critical role of IL-1α in IL-1β–induced inflammatory responses: cooperation with NF-κBp65 in transcriptional regulation

    doi: 10.1096/fj.201801513R

    Figure Lengend Snippet: Effects of IL-1α knockdown. A ) IL-1α expression was knocked down using IL-1α siRNA for 48 h prior to IL-1β stimulation for 24 h. We observed that knocking down IL-1α also reduced IL-1β expression. B ) We reconfirmed the knockdown of IL-1α using an IL-1α ELISA and stimulating RASFs with ATP (5 nM) and LPS (10 μg/ml) to trigger IL-1α release into conditioned medium. C ) Upon confirming IL-1α has been knocked down, we observed a statistically significant decrease of IL-6 production upon IL-1β stimulation. D ) We also observed a decrease in IL-8 production; however, this was not statistically significant. E ) In addition to released proteins, we analyzed expression of cyclooxygenases 1 and 2 (COX-1 and -2) and saw a statistically significant reduction in IL-1β–induced COX-2 and intercellular adhesion molecule 1 expression in IL-1α knockdown samples. * P

    Article Snippet: Small interfering RNA (siRNA) sequences for IL-1α were purchased from MilliporeSigma (eMission predesigned siRNA sequences SASI_Hs01_00167953 and SASI_Hs02_00302829).

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay

    Cell fractionation and ChIP findings provide evidence for the role of NF-κBp65 in transcriptional regulation of IL-1α and IL-1β genes. A ) RASFs were stimulated with IL-1β for 24 h prior to collecting, cytoplasmic, nuclear, chromatin bound, and insoluble nuclear fractions which were probed for both mature and pro forms of IL-1β and IL-1α. B ) RASFs lysate (600 µg) from each treated sample was incubated with 30 µl of protein G Sepharose beads along with 3 µg IL-1α pAb. Pull-down proteins were eluted with 2× SDS and loaded onto acrylamide gel for Western blot analysis for interacting partners. C , D ) We performed ChIP assay on the 3 different promoter regions of IL-1α (−418 to −574 upstream of TSS) ( C ) and IL-1β (−64 to −222 upstream of TSS) ( D ) to identify the role of IL-1α and NF-κBp65. NS, nonstimulated.

    Journal: The FASEB Journal

    Article Title: Critical role of IL-1α in IL-1β–induced inflammatory responses: cooperation with NF-κBp65 in transcriptional regulation

    doi: 10.1096/fj.201801513R

    Figure Lengend Snippet: Cell fractionation and ChIP findings provide evidence for the role of NF-κBp65 in transcriptional regulation of IL-1α and IL-1β genes. A ) RASFs were stimulated with IL-1β for 24 h prior to collecting, cytoplasmic, nuclear, chromatin bound, and insoluble nuclear fractions which were probed for both mature and pro forms of IL-1β and IL-1α. B ) RASFs lysate (600 µg) from each treated sample was incubated with 30 µl of protein G Sepharose beads along with 3 µg IL-1α pAb. Pull-down proteins were eluted with 2× SDS and loaded onto acrylamide gel for Western blot analysis for interacting partners. C , D ) We performed ChIP assay on the 3 different promoter regions of IL-1α (−418 to −574 upstream of TSS) ( C ) and IL-1β (−64 to −222 upstream of TSS) ( D ) to identify the role of IL-1α and NF-κBp65. NS, nonstimulated.

    Article Snippet: Small interfering RNA (siRNA) sequences for IL-1α were purchased from MilliporeSigma (eMission predesigned siRNA sequences SASI_Hs01_00167953 and SASI_Hs02_00302829).

    Techniques: Cell Fractionation, Chromatin Immunoprecipitation, Incubation, Acrylamide Gel Assay, Western Blot

    Signaling kinetics of IL-1α and IL-1β corroborate differences in the interaction with IL-1R. A ) RASFs were stimulated with 5 ng/ml of IL-1β or IL-1α for 5–30 min. Key signaling proteins in the IL-1 signaling panel were analyzed using Western immunoblotting. B , C ) Electrostatic potential map and molecular docking studies of the surface of IL-1R protein receptor bound to IL-1α ( B ) and IL-1β ( C ). The electrostatic potential map of interacting surface was made using the last frame after 100 ns molecular dynamics simulation. The interacting surface of the conformation of IL-1R (green) and IL-1α or IL-1β (gray) complex at 100 ns molecular dynamics simulation. The electrostatic potential map of interacting surface of receptor shown in red, blue, and white represent electronegative, electropositive, and hydrophobic surface area of the binding sites, respectively. NS, nonstimulated.

    Journal: The FASEB Journal

    Article Title: Critical role of IL-1α in IL-1β–induced inflammatory responses: cooperation with NF-κBp65 in transcriptional regulation

    doi: 10.1096/fj.201801513R

    Figure Lengend Snippet: Signaling kinetics of IL-1α and IL-1β corroborate differences in the interaction with IL-1R. A ) RASFs were stimulated with 5 ng/ml of IL-1β or IL-1α for 5–30 min. Key signaling proteins in the IL-1 signaling panel were analyzed using Western immunoblotting. B , C ) Electrostatic potential map and molecular docking studies of the surface of IL-1R protein receptor bound to IL-1α ( B ) and IL-1β ( C ). The electrostatic potential map of interacting surface was made using the last frame after 100 ns molecular dynamics simulation. The interacting surface of the conformation of IL-1R (green) and IL-1α or IL-1β (gray) complex at 100 ns molecular dynamics simulation. The electrostatic potential map of interacting surface of receptor shown in red, blue, and white represent electronegative, electropositive, and hydrophobic surface area of the binding sites, respectively. NS, nonstimulated.

    Article Snippet: Small interfering RNA (siRNA) sequences for IL-1α were purchased from MilliporeSigma (eMission predesigned siRNA sequences SASI_Hs01_00167953 and SASI_Hs02_00302829).

    Techniques: Western Blot, Binding Assay

    IL-1β selectively induces IL-1α expression in RASFs. A ) Human samples for RA and normal (NL) synovial tissues (STs) were prepared and analyzed for IL-1α and IL-1β expression. IL-1α and IL-1β expression were significantly higher in RASTs than nondiseased synovial tissues (NLSTs). B ) RASFs were stimulated with IL-1β (10 ng/ml), TNF-α (20 ng/ml), and LPS (5 µg/ml) for 6 h for IL-1α mRNA analysis using quantitative RT-PCR. IL-1β stimulation increases IL-1α mRNA expression significantly. C ) RNA analysis was confirmed using immunofluorescence studies for IL-1α expression. Images were taken using constant exposure was applied across all the samples using Leica immunofluorescence microscope. D ) RASFs were stimulated with varying concentrations of IL-1β (0.5–10 ng/ml) for 24 h then analyzed for IL-1α and IL-1β expression. IL-1α expression increases with as little as 0.5 ng/ml IL-1β. * P

    Journal: The FASEB Journal

    Article Title: Critical role of IL-1α in IL-1β–induced inflammatory responses: cooperation with NF-κBp65 in transcriptional regulation

    doi: 10.1096/fj.201801513R

    Figure Lengend Snippet: IL-1β selectively induces IL-1α expression in RASFs. A ) Human samples for RA and normal (NL) synovial tissues (STs) were prepared and analyzed for IL-1α and IL-1β expression. IL-1α and IL-1β expression were significantly higher in RASTs than nondiseased synovial tissues (NLSTs). B ) RASFs were stimulated with IL-1β (10 ng/ml), TNF-α (20 ng/ml), and LPS (5 µg/ml) for 6 h for IL-1α mRNA analysis using quantitative RT-PCR. IL-1β stimulation increases IL-1α mRNA expression significantly. C ) RNA analysis was confirmed using immunofluorescence studies for IL-1α expression. Images were taken using constant exposure was applied across all the samples using Leica immunofluorescence microscope. D ) RASFs were stimulated with varying concentrations of IL-1β (0.5–10 ng/ml) for 24 h then analyzed for IL-1α and IL-1β expression. IL-1α expression increases with as little as 0.5 ng/ml IL-1β. * P

    Article Snippet: Small interfering RNA (siRNA) sequences for IL-1α were purchased from MilliporeSigma (eMission predesigned siRNA sequences SASI_Hs01_00167953 and SASI_Hs02_00302829).

    Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Microscopy

    (a) Ratio between receptor activator of nuclear factor kappa B ligand (RANKL) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA expression in four cell lines of human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (PDL) stimulated with 10 ng/ml interleukin (IL)-1α. RNA was extracted from HGF and PDL, first-strand cDNA synthesis and real-time polymerase chain reaction (PCR) analysis for osteoprotegerin (OPG) and GAPDH was carried out as described in Materials and methods. Data are expressed as a ratio between the amount of RANKL and GAPDH mRNA expression ± s.d. of four experiments. An asterisk (*) represents statistical significance ( P

    Journal: Clinical and Experimental Immunology

    Article Title: Protein kinase-A-dependent osteoprotegerin production on interleukin-1 stimulation in human gingival fibroblasts is distinct from periodontal ligament fibroblasts

    doi: 10.1111/j.1365-2249.2005.02937.x

    Figure Lengend Snippet: (a) Ratio between receptor activator of nuclear factor kappa B ligand (RANKL) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA expression in four cell lines of human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (PDL) stimulated with 10 ng/ml interleukin (IL)-1α. RNA was extracted from HGF and PDL, first-strand cDNA synthesis and real-time polymerase chain reaction (PCR) analysis for osteoprotegerin (OPG) and GAPDH was carried out as described in Materials and methods. Data are expressed as a ratio between the amount of RANKL and GAPDH mRNA expression ± s.d. of four experiments. An asterisk (*) represents statistical significance ( P

    Article Snippet: Cycloheximide (CHX), human recombinant IL-1α, phorbol 12,13-didecanoate (PDD), forskolin (For) and Staurosporine were purchased from Sigma Chemicals (St Louis, MO, USA).

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

    Ratio between osteoprotegerin (OPG) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA expression in four cell lines of human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (PDL) stimulated with 10 ng/ml interleukin (IL)-1α with or without 1% cycloheximide (CHX). RNA was extracted from both HGF and PDL, first-strand cDNA synthesis and real-time polymerase chain reaction (PCR) analysis for osteoprotegerin (OPG) and GAPDH was carried out as described in Materials and methods. Data are expressed as a ratio between the amount of OPG and GAPDH mRNA expression ± s.d. of four experiments. An asterisk (*) represents statistical significance ( P

    Journal: Clinical and Experimental Immunology

    Article Title: Protein kinase-A-dependent osteoprotegerin production on interleukin-1 stimulation in human gingival fibroblasts is distinct from periodontal ligament fibroblasts

    doi: 10.1111/j.1365-2249.2005.02937.x

    Figure Lengend Snippet: Ratio between osteoprotegerin (OPG) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA expression in four cell lines of human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (PDL) stimulated with 10 ng/ml interleukin (IL)-1α with or without 1% cycloheximide (CHX). RNA was extracted from both HGF and PDL, first-strand cDNA synthesis and real-time polymerase chain reaction (PCR) analysis for osteoprotegerin (OPG) and GAPDH was carried out as described in Materials and methods. Data are expressed as a ratio between the amount of OPG and GAPDH mRNA expression ± s.d. of four experiments. An asterisk (*) represents statistical significance ( P

    Article Snippet: Cycloheximide (CHX), human recombinant IL-1α, phorbol 12,13-didecanoate (PDD), forskolin (For) and Staurosporine were purchased from Sigma Chemicals (St Louis, MO, USA).

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

    CD11a hi FcγRIII hi B cells produce a high level of IFN-γ in response to CD40 ligation. C57BL/6 mice were infected with 2 × 10 6 LM. (A) The intracellular expression of IL-1α, IL-2, IL-6, IL-10 and IFN-γ in CD11a hi FcγRIII hi and conventional B cells was assayed on day 3 post-infection. (B - G) Splenic CD11a hi FcγRIII hi and conventional B cells were sorted on day 3 post-infection. IL-1β, IL-2, IL-6, IL-12p70, IFN-γ, or TNF-α secretion by CD11a hi FcγRIII hi and conventional B cells was detected by ELISA. Data shown represent the mean ± SD of triplicate experiments. * P

    Journal: Cell Research

    Article Title: Identification of IFN-?-producing innate B cells

    doi: 10.1038/cr.2013.155

    Figure Lengend Snippet: CD11a hi FcγRIII hi B cells produce a high level of IFN-γ in response to CD40 ligation. C57BL/6 mice were infected with 2 × 10 6 LM. (A) The intracellular expression of IL-1α, IL-2, IL-6, IL-10 and IFN-γ in CD11a hi FcγRIII hi and conventional B cells was assayed on day 3 post-infection. (B - G) Splenic CD11a hi FcγRIII hi and conventional B cells were sorted on day 3 post-infection. IL-1β, IL-2, IL-6, IL-12p70, IFN-γ, or TNF-α secretion by CD11a hi FcγRIII hi and conventional B cells was detected by ELISA. Data shown represent the mean ± SD of triplicate experiments. * P

    Article Snippet: Fluorescence-conjugated mAbs against CD4 (RM4-5), CD5 (53-7.3), CD8 (53-6.7), CD11a (2D7), CD11b (M1/70), CD11c (HL3), CD16/32 (2.4G2), CD19 (1D3), CD21/CD35 (7G6), CD23 (B3B4), CD40 (3/23), CD40L (TRAP1), CD43 (S7), CD45R/B220 (RA3-6B2), CD80 (16-10A1), CD86 (GL1), CD93 (C1qRp) (R139), F4/80 (6F12), NK1.1 (PK136), I-A/I-E (2G9), IgD (11-26c.2a), IgM (G155-228), IL-1α (364-3B3-14), IL-2 (MQ1-17H12), IL-6 (MP5-20F3), IL-10 (JES3-19F1), IFN-γ (B27), IgD (11-26c.2a) and IgM (G155-228) and agonistic anti-CD40 (HM40-3) and neutralizing CD11a mAbs (M17/4) were purchased from BD Biosciences Pharmingen (San Diego, CA, USA).

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

    Tissue cytokine changes in hippocampal organotypic cultures after SD. Tissue cytokine measurements from sham-treated (light-gray bars) and SD-treated (dark-gray bars) cultures were made after recovery of 6 hours, 1 day, and 3 days (left, middle, and right of each cytokine panel, respectively). Units of gray bars in each panel are log picograms per milligram. Right axis units are linear × 1,000 pg/mg. Colored bars in each panel emphasize the difference between SD- and sham-treated cytokine levels for each time point. For example, in the top left panel, the cytokine levels of IL-1α at 6 hours of recovery are 1,081 ± 411 and 2,488 ± 1,003 for sham-and SD-treated cultures, respectively. Subtracting the sham value from the SD value and dividing by 1,000 equals 1.41, the value represented by the blue bar. Each measurement represents tissue samples from a total of five to nine cultures. Significant differences ( P

    Journal: Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism

    Article Title: Multiplexed Cytokine Protein Expression Profiles From Spreading Depression in Hippocampal Organotypic Cultures

    doi: 10.1097/01.WCB.0000126566.34753.30

    Figure Lengend Snippet: Tissue cytokine changes in hippocampal organotypic cultures after SD. Tissue cytokine measurements from sham-treated (light-gray bars) and SD-treated (dark-gray bars) cultures were made after recovery of 6 hours, 1 day, and 3 days (left, middle, and right of each cytokine panel, respectively). Units of gray bars in each panel are log picograms per milligram. Right axis units are linear × 1,000 pg/mg. Colored bars in each panel emphasize the difference between SD- and sham-treated cytokine levels for each time point. For example, in the top left panel, the cytokine levels of IL-1α at 6 hours of recovery are 1,081 ± 411 and 2,488 ± 1,003 for sham-and SD-treated cultures, respectively. Subtracting the sham value from the SD value and dividing by 1,000 equals 1.41, the value represented by the blue bar. Each measurement represents tissue samples from a total of five to nine cultures. Significant differences ( P

    Article Snippet: The cultures were then incubated overnight at 4°C in blocking solution containing either OX-42 (1:1,000 dilution; Serotec, Raleigh, NC, U.S.A.), IL-1α (1:500; Serotec), IL-1β (1:200; Serotec), or IFN-γ (1:200; Biosource International, Camarillo, CA, U.S.A.).

    Techniques:

    Representative photomicrographs showing immunostaining changes in hippocampal organotypic culture after SD treatment and recovery. Top panel of images shows OX-42 immunostaining in normal HOTC ( A ), 1-day sham recovery ( B ), 1-day SD recovery ( C ), and 3-day SD recovery ( D ). Bottom panel of images shows representative immunostaining for IL-1α ( E ), IL-1β ( F ), and IFN-γ ( G ) after recurrent SD and 6 hours of recovery. OX-42–positive microglia in normal HOTC ( A ) display finely branched processes typical of ramified resting microglia. After sham or SD treatment ( B and C ), microglia became activated; i.e., cell processes were shorter and cell bodies somewhat enlarged. The latter changes were even more evident by 3 days after SD ( D ). Immunostaining for selected cytokines (IL-1α, IL-1β, and IFN-γ) was positive after SD in cells morphologically resembling microglia. This supports the results from multiplexed MFCA measurements and suggests that the main source for increased expression of these cytokines is microglia. Calibration bar is 10 μM.

    Journal: Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism

    Article Title: Multiplexed Cytokine Protein Expression Profiles From Spreading Depression in Hippocampal Organotypic Cultures

    doi: 10.1097/01.WCB.0000126566.34753.30

    Figure Lengend Snippet: Representative photomicrographs showing immunostaining changes in hippocampal organotypic culture after SD treatment and recovery. Top panel of images shows OX-42 immunostaining in normal HOTC ( A ), 1-day sham recovery ( B ), 1-day SD recovery ( C ), and 3-day SD recovery ( D ). Bottom panel of images shows representative immunostaining for IL-1α ( E ), IL-1β ( F ), and IFN-γ ( G ) after recurrent SD and 6 hours of recovery. OX-42–positive microglia in normal HOTC ( A ) display finely branched processes typical of ramified resting microglia. After sham or SD treatment ( B and C ), microglia became activated; i.e., cell processes were shorter and cell bodies somewhat enlarged. The latter changes were even more evident by 3 days after SD ( D ). Immunostaining for selected cytokines (IL-1α, IL-1β, and IFN-γ) was positive after SD in cells morphologically resembling microglia. This supports the results from multiplexed MFCA measurements and suggests that the main source for increased expression of these cytokines is microglia. Calibration bar is 10 μM.

    Article Snippet: The cultures were then incubated overnight at 4°C in blocking solution containing either OX-42 (1:1,000 dilution; Serotec, Raleigh, NC, U.S.A.), IL-1α (1:500; Serotec), IL-1β (1:200; Serotec), or IFN-γ (1:200; Biosource International, Camarillo, CA, U.S.A.).

    Techniques: Immunostaining, Expressing

    Kinetics of cytokine secretion in Max Planck Institute (MPI) cells. Production of interleukin 6 (IL-6), TNF-α, IL-1α, IL-1β, and IL-10 by MPI cells (P32) in response to (A) heat-killed (HK) Mycobacterium tuberculosis ( Mtb ) or 10 ng/mL lipopolysaccharide (LPS) or (B) live Mtb infection challenge. Mtb was used at different multiplicity of infection (MOI) and cytokines were quantified at 24 h post-treatment for LPS, 4, 8, 24, and 48 h post-infection with HK bacteria and 4, 8, 24, and 48 h (MOI 5) or 4, 8, and 24 h (MOI 10 and 20) post-infection with live bacteria. Data are representative of three independent experiments and expressed as mean ± SD for three biological replicates. Statistical significance between time points (black asterisks) or between stimulated and non-stimulated cells at corresponding time points (red asterisks) were determined by Prism software using the unpaired Student’s t -test. *, p -value

    Journal: Frontiers in Immunology

    Article Title: Mycobacterium tuberculosis Infection and Innate Responses in a New Model of Lung Alveolar Macrophages

    doi: 10.3389/fimmu.2018.00438

    Figure Lengend Snippet: Kinetics of cytokine secretion in Max Planck Institute (MPI) cells. Production of interleukin 6 (IL-6), TNF-α, IL-1α, IL-1β, and IL-10 by MPI cells (P32) in response to (A) heat-killed (HK) Mycobacterium tuberculosis ( Mtb ) or 10 ng/mL lipopolysaccharide (LPS) or (B) live Mtb infection challenge. Mtb was used at different multiplicity of infection (MOI) and cytokines were quantified at 24 h post-treatment for LPS, 4, 8, 24, and 48 h post-infection with HK bacteria and 4, 8, 24, and 48 h (MOI 5) or 4, 8, and 24 h (MOI 10 and 20) post-infection with live bacteria. Data are representative of three independent experiments and expressed as mean ± SD for three biological replicates. Statistical significance between time points (black asterisks) or between stimulated and non-stimulated cells at corresponding time points (red asterisks) were determined by Prism software using the unpaired Student’s t -test. *, p -value

    Article Snippet: Cytokines were titrated by ELISA, using antibody pairs directed against interleukin 6 (IL-6), TNF-α, IL-1β, IL-10 (ThemoFisher Scientific), and IL-1α (Peprotech), following manufacturers’ recommendations.

    Techniques: Infection, Software

    IL-1R signaling instructs AECII to produce GM-CSF during pulmonary Legionella infection (A-C) GM-CSF levels in the BAL of C57BL/6 mice at 24, 48, and 72 hpi (A), WT, Il1a -/- , Il1b -/- and Il1r1 -/- mice at 24 hpi (B), and WT: Csf2 -/- BM chimeras at 24 hpi (C). (D) Csf2 transcript levels in the indicated cell types isolated from the lungs of naïve and L . p .-infected C57BL/6 mice at 24 hpi. (E) Representative flow cytometric plots showing intracellular staining for GM-CSF in AECII and AECI from the lungs of naïve and L . p .-infected C57BL/6 mice at 24 hpi. Frequency and total number of GM-CSF-producing AECII and AECI. (F-H) GM-CSF levels in the BAL of WT: Il1r1 -/- BM chimeras (F), Il1r1 fl/fl ;Spc-cre ERT2 and Il1r1 fl/fl mice (G), and Il1r1 r/r ;Spc-cre ERT2 and Il1r1 r/r mice (H) at 24 hpi. (I) GM-CSF levels in the supernatants of primary WT or Il1r1 -/- AECII treated with 10 ng/ml each of recombinant IL-1α and IL-1β (rIL-1α/β) or PBS vehicle control and infected with L . p . (MOI=5) at 12 hpi. Data shown are the pooled results of three (A, B, G, and H) or two (C-F) independent experiments with 3-4 mice per condition in each experiment. Data shown in I are the pooled results of two independent experiments with 3 mice and triplicate wells per condition in each experiment. NS, not significant; *p

    Journal: bioRxiv

    Article Title: Infected macrophages engage alveolar epithelium to metabolically reprogram myeloid cells and promote antibacterial inflammation

    doi: 10.1101/2020.02.29.970004

    Figure Lengend Snippet: IL-1R signaling instructs AECII to produce GM-CSF during pulmonary Legionella infection (A-C) GM-CSF levels in the BAL of C57BL/6 mice at 24, 48, and 72 hpi (A), WT, Il1a -/- , Il1b -/- and Il1r1 -/- mice at 24 hpi (B), and WT: Csf2 -/- BM chimeras at 24 hpi (C). (D) Csf2 transcript levels in the indicated cell types isolated from the lungs of naïve and L . p .-infected C57BL/6 mice at 24 hpi. (E) Representative flow cytometric plots showing intracellular staining for GM-CSF in AECII and AECI from the lungs of naïve and L . p .-infected C57BL/6 mice at 24 hpi. Frequency and total number of GM-CSF-producing AECII and AECI. (F-H) GM-CSF levels in the BAL of WT: Il1r1 -/- BM chimeras (F), Il1r1 fl/fl ;Spc-cre ERT2 and Il1r1 fl/fl mice (G), and Il1r1 r/r ;Spc-cre ERT2 and Il1r1 r/r mice (H) at 24 hpi. (I) GM-CSF levels in the supernatants of primary WT or Il1r1 -/- AECII treated with 10 ng/ml each of recombinant IL-1α and IL-1β (rIL-1α/β) or PBS vehicle control and infected with L . p . (MOI=5) at 12 hpi. Data shown are the pooled results of three (A, B, G, and H) or two (C-F) independent experiments with 3-4 mice per condition in each experiment. Data shown in I are the pooled results of two independent experiments with 3 mice and triplicate wells per condition in each experiment. NS, not significant; *p

    Article Snippet: For intracellular cytokine staining, cell suspensions were incubated with Brefeldin A (0.1%) and monensin (0.066%) solution for 4 hours at 37°C prior to all staining, treated with BD Cytofix/Cytoperm™ buffer for 20 min at 4°C, and stained with antibodies specific for TNFα (Thermo Fisher Scientific, clone MP6-XT22), IL-12 (BioLegend, clone C15.6), GM-CSF (BioLegend, clone MP1-31G6), IL-1α (Thermo Fisher Scientific, clone ALF-161), or IL-1β (Thermo Fisher Scientific, clone NJTEN3) in perm/wash buffer for 30 min at 4°C.

    Techniques: Infection, Mouse Assay, Isolation, Staining, Recombinant

    GM-CSF is required for maximal inflammatory cytokine production and bacterial clearance (A and B) IL-1β, TNF, IL-12, and IFNγ levels in the BAL at 24 and 48 hpi (A) and L . p . CFUs in the lungs at 72 hpi (B) of WT and Csf2 -/- mice. (C and D) C57BL/6 mice injected with anti-GM-CSF or isotype control antibodies (Ab) prior to L . p . infection (see Method Details). IL-1β, TNF, IL-12, and IFNγ levels in the BAL at 24 hpi and 48 hpi (C). L . p . CFUs in the lungs at 72 hpi (D). (E and F) Representative flow cytometric plots showing intracellular staining for IL-1α and IL-1β in Ly6C hi MCs (E) and cDCs (F) from the lungs of anti-GM-CSF or isotype control Ab-treated mice infected with L . p . at 24 hpi. Frequency and total number of IL-1α- and IL-1β-producing MCs (E) and cDCs (F) are shown. (G) L . p . CFUs in the lungs at 72 hpi of C57BL/6 mice intraperitoneally injected with anti-GM-CSF or isotype control Ab prior to L . p . infection or a third group injected with anti-GM-CSF Ab provided 500 ng recombinant IL-12p70 (rIL-12) intranasally at the time of infection and at day 2 post-infection (see Method Details). Data shown are the pooled results of two (A-C and G) or three (D-F) independent experiments or with 3-4 mice per condition for each experiment. NS, not significant; *p

    Journal: bioRxiv

    Article Title: Infected macrophages engage alveolar epithelium to metabolically reprogram myeloid cells and promote antibacterial inflammation

    doi: 10.1101/2020.02.29.970004

    Figure Lengend Snippet: GM-CSF is required for maximal inflammatory cytokine production and bacterial clearance (A and B) IL-1β, TNF, IL-12, and IFNγ levels in the BAL at 24 and 48 hpi (A) and L . p . CFUs in the lungs at 72 hpi (B) of WT and Csf2 -/- mice. (C and D) C57BL/6 mice injected with anti-GM-CSF or isotype control antibodies (Ab) prior to L . p . infection (see Method Details). IL-1β, TNF, IL-12, and IFNγ levels in the BAL at 24 hpi and 48 hpi (C). L . p . CFUs in the lungs at 72 hpi (D). (E and F) Representative flow cytometric plots showing intracellular staining for IL-1α and IL-1β in Ly6C hi MCs (E) and cDCs (F) from the lungs of anti-GM-CSF or isotype control Ab-treated mice infected with L . p . at 24 hpi. Frequency and total number of IL-1α- and IL-1β-producing MCs (E) and cDCs (F) are shown. (G) L . p . CFUs in the lungs at 72 hpi of C57BL/6 mice intraperitoneally injected with anti-GM-CSF or isotype control Ab prior to L . p . infection or a third group injected with anti-GM-CSF Ab provided 500 ng recombinant IL-12p70 (rIL-12) intranasally at the time of infection and at day 2 post-infection (see Method Details). Data shown are the pooled results of two (A-C and G) or three (D-F) independent experiments or with 3-4 mice per condition for each experiment. NS, not significant; *p

    Article Snippet: For intracellular cytokine staining, cell suspensions were incubated with Brefeldin A (0.1%) and monensin (0.066%) solution for 4 hours at 37°C prior to all staining, treated with BD Cytofix/Cytoperm™ buffer for 20 min at 4°C, and stained with antibodies specific for TNFα (Thermo Fisher Scientific, clone MP6-XT22), IL-12 (BioLegend, clone C15.6), GM-CSF (BioLegend, clone MP1-31G6), IL-1α (Thermo Fisher Scientific, clone ALF-161), or IL-1β (Thermo Fisher Scientific, clone NJTEN3) in perm/wash buffer for 30 min at 4°C.

    Techniques: Mouse Assay, Injection, Infection, Staining, Recombinant

    Cell-intrinsic GM-CSF receptor signaling is required for inflammatory cytokine production by myeloid cells WT→WT, Csf2rb -/- →WT, and 50% WT/50% Csf2rb -/- →WT BM chimeras intranasally infected with L . p . (A) IL-1α, IL-1β, IL-6, TNF, and IL-12 levels in the BAL at 24 hpi and IFNγ at 48 hpi. (B) L . p . CFUs in the lungs of chimeric WT→WT and Csf2rb -/- →WT mice at 72 hpi. (C and D) Representative flow cytometric plots and graphs depicting the frequency of IL-1α+ or IL-1β+ WT or Csf2rb -/- MCs (C) and cDCs (D) from the lungs of 50% WT/50% Csf2rb -/- →WT mixed BM chimeras at 24 hpi. Each line represents the paired values of WT and Csf2rb -/- cells from a given mouse. (E) Il1a, Il1b, Il6, Il12a, Il12b, Tnf , and Nox2 transcript levels in Ly6C hi MCs and Ly6C + CD11b + cDCs isolated from the lungs of 50% WT/50% Csf2rb -/- →WT mixed BM chimeras at 24 hpi, as quantified by qRT-PCR. Each line represents the paired values of WT and Csf2rb -/- cells from a given mouse. Data shown are the pooled results of two (A, B, and E) or three (C and D) independent experiments with 3 mice per experiment. NS, not significant; *p

    Journal: bioRxiv

    Article Title: Infected macrophages engage alveolar epithelium to metabolically reprogram myeloid cells and promote antibacterial inflammation

    doi: 10.1101/2020.02.29.970004

    Figure Lengend Snippet: Cell-intrinsic GM-CSF receptor signaling is required for inflammatory cytokine production by myeloid cells WT→WT, Csf2rb -/- →WT, and 50% WT/50% Csf2rb -/- →WT BM chimeras intranasally infected with L . p . (A) IL-1α, IL-1β, IL-6, TNF, and IL-12 levels in the BAL at 24 hpi and IFNγ at 48 hpi. (B) L . p . CFUs in the lungs of chimeric WT→WT and Csf2rb -/- →WT mice at 72 hpi. (C and D) Representative flow cytometric plots and graphs depicting the frequency of IL-1α+ or IL-1β+ WT or Csf2rb -/- MCs (C) and cDCs (D) from the lungs of 50% WT/50% Csf2rb -/- →WT mixed BM chimeras at 24 hpi. Each line represents the paired values of WT and Csf2rb -/- cells from a given mouse. (E) Il1a, Il1b, Il6, Il12a, Il12b, Tnf , and Nox2 transcript levels in Ly6C hi MCs and Ly6C + CD11b + cDCs isolated from the lungs of 50% WT/50% Csf2rb -/- →WT mixed BM chimeras at 24 hpi, as quantified by qRT-PCR. Each line represents the paired values of WT and Csf2rb -/- cells from a given mouse. Data shown are the pooled results of two (A, B, and E) or three (C and D) independent experiments with 3 mice per experiment. NS, not significant; *p

    Article Snippet: For intracellular cytokine staining, cell suspensions were incubated with Brefeldin A (0.1%) and monensin (0.066%) solution for 4 hours at 37°C prior to all staining, treated with BD Cytofix/Cytoperm™ buffer for 20 min at 4°C, and stained with antibodies specific for TNFα (Thermo Fisher Scientific, clone MP6-XT22), IL-12 (BioLegend, clone C15.6), GM-CSF (BioLegend, clone MP1-31G6), IL-1α (Thermo Fisher Scientific, clone ALF-161), or IL-1β (Thermo Fisher Scientific, clone NJTEN3) in perm/wash buffer for 30 min at 4°C.

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

    GM-CSF metabolically reprograms monocytes to undergo increased aerobic glycolysis, which enhances inflammatory cytokine production (A) Isolated WT MCs were uninfected or infected with L . p . (MOI=5) and given 10 ng/mL rGM-CSF or PBS vehicle control. IL-1α, IL-1β, TNF, and IL-12 levels at 12 hpi are shown. (B) Extracellular acidification rate (ECAR) of uninfected, L . p . - infected (MOI=5), or LPS-treated (10 ng/ml) MCs incubated with rGM-CSF (10 ng/ml) or PBS vehicle control for 12 h before and after sequential treatment with glucose (10mM), oligomycin (Oligo) (1μM), and 2-DG (50 mM). (C) Heatmap depicting the fold-change in transcript levels of genes encoding glucose transporters and glycolytic enzymes in LPS- or L . p . - treated MCs incubated with or without rGM-CSF for 10 h relative to uninfected cells without rGM-CSF. Graphical model depicts an abbreviated version of the glycolytic pathway showing upregulated GLUT1, HK2 and PFKP expression following rGM-CSF treatment. (D) Immunoblot analysis of GLUT1, HK2, PFKP, pro-IL-1β, and β-actin in the lysates of LPS or L . p . - treated MCs incubated with or without rGM-CSF at 10 and 20 hpi. (E) Glut1, HK2 and Pfkp transcript levels in WT and Csf2rb -/- Ly6C hi MCs from the lungs of 50% WT/50% Csf2rb -/- →WT mixed BM chimeras at 24 hpi. Each line represents the paired values of WT and Csf2rb -/- MCs from a given mouse. (F) Il1b, Tnf , and Il12a transcript levels in LPS- or L . p . - treated MCs treated with 10 mM 2-DG or vehicle control in the presence of rGM-CSF or PBS vehicle control at 6 hpi. (G) Immunoblot analysis of pro-IL-1β and β-actin in the lysates of WT MCs incubated in media containing glucose (10 mM) or galactose (10 mM) that were then uninfected, infected with L . p . (MOI=5), or treated with LPS (10 ng/ml) in the presence of rGM-CSF or PBS vehicle control for 10 hours.. (H) TNF and IL-12 levels in the supernatants of WT MCs incubated in media containing glucose or galactose, infected with L . p ., and treated with rGM-CSF or PBS vehicle control at 10 hpi. (I) Graphical model depicting that GM-CSF/JAK2/STAT5 signaling upregulates Glut1, HK2 and Pfkp expression and promotes aerobic glycolysis, which is critical for maximal inflammatory cytokine expression. Data shown are the pooled results of three (A, B, F and H) or two (C) independent experiments with triplicate wells per condition in each experiment. Data shown in E are the pooled results of two independent experiments with 3 mice per experiment. Data shown in D and G are representative of two independent experiments. NS, not significant; *p

    Journal: bioRxiv

    Article Title: Infected macrophages engage alveolar epithelium to metabolically reprogram myeloid cells and promote antibacterial inflammation

    doi: 10.1101/2020.02.29.970004

    Figure Lengend Snippet: GM-CSF metabolically reprograms monocytes to undergo increased aerobic glycolysis, which enhances inflammatory cytokine production (A) Isolated WT MCs were uninfected or infected with L . p . (MOI=5) and given 10 ng/mL rGM-CSF or PBS vehicle control. IL-1α, IL-1β, TNF, and IL-12 levels at 12 hpi are shown. (B) Extracellular acidification rate (ECAR) of uninfected, L . p . - infected (MOI=5), or LPS-treated (10 ng/ml) MCs incubated with rGM-CSF (10 ng/ml) or PBS vehicle control for 12 h before and after sequential treatment with glucose (10mM), oligomycin (Oligo) (1μM), and 2-DG (50 mM). (C) Heatmap depicting the fold-change in transcript levels of genes encoding glucose transporters and glycolytic enzymes in LPS- or L . p . - treated MCs incubated with or without rGM-CSF for 10 h relative to uninfected cells without rGM-CSF. Graphical model depicts an abbreviated version of the glycolytic pathway showing upregulated GLUT1, HK2 and PFKP expression following rGM-CSF treatment. (D) Immunoblot analysis of GLUT1, HK2, PFKP, pro-IL-1β, and β-actin in the lysates of LPS or L . p . - treated MCs incubated with or without rGM-CSF at 10 and 20 hpi. (E) Glut1, HK2 and Pfkp transcript levels in WT and Csf2rb -/- Ly6C hi MCs from the lungs of 50% WT/50% Csf2rb -/- →WT mixed BM chimeras at 24 hpi. Each line represents the paired values of WT and Csf2rb -/- MCs from a given mouse. (F) Il1b, Tnf , and Il12a transcript levels in LPS- or L . p . - treated MCs treated with 10 mM 2-DG or vehicle control in the presence of rGM-CSF or PBS vehicle control at 6 hpi. (G) Immunoblot analysis of pro-IL-1β and β-actin in the lysates of WT MCs incubated in media containing glucose (10 mM) or galactose (10 mM) that were then uninfected, infected with L . p . (MOI=5), or treated with LPS (10 ng/ml) in the presence of rGM-CSF or PBS vehicle control for 10 hours.. (H) TNF and IL-12 levels in the supernatants of WT MCs incubated in media containing glucose or galactose, infected with L . p ., and treated with rGM-CSF or PBS vehicle control at 10 hpi. (I) Graphical model depicting that GM-CSF/JAK2/STAT5 signaling upregulates Glut1, HK2 and Pfkp expression and promotes aerobic glycolysis, which is critical for maximal inflammatory cytokine expression. Data shown are the pooled results of three (A, B, F and H) or two (C) independent experiments with triplicate wells per condition in each experiment. Data shown in E are the pooled results of two independent experiments with 3 mice per experiment. Data shown in D and G are representative of two independent experiments. NS, not significant; *p

    Article Snippet: For intracellular cytokine staining, cell suspensions were incubated with Brefeldin A (0.1%) and monensin (0.066%) solution for 4 hours at 37°C prior to all staining, treated with BD Cytofix/Cytoperm™ buffer for 20 min at 4°C, and stained with antibodies specific for TNFα (Thermo Fisher Scientific, clone MP6-XT22), IL-12 (BioLegend, clone C15.6), GM-CSF (BioLegend, clone MP1-31G6), IL-1α (Thermo Fisher Scientific, clone ALF-161), or IL-1β (Thermo Fisher Scientific, clone NJTEN3) in perm/wash buffer for 30 min at 4°C.

    Techniques: Metabolic Labelling, Isolation, Infection, Incubation, Expressing, Mouse Assay

    CIK Cell Culture, Identification, and Adenovirus-Binding Ability Detection (A–C) CIK cells were isolated from human peripheral blood and differentiated by adding IFN-γ, anti-CD3 mAb, IL-1α, and IL-2 in different culture stages. CIK cells were observed under a phase contrast microscope at 200× after culturing for 5 (A), 10 (B), and 14 (C) days. Microscope observation showed that the cell number increased gradually and reached its peak and formed a cell colony on day 14, which is a defining characteristic of CIK cells. (D–I) CIK cells were harvested on day 14 and embedded in wax blocks. (D) CIK cells were sectioned and stained with H E, and they were observed under a microscope at 400× magnification. Microscope observation showed morphologically regular cells, with larger nuclei and less cytoplasm. (E and F) CIK cell markers (E, CD3 and F, CD56) were detected by immunohistochemistry and found to be expressed on CIK cell membranes at 400× magnification. (G) CD46 protein (the receptor for KGHV500 and KGHV400 adenoviruses) was also detected on CIK cell surfaces at 400× magnification. (H and I) KGHV500 (H) and KGHV400 (I) adenovirus hexon was detected on CIK cell membranes at 400× magnification.

    Journal: Molecular Therapy Oncolytics

    Article Title: Recombinant Adenovirus KGHV500 and CIK Cells Codeliver Anti-p21-Ras scFv for the Treatment of Gastric Cancer with Wild-Type Ras Overexpression

    doi: 10.1016/j.omto.2018.10.003

    Figure Lengend Snippet: CIK Cell Culture, Identification, and Adenovirus-Binding Ability Detection (A–C) CIK cells were isolated from human peripheral blood and differentiated by adding IFN-γ, anti-CD3 mAb, IL-1α, and IL-2 in different culture stages. CIK cells were observed under a phase contrast microscope at 200× after culturing for 5 (A), 10 (B), and 14 (C) days. Microscope observation showed that the cell number increased gradually and reached its peak and formed a cell colony on day 14, which is a defining characteristic of CIK cells. (D–I) CIK cells were harvested on day 14 and embedded in wax blocks. (D) CIK cells were sectioned and stained with H E, and they were observed under a microscope at 400× magnification. Microscope observation showed morphologically regular cells, with larger nuclei and less cytoplasm. (E and F) CIK cell markers (E, CD3 and F, CD56) were detected by immunohistochemistry and found to be expressed on CIK cell membranes at 400× magnification. (G) CD46 protein (the receptor for KGHV500 and KGHV400 adenoviruses) was also detected on CIK cell surfaces at 400× magnification. (H and I) KGHV500 (H) and KGHV400 (I) adenovirus hexon was detected on CIK cell membranes at 400× magnification.

    Article Snippet: After 24-hr incubation, 50 ng/mL anti-CD3 monoclonal antibody (mAb) (OKT eBioscience, USA), 100 U/mL recombinant human IL-1α (PeproTech, 200-01A, USA), and 300 U/mL recombinant human IL-2 (PeproTech, 200-02, USA) were added.

    Techniques: Cell Culture, Binding Assay, Isolation, Microscopy, Staining, Immunohistochemistry

    TRAIL expression and modulation in pIL6-TRAIL + -GFP + -UC-MSCs. a Left: transfection efficiency of UC-MSCs evaluated by flow cytometry for GFP fluorescence. Wildtype UC-MSCs were negative as compared to GFP + -UC-MSCs (red histogram: 83.5%) in a similar amount as for pIL6-TRAIL + -GFP + -UC-MSCs (blue histogram: 87.6%). Right: FACS analysis revealed constitutive TRAIL expression in transduced UC-MSCs (green histogram: 62.5%) that was significantly enhanced by the conditioned medium from U-266 cells (blue histogram: 80.3%) as well as in response to IL-1α/IL-1β (red histogram: 91.2%). b Confocal microscopy of pIL6-TRAIL + -GFP + -UC-MSCs confirmed the upregulation of TRAIL (FITC, green) induced by either U-266 supernatant or IL-1α/IL-1β treatment. Phalloidin staining was used to show actin (TRITC-red); nuclei were counterstained with Hoechst 33342 (blue). Magnification: 100×. c Evaluation of TRAIL expression in transduced cells by RT-qPCR, western blot analysis, and ELISA. (i) After either U-266 or IL-1α/IL-1β treatment, TRAIL mRNA levels in pIL6-TRAIL + -GFP + -UC-MSCs increased up to 2.2-fold and 2.5-fold compared to basal condition. Data are relative amount of mRNA expression normalized to GAPDH and are presented as mean ± SD of three independent experiments. * p

    Journal: Stem Cell Research & Therapy

    Article Title: pIL6-TRAIL-engineered umbilical cord mesenchymal/stromal stem cells are highly cytotoxic for myeloma cells both in vitro and in vivo

    doi: 10.1186/s13287-017-0655-6

    Figure Lengend Snippet: TRAIL expression and modulation in pIL6-TRAIL + -GFP + -UC-MSCs. a Left: transfection efficiency of UC-MSCs evaluated by flow cytometry for GFP fluorescence. Wildtype UC-MSCs were negative as compared to GFP + -UC-MSCs (red histogram: 83.5%) in a similar amount as for pIL6-TRAIL + -GFP + -UC-MSCs (blue histogram: 87.6%). Right: FACS analysis revealed constitutive TRAIL expression in transduced UC-MSCs (green histogram: 62.5%) that was significantly enhanced by the conditioned medium from U-266 cells (blue histogram: 80.3%) as well as in response to IL-1α/IL-1β (red histogram: 91.2%). b Confocal microscopy of pIL6-TRAIL + -GFP + -UC-MSCs confirmed the upregulation of TRAIL (FITC, green) induced by either U-266 supernatant or IL-1α/IL-1β treatment. Phalloidin staining was used to show actin (TRITC-red); nuclei were counterstained with Hoechst 33342 (blue). Magnification: 100×. c Evaluation of TRAIL expression in transduced cells by RT-qPCR, western blot analysis, and ELISA. (i) After either U-266 or IL-1α/IL-1β treatment, TRAIL mRNA levels in pIL6-TRAIL + -GFP + -UC-MSCs increased up to 2.2-fold and 2.5-fold compared to basal condition. Data are relative amount of mRNA expression normalized to GAPDH and are presented as mean ± SD of three independent experiments. * p

    Article Snippet: Finally, soluble TRAIL was also measured in supernatants of pIL6-TRAIL + -GFP + -UC-MSCs after treatment with U-266 conditioned medium, or IL-1α and IL-1β, by dedicated ELISA kit (Abcam).

    Techniques: Expressing, Transfection, Flow Cytometry, Cytometry, Fluorescence, FACS, Confocal Microscopy, Staining, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

    In vitro apoptosis of U-266 cells induced by pIL6-TRAIL + -GFP + -UC-MSCs. a Apoptosis in U-266 cells by transduced UC-MSCs was measured by Annexin V/PI staining using flow cytometry. Representative dot plots revealed that the apoptosis extent was significantly increased (43.8%) after 24 h of coculture with pIL6-TRAIL + -GFP + -UC-MSCs with respect to control UC-MSCs (19.4%) and GFP + -UC-MSCs (20.2%). The effect was also enhanced when IL-1α and IL-1β were added to the cocultures (70.9% of U-266 cell apoptosis). b Active caspase-8 in U-266 cells as signature of TRAIL-induced apoptosis was measured by flow cytometry after 24 h of coculture with UC-MSCs, GFP + -UC-MSCs, and pIL6-TRAIL + -GFP + -UC-MSCs. This representative experiment depicts the activity of caspase-8 in 97.8% of U266 cells cocultured with pIL6-TRAIL + -GFP + -UC-MSCs as compared to 17.9% of control UC-MSCs, and 22% of GFP + -UC-MSCs. Such high levels of active caspase-8 were not further modified by supplementing the cultures with IL-1α/IL-1β (98.9% of positive cells). GFP green fluorescent protein, MSC mesenchymal/stromal stem cell, pIL6 interleukin-6 promoter, TRAIL tumor necrosis factor related apoptosis inducing ligand, UC umbilical cord, WT wildtype

    Journal: Stem Cell Research & Therapy

    Article Title: pIL6-TRAIL-engineered umbilical cord mesenchymal/stromal stem cells are highly cytotoxic for myeloma cells both in vitro and in vivo

    doi: 10.1186/s13287-017-0655-6

    Figure Lengend Snippet: In vitro apoptosis of U-266 cells induced by pIL6-TRAIL + -GFP + -UC-MSCs. a Apoptosis in U-266 cells by transduced UC-MSCs was measured by Annexin V/PI staining using flow cytometry. Representative dot plots revealed that the apoptosis extent was significantly increased (43.8%) after 24 h of coculture with pIL6-TRAIL + -GFP + -UC-MSCs with respect to control UC-MSCs (19.4%) and GFP + -UC-MSCs (20.2%). The effect was also enhanced when IL-1α and IL-1β were added to the cocultures (70.9% of U-266 cell apoptosis). b Active caspase-8 in U-266 cells as signature of TRAIL-induced apoptosis was measured by flow cytometry after 24 h of coculture with UC-MSCs, GFP + -UC-MSCs, and pIL6-TRAIL + -GFP + -UC-MSCs. This representative experiment depicts the activity of caspase-8 in 97.8% of U266 cells cocultured with pIL6-TRAIL + -GFP + -UC-MSCs as compared to 17.9% of control UC-MSCs, and 22% of GFP + -UC-MSCs. Such high levels of active caspase-8 were not further modified by supplementing the cultures with IL-1α/IL-1β (98.9% of positive cells). GFP green fluorescent protein, MSC mesenchymal/stromal stem cell, pIL6 interleukin-6 promoter, TRAIL tumor necrosis factor related apoptosis inducing ligand, UC umbilical cord, WT wildtype

    Article Snippet: Finally, soluble TRAIL was also measured in supernatants of pIL6-TRAIL + -GFP + -UC-MSCs after treatment with U-266 conditioned medium, or IL-1α and IL-1β, by dedicated ELISA kit (Abcam).

    Techniques: In Vitro, Staining, Flow Cytometry, Cytometry, Activity Assay, Modification

    Experimental evidence that regulation of parkin expression and phosphorylation is related to glial activation and excess IL-1β in Alzheimer brain and in AD Model Systems. a Parkin in neurons in brains of age-matched control patients (AMC) is diffusely distributed within the cytoplasm whereas parkin in neurons in brains of Alzheimer patients (AD) is present in rosette-like aggregates ( b ). c Parkin in Alzheimer brain is colocalized with tau aggregates. d Activated glia overexpressing IL-1α (brown) are immediately adjacent to parkin-immunoreactive neurons (blue), scale bar 20 μM. e Comparative levels of IL-1α, IL-1β, and parkin mRNAs in wild-type littermates or PD-APP mice. f Representative western immunoblot showing proteins from NT2 cells either treated, or not, overnight with IL-1β. g Phospho-parkin levels (P-parkin Ser 65 ) in two representative western immunoblots of proteins from NT2 cell and rat primary neuron cultures treated, or not, with IL-1β. h Quantification of parkin in untreated and treated cell cultures ( n = 6), data is mean ± SEM (** p

    Journal: Journal of Neuroinflammation

    Article Title: Interleukin-1β drives NEDD8 nuclear-to-cytoplasmic translocation, fostering parkin activation via NEDD8 binding to the P-ubiquitin activating site

    doi: 10.1186/s12974-019-1669-z

    Figure Lengend Snippet: Experimental evidence that regulation of parkin expression and phosphorylation is related to glial activation and excess IL-1β in Alzheimer brain and in AD Model Systems. a Parkin in neurons in brains of age-matched control patients (AMC) is diffusely distributed within the cytoplasm whereas parkin in neurons in brains of Alzheimer patients (AD) is present in rosette-like aggregates ( b ). c Parkin in Alzheimer brain is colocalized with tau aggregates. d Activated glia overexpressing IL-1α (brown) are immediately adjacent to parkin-immunoreactive neurons (blue), scale bar 20 μM. e Comparative levels of IL-1α, IL-1β, and parkin mRNAs in wild-type littermates or PD-APP mice. f Representative western immunoblot showing proteins from NT2 cells either treated, or not, overnight with IL-1β. g Phospho-parkin levels (P-parkin Ser 65 ) in two representative western immunoblots of proteins from NT2 cell and rat primary neuron cultures treated, or not, with IL-1β. h Quantification of parkin in untreated and treated cell cultures ( n = 6), data is mean ± SEM (** p

    Article Snippet: For double staining with IL-1α (sc-1279, Santa Cruz Biotechnology) and parkin, endogenous background was blocked with 100% normal horse serum for 30 min at RT.

    Techniques: Expressing, Activation Assay, Mouse Assay, Western Blot

    Flow cytometric analysis of P-selectin on HDMECs. A: Basal expression of P-selectin was minimal or undetectable. When HDMECs were incubated with histamine (10 −5 mol/L), transient expression was observed 5 and 10 minutes after histamine treatment. B: HDMECs were incubated with IL-1α (10 ng/ml), TNF-α (10 ng/ml), interferon-γ (10 ng/ml), IL-4 (10 ng/ml), IL-13 (10 ng/ml), or substance P (100 nmol/L) for 24 hours. Whereas IL-4, IL-13, and substance P increased P-selectin expression, TNF-α had no effect on P-selectin expression. The concentration of each cytokine that induced maximal response was determined in preliminary experiments. C and D: Time course changes in P-selectin expression by HDMECs ( C ) and HUVECs ( D ) after treatment with IL-4. Surface P-selectin expression in HDMECs peaked 24 hours after treatment with IL-4. Black line , control; green line , treated with stimulants. Results are from a single representative of three separate experiments.

    Journal: The American Journal of Pathology

    Article Title: STAT-6-Mediated Control of P-Selectin by Substance P and Interleukin-4 in Human Dermal Endothelial Cells

    doi: 10.2353/ajpath.2006.051211

    Figure Lengend Snippet: Flow cytometric analysis of P-selectin on HDMECs. A: Basal expression of P-selectin was minimal or undetectable. When HDMECs were incubated with histamine (10 −5 mol/L), transient expression was observed 5 and 10 minutes after histamine treatment. B: HDMECs were incubated with IL-1α (10 ng/ml), TNF-α (10 ng/ml), interferon-γ (10 ng/ml), IL-4 (10 ng/ml), IL-13 (10 ng/ml), or substance P (100 nmol/L) for 24 hours. Whereas IL-4, IL-13, and substance P increased P-selectin expression, TNF-α had no effect on P-selectin expression. The concentration of each cytokine that induced maximal response was determined in preliminary experiments. C and D: Time course changes in P-selectin expression by HDMECs ( C ) and HUVECs ( D ) after treatment with IL-4. Surface P-selectin expression in HDMECs peaked 24 hours after treatment with IL-4. Black line , control; green line , treated with stimulants. Results are from a single representative of three separate experiments.

    Article Snippet: Briefly, cells were incubated with histamine (Sigma), IL-4 (Genzyme Techne, Boston, MA), IL-13 (Genzyme Techne), tumor necrosis factor (TNF)-α (Genzyme Techne), IL-1α (Genzyme Techne), interferon-γ (Genzyme Techne), or substance P (Sigma) for the indicated times.

    Techniques: Flow Cytometry, Expressing, Incubation, Concentration Assay

    Priming by C5a induces expression of pro-IL-1β protein. Expression of pro-IL-1β protein (36 kDa) was assessed in ARPE-19 cells following priming with IL-1α, complement-competent normal human serum (NHS), recombinant human C5a,

    Journal: The Journal of Biological Chemistry

    Article Title: Complement Component C5a Primes Retinal Pigment Epithelial Cells for Inflammasome Activation by Lipofuscin-mediated Photooxidative Damage *

    doi: 10.1074/jbc.M115.671180

    Figure Lengend Snippet: Priming by C5a induces expression of pro-IL-1β protein. Expression of pro-IL-1β protein (36 kDa) was assessed in ARPE-19 cells following priming with IL-1α, complement-competent normal human serum (NHS), recombinant human C5a,

    Article Snippet: For priming with interleukins, cells were treated with 4 ng/ml recombinant human IL-1α (R & D Systems, Wiesbaden, Germany) ( ) or 50 pg/ml recombinant human IL-1β (R & D Systems).

    Techniques: Expressing, Recombinant

    Inflammasome priming in RPE cells is enhanced by a paracrine amplification loop. A , inflammasome activation was induced in IL-1α-primed ARPE-19 cells by HNE-POS/blue light treatment or incubation with Leu-Leu-OMe. Cells treated with HNE-POS but

    Journal: The Journal of Biological Chemistry

    Article Title: Complement Component C5a Primes Retinal Pigment Epithelial Cells for Inflammasome Activation by Lipofuscin-mediated Photooxidative Damage *

    doi: 10.1074/jbc.M115.671180

    Figure Lengend Snippet: Inflammasome priming in RPE cells is enhanced by a paracrine amplification loop. A , inflammasome activation was induced in IL-1α-primed ARPE-19 cells by HNE-POS/blue light treatment or incubation with Leu-Leu-OMe. Cells treated with HNE-POS but

    Article Snippet: For priming with interleukins, cells were treated with 4 ng/ml recombinant human IL-1α (R & D Systems, Wiesbaden, Germany) ( ) or 50 pg/ml recombinant human IL-1β (R & D Systems).

    Techniques: Amplification, Activation Assay, Incubation

    Neutrophil-recruiting chemokines in the oral mucosa are reduced in absence of IL-1R signaling. ( A ) Chemokine expression in the tongues of naïve (n) and infected (inf) WT mice was measured by qRT-PCR 24 hours post-infection. ( B ) Chemokine expression in the tongues of naïve and infected WT and infected Il1r1 -/- mice 24 hours post-infection. ( C ) CD45 + leukocytes, CD45 - EpCAM + CD31 - epithelial cells, and CD45 - EpCAM - CD31 + endothelial cells were isolated from the tongues of naïve and infected WT mice by FACS sorting 24 hours post-infection, and Cxcl1 mRNA was quantified by qRT-PCR. ( D ) The tongue-derived keratinocyte (TDK) cell line was treated with recombinant IL-1α (20 ng/ml), anakinra (250 μg/ml), or PBS, and CXCL1 levels in the culture supernatant were determined by ELISA. Bar graphs show the group mean + SD. Data are representative of two (A, C–D) or pooled from two (B) independent experiments, with the exception of the naïve group in B, which is from one experiment. Statistical analysis was performed using log 10 transformation and Student’s t-test with Welch’s correction (A) or one-way ANOVA with Dunnett’s test (B, D).

    Journal: PLoS Pathogens

    Article Title: IL-1 Coordinates the Neutrophil Response to C. albicans in the Oral Mucosa

    doi: 10.1371/journal.ppat.1005882

    Figure Lengend Snippet: Neutrophil-recruiting chemokines in the oral mucosa are reduced in absence of IL-1R signaling. ( A ) Chemokine expression in the tongues of naïve (n) and infected (inf) WT mice was measured by qRT-PCR 24 hours post-infection. ( B ) Chemokine expression in the tongues of naïve and infected WT and infected Il1r1 -/- mice 24 hours post-infection. ( C ) CD45 + leukocytes, CD45 - EpCAM + CD31 - epithelial cells, and CD45 - EpCAM - CD31 + endothelial cells were isolated from the tongues of naïve and infected WT mice by FACS sorting 24 hours post-infection, and Cxcl1 mRNA was quantified by qRT-PCR. ( D ) The tongue-derived keratinocyte (TDK) cell line was treated with recombinant IL-1α (20 ng/ml), anakinra (250 μg/ml), or PBS, and CXCL1 levels in the culture supernatant were determined by ELISA. Bar graphs show the group mean + SD. Data are representative of two (A, C–D) or pooled from two (B) independent experiments, with the exception of the naïve group in B, which is from one experiment. Statistical analysis was performed using log 10 transformation and Student’s t-test with Welch’s correction (A) or one-way ANOVA with Dunnett’s test (B, D).

    Article Snippet: Prior to stimulation experiments, TDK and MS1 cells were rested for 48 hours and then stimulated with recombinant IL-1α (Peprotech, 20 ng/ml), IL-1β (Peprotech, 20 ng/ml), anakinra (250 μg/ml), zymosan (20 μg/ml), curdlan (200 μg/ml), LPS (100 ng/ml) or C . albicans at MOI = 3 for 24 hours.

    Techniques: Expressing, Infection, Mouse Assay, Quantitative RT-PCR, Isolation, FACS, Derivative Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Transformation Assay

    TDKs release IL-1α in response to C . albicans . ( A—B ) TDKs were stimulated with C . albicans , zymosan, curdlan or left untreated (PBS), and IL-1α (A) and IL-1β (B) levels were determined in the supernatant by cytometric bead array. ( C ) IL-1α release from TDKs was determined as in (A) after stimulation with live or heat-killed (HK) C . albicans strain SC5314, with the yeast-locked strain hgc1 Δ/Δ, its revertant hgc1 Δ/Δ:: hgc1 , or with preformed hyphae prepared from strain SC5314. ( D ) TDKs were stimulated with C . albicans or left unstimulated (PBS). Amphotericin B was added after 8 hours of stimulation to prevent hyphal overgrowth. IL-1α levels were determined in the supernatant and in lysates prepared from the cells in the same wells. As a control, triton was added to separate wells containing cells and supernatant to quantify total amounts of IL-1α per well (total). Bar graphs show the group mean + SD. Data are representative of two independent experiments. Statistical analysis was performed using one-way ANOVA with Dunnett’s test (A–B) or Tukey’s test (C).

    Journal: PLoS Pathogens

    Article Title: IL-1 Coordinates the Neutrophil Response to C. albicans in the Oral Mucosa

    doi: 10.1371/journal.ppat.1005882

    Figure Lengend Snippet: TDKs release IL-1α in response to C . albicans . ( A—B ) TDKs were stimulated with C . albicans , zymosan, curdlan or left untreated (PBS), and IL-1α (A) and IL-1β (B) levels were determined in the supernatant by cytometric bead array. ( C ) IL-1α release from TDKs was determined as in (A) after stimulation with live or heat-killed (HK) C . albicans strain SC5314, with the yeast-locked strain hgc1 Δ/Δ, its revertant hgc1 Δ/Δ:: hgc1 , or with preformed hyphae prepared from strain SC5314. ( D ) TDKs were stimulated with C . albicans or left unstimulated (PBS). Amphotericin B was added after 8 hours of stimulation to prevent hyphal overgrowth. IL-1α levels were determined in the supernatant and in lysates prepared from the cells in the same wells. As a control, triton was added to separate wells containing cells and supernatant to quantify total amounts of IL-1α per well (total). Bar graphs show the group mean + SD. Data are representative of two independent experiments. Statistical analysis was performed using one-way ANOVA with Dunnett’s test (A–B) or Tukey’s test (C).

    Article Snippet: Prior to stimulation experiments, TDK and MS1 cells were rested for 48 hours and then stimulated with recombinant IL-1α (Peprotech, 20 ng/ml), IL-1β (Peprotech, 20 ng/ml), anakinra (250 μg/ml), zymosan (20 μg/ml), curdlan (200 μg/ml), LPS (100 ng/ml) or C . albicans at MOI = 3 for 24 hours.

    Techniques:

    Keratinocyte-derived IL-1α regulates G-CSF production during OPC. ( A ) Il1a and Il1b mRNA was quantified in the tongues of naïve (n) and infected (inf) WT mice by qRT-PCR 24 hours post-infection. ( B–C ) WT, Il1a -/- and Il1b -/- mice were infected with C . albicans . Csf3 mRNA levels in naïve and infected tongue tissue were determined by qRT-PCR (B), and G-CSF serum levels were measured by ELISA (C) before infection (open bars) and 24 hours post-infection (closed bars). ( D ) Immunofluorescent staining of sagittal tongue sections from naïve and infected WT and Il1a -/- mice stained for IL-1α (yellow) and DAPI (blue) 24 hours post-infection. ( E ) Immunofluorescent staining of sagittal tongue sections from infected WT mice stained for keratin-6 (K6, left) or keratin-14 (K14, right) in red, as well as IL-1α (yellow) and DAPI (blue) 24 hours post-infection. Note that the IL-1α signal is absent in neutrophil-rich areas. The white arrow serves as orientation. ( F ) IL-1α and IL-1β mRNA expression in the tongues of naïve WT and infected WT, Il1a -/- and Il1b -/- mice was measured by qRT-PCR 24 hours post-infection. The detection limit, which was calculated using the average Ct ( Actb ) of all samples and Ct ( Il1a ) or Ct ( Il1b ) = 50, is depicted by a dotted line. Each symbol represents an individual mouse (A–B, F) and the lines represent the geometric mean of each group. The bar graph in C shows the group mean + SD. Data are representative of two independent experiments (A, D–E), or pooled from two independent experiments (B–C, F), with the exception of the naïve groups in C, which are the mean + SD of 4 (WT) or 3 ( Il1a -/- , Il1b -/- ) animals from one experiment. Statistical analysis was performed using log 10 transformation (A–B, F) and Student’s t-test with Welch’s correction (A), a one-way ANOVA with Dunnett’s test (B, F) or two-way ANOVA with Tukey’s test (C).

    Journal: PLoS Pathogens

    Article Title: IL-1 Coordinates the Neutrophil Response to C. albicans in the Oral Mucosa

    doi: 10.1371/journal.ppat.1005882

    Figure Lengend Snippet: Keratinocyte-derived IL-1α regulates G-CSF production during OPC. ( A ) Il1a and Il1b mRNA was quantified in the tongues of naïve (n) and infected (inf) WT mice by qRT-PCR 24 hours post-infection. ( B–C ) WT, Il1a -/- and Il1b -/- mice were infected with C . albicans . Csf3 mRNA levels in naïve and infected tongue tissue were determined by qRT-PCR (B), and G-CSF serum levels were measured by ELISA (C) before infection (open bars) and 24 hours post-infection (closed bars). ( D ) Immunofluorescent staining of sagittal tongue sections from naïve and infected WT and Il1a -/- mice stained for IL-1α (yellow) and DAPI (blue) 24 hours post-infection. ( E ) Immunofluorescent staining of sagittal tongue sections from infected WT mice stained for keratin-6 (K6, left) or keratin-14 (K14, right) in red, as well as IL-1α (yellow) and DAPI (blue) 24 hours post-infection. Note that the IL-1α signal is absent in neutrophil-rich areas. The white arrow serves as orientation. ( F ) IL-1α and IL-1β mRNA expression in the tongues of naïve WT and infected WT, Il1a -/- and Il1b -/- mice was measured by qRT-PCR 24 hours post-infection. The detection limit, which was calculated using the average Ct ( Actb ) of all samples and Ct ( Il1a ) or Ct ( Il1b ) = 50, is depicted by a dotted line. Each symbol represents an individual mouse (A–B, F) and the lines represent the geometric mean of each group. The bar graph in C shows the group mean + SD. Data are representative of two independent experiments (A, D–E), or pooled from two independent experiments (B–C, F), with the exception of the naïve groups in C, which are the mean + SD of 4 (WT) or 3 ( Il1a -/- , Il1b -/- ) animals from one experiment. Statistical analysis was performed using log 10 transformation (A–B, F) and Student’s t-test with Welch’s correction (A), a one-way ANOVA with Dunnett’s test (B, F) or two-way ANOVA with Tukey’s test (C).

    Article Snippet: Prior to stimulation experiments, TDK and MS1 cells were rested for 48 hours and then stimulated with recombinant IL-1α (Peprotech, 20 ng/ml), IL-1β (Peprotech, 20 ng/ml), anakinra (250 μg/ml), zymosan (20 μg/ml), curdlan (200 μg/ml), LPS (100 ng/ml) or C . albicans at MOI = 3 for 24 hours.

    Techniques: Derivative Assay, Infection, Mouse Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Transformation Assay

    TDK-derived IL-1α induces G-CSF secretion by endothelial cells. ( A ) Schematic overview of the experimental setup used in panels B–J. ( B–J ) G-CSF levels in the supernatants of TDKs (B) and MS1 cells (C–J) were determined by ELISA 24 hours after stimulation. TDKs (B) and MS1 cells (C) were stimulated with C . albicans , zymosan, curdlan, LPS or left unstimulated (PBS) as indicated. ( D ) MS1 cells were stimulated with the supernatants of stimulated TDKs from (A) (diluted 3-fold in MS1 culture medium). ( E ) MS1 cells were stimulated with the serially diluted supernatant of C . albicans -stimulated TDKs. ( F ) MS1 cells were stimulated with the TDK supernatants from Fig 7C . ( G ) MS1 cells were pretreated with anakinra as indicated prior to the addition of the supernatant of C . albicans -stimulated TDKs. ( H—I ) The supernatant of C . albicans -stimulated TDKs was treated with anti-IL-1α and/or anti-IL-1β as indicated before being transferred to MS1 cells. ( J ) MS1 cells were treated with recombinant IL-1α, IL-1β, or left untreated. The conditions of G-CSF induction shown in (F–I) were measured all in one experiment but are displayed in several individual graphs for better comprehension. Data (B–J) are all representative of at least two independent experiments. Bar graphs show the group mean + SD. Statistical analysis was performed using one-way ANOVA with Dunnett’s test.

    Journal: PLoS Pathogens

    Article Title: IL-1 Coordinates the Neutrophil Response to C. albicans in the Oral Mucosa

    doi: 10.1371/journal.ppat.1005882

    Figure Lengend Snippet: TDK-derived IL-1α induces G-CSF secretion by endothelial cells. ( A ) Schematic overview of the experimental setup used in panels B–J. ( B–J ) G-CSF levels in the supernatants of TDKs (B) and MS1 cells (C–J) were determined by ELISA 24 hours after stimulation. TDKs (B) and MS1 cells (C) were stimulated with C . albicans , zymosan, curdlan, LPS or left unstimulated (PBS) as indicated. ( D ) MS1 cells were stimulated with the supernatants of stimulated TDKs from (A) (diluted 3-fold in MS1 culture medium). ( E ) MS1 cells were stimulated with the serially diluted supernatant of C . albicans -stimulated TDKs. ( F ) MS1 cells were stimulated with the TDK supernatants from Fig 7C . ( G ) MS1 cells were pretreated with anakinra as indicated prior to the addition of the supernatant of C . albicans -stimulated TDKs. ( H—I ) The supernatant of C . albicans -stimulated TDKs was treated with anti-IL-1α and/or anti-IL-1β as indicated before being transferred to MS1 cells. ( J ) MS1 cells were treated with recombinant IL-1α, IL-1β, or left untreated. The conditions of G-CSF induction shown in (F–I) were measured all in one experiment but are displayed in several individual graphs for better comprehension. Data (B–J) are all representative of at least two independent experiments. Bar graphs show the group mean + SD. Statistical analysis was performed using one-way ANOVA with Dunnett’s test.

    Article Snippet: Prior to stimulation experiments, TDK and MS1 cells were rested for 48 hours and then stimulated with recombinant IL-1α (Peprotech, 20 ng/ml), IL-1β (Peprotech, 20 ng/ml), anakinra (250 μg/ml), zymosan (20 μg/ml), curdlan (200 μg/ml), LPS (100 ng/ml) or C . albicans at MOI = 3 for 24 hours.

    Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Recombinant

    Secreted cytokines induced by Helicobacter pylori (Hp) exosomes using a protein array. (a) Map of antibodies against cytokines on the RayBiotech human inflammation antibody array. (b) Cell culture supernatants from gastric epithelial cells (GES)‐1 cells treated with exosomes were used in the array. Bound cytokines were recognized by a pool of anti‐cytokine antibodies corresponding to the antibodies spotted on the array. The Hp exosome group revealed increased expression of IL‐1α and sIL‐6R. (c) Semi‐quantification of scanned antibody arrays. The levels were normalized to internal positive controls present in the membrane. Semiquantitative levels are represented in the heat map.

    Journal: Clinical and Experimental Immunology

    Article Title: Serum exosomes of chronic gastritis patients infected with Helicobacter pylori mediate IL‐1α expression via IL‐6 trans‐signalling in gastric epithelial cells

    doi: 10.1111/cei.13200

    Figure Lengend Snippet: Secreted cytokines induced by Helicobacter pylori (Hp) exosomes using a protein array. (a) Map of antibodies against cytokines on the RayBiotech human inflammation antibody array. (b) Cell culture supernatants from gastric epithelial cells (GES)‐1 cells treated with exosomes were used in the array. Bound cytokines were recognized by a pool of anti‐cytokine antibodies corresponding to the antibodies spotted on the array. The Hp exosome group revealed increased expression of IL‐1α and sIL‐6R. (c) Semi‐quantification of scanned antibody arrays. The levels were normalized to internal positive controls present in the membrane. Semiquantitative levels are represented in the heat map.

    Article Snippet: The following specific primary antibodies were used: anti‐human IL‐6R (1 : 1000; R & D Systems), anti‐IL‐1α (1 : 1000; Santa Cruz), anti‐calnexin (1 : 1000; Cell Signaling Technology, Danvers, MA, USA), anti‐CD63 (1:1000; Abcam, Cambridge, MA, USA) and anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (1 : 1000; Sangon Biotech, Shanghai, China).

    Techniques: Protein Array, Ab Array, Cell Culture, Expressing

    Hypothetical schematic of the regulation of interleukin (IL)‐1α by soluble interleukin‐6 receptor (sIL‐6R) in gastric epithelial cells (GES)‐1 cells in response to Helicobacter pylori (Hp) exosome treatment.

    Journal: Clinical and Experimental Immunology

    Article Title: Serum exosomes of chronic gastritis patients infected with Helicobacter pylori mediate IL‐1α expression via IL‐6 trans‐signalling in gastric epithelial cells

    doi: 10.1111/cei.13200

    Figure Lengend Snippet: Hypothetical schematic of the regulation of interleukin (IL)‐1α by soluble interleukin‐6 receptor (sIL‐6R) in gastric epithelial cells (GES)‐1 cells in response to Helicobacter pylori (Hp) exosome treatment.

    Article Snippet: The following specific primary antibodies were used: anti‐human IL‐6R (1 : 1000; R & D Systems), anti‐IL‐1α (1 : 1000; Santa Cruz), anti‐calnexin (1 : 1000; Cell Signaling Technology, Danvers, MA, USA), anti‐CD63 (1:1000; Abcam, Cambridge, MA, USA) and anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (1 : 1000; Sangon Biotech, Shanghai, China).

    Techniques:

    IL-1β/α induction by 25-HC is highly specific. The levels of secreted IL-1β ( a ), IL-1α ( b ), or IL-6 ( c ) in the medium of primary microglia treated with LPS (10 ng/ml) in the presence of 25-HC (10 μg/ml), cholesterol (10μg/ml), or 7α-HC (10 μg/ml) for 24 h. d Ent-25-HC (10 μg/ml) has much weaker effects in augmenting IL-1β production in primary microglia treated with LPS (10 ng/ml) for 24 h. The levels of cytokines were determined by ELISA. Statistical significances were determined by ordinary two-way ANOVA with Tukey multiple comparisons test. * p

    Journal: Journal of Neuroinflammation

    Article Title: 25-Hydroxycholesterol amplifies microglial IL-1β production in an apoE isoform-dependent manner

    doi: 10.1186/s12974-020-01869-3

    Figure Lengend Snippet: IL-1β/α induction by 25-HC is highly specific. The levels of secreted IL-1β ( a ), IL-1α ( b ), or IL-6 ( c ) in the medium of primary microglia treated with LPS (10 ng/ml) in the presence of 25-HC (10 μg/ml), cholesterol (10μg/ml), or 7α-HC (10 μg/ml) for 24 h. d Ent-25-HC (10 μg/ml) has much weaker effects in augmenting IL-1β production in primary microglia treated with LPS (10 ng/ml) for 24 h. The levels of cytokines were determined by ELISA. Statistical significances were determined by ordinary two-way ANOVA with Tukey multiple comparisons test. * p

    Article Snippet: Cytokine ELISAsSupernatants from cell cultures were collected and the concentrations of IL-1β (BioLegend#432601), IL-1α (Biolegend#433401), IL-6 (Bon Opus Biosciences#BE010059B), and TNFα (Biolegend#430901) were determined by ELISA according to the manufacturer’s instructions.

    Techniques: Enzyme-linked Immunosorbent Assay

    25-HC selectively amplifies LPS-induced IL-1β expression and secretion. a - c WT and CH25H KO primary microglia were treated with LPS (0, 0.1, 1, 10, 100 ng/ml) for 24 h. The levels of secreted IL-1β ( a ), IL-1α ( b ), and TNFα (c) in the medium were measured by ELISA. d The levels of secreted IL1β from WT and CH25H KO microglia treated with LPS (10 ng/ml) with or without 25-HC (10μg/ml) were measured by ELISA. e Primary microglia were treated with 10 ng/ml LPS in the presence of different concentrations of 25-HC for 24 h. The levels of IL-1β in the media were determined by ELISA and ( f ) the levels of intracellular pro-IL1β and mature IL1β secreted in the media as measured by Western blotting. Statistical analyses were determined by multiple t test in a , b , c , d ; one-way ANOVA in e . * p

    Journal: Journal of Neuroinflammation

    Article Title: 25-Hydroxycholesterol amplifies microglial IL-1β production in an apoE isoform-dependent manner

    doi: 10.1186/s12974-020-01869-3

    Figure Lengend Snippet: 25-HC selectively amplifies LPS-induced IL-1β expression and secretion. a - c WT and CH25H KO primary microglia were treated with LPS (0, 0.1, 1, 10, 100 ng/ml) for 24 h. The levels of secreted IL-1β ( a ), IL-1α ( b ), and TNFα (c) in the medium were measured by ELISA. d The levels of secreted IL1β from WT and CH25H KO microglia treated with LPS (10 ng/ml) with or without 25-HC (10μg/ml) were measured by ELISA. e Primary microglia were treated with 10 ng/ml LPS in the presence of different concentrations of 25-HC for 24 h. The levels of IL-1β in the media were determined by ELISA and ( f ) the levels of intracellular pro-IL1β and mature IL1β secreted in the media as measured by Western blotting. Statistical analyses were determined by multiple t test in a , b , c , d ; one-way ANOVA in e . * p

    Article Snippet: Cytokine ELISAsSupernatants from cell cultures were collected and the concentrations of IL-1β (BioLegend#432601), IL-1α (Biolegend#433401), IL-6 (Bon Opus Biosciences#BE010059B), and TNFα (Biolegend#430901) were determined by ELISA according to the manufacturer’s instructions.

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

    PPARγ loss of function is associated with impaired IL-1 cytokine release in primary macrophages. ( A-C ) Peritoneal macrophages (pMACs) isolated from WT (open bars) or mPPARγKO mice (filled bars) were treated with control (BSA-LPS), palm (250 μM)-LPS (100 ng), or stearate (150 μM)-LPS for 20h and the release of IL-1β ( A ), ( B ) IL-1α, and ( C ) TNFα was determined by ELISA. ( D-F ) pMACs isolated from WT (open bars) or mPPARγKO mice (filled bars) were treated with LPS and ATP, alum or silica as described in the methods and IL-1β ( D ), ( E ) IL-1α, and ( F ) TNFα was determined by ELISA. WT pMACs were treated with veh (open bars) or T0070907 (T007; gray bars) for 24h after which they received the indicated stimuli. The release of IL-1β ( G ), ( H ) IL-1α, and ( I ) TNFα was determined by ELISA. Bar graphs report the mean ± standard error (SE) for a minimum of 3 experiments, each performed in triplicate. *, p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: PPARγ Deficiency Suppresses the Release of IL-1β and α in Macrophages via a Type 1 Interferon Dependent Mechanism

    doi: 10.4049/jimmunol.1800224

    Figure Lengend Snippet: PPARγ loss of function is associated with impaired IL-1 cytokine release in primary macrophages. ( A-C ) Peritoneal macrophages (pMACs) isolated from WT (open bars) or mPPARγKO mice (filled bars) were treated with control (BSA-LPS), palm (250 μM)-LPS (100 ng), or stearate (150 μM)-LPS for 20h and the release of IL-1β ( A ), ( B ) IL-1α, and ( C ) TNFα was determined by ELISA. ( D-F ) pMACs isolated from WT (open bars) or mPPARγKO mice (filled bars) were treated with LPS and ATP, alum or silica as described in the methods and IL-1β ( D ), ( E ) IL-1α, and ( F ) TNFα was determined by ELISA. WT pMACs were treated with veh (open bars) or T0070907 (T007; gray bars) for 24h after which they received the indicated stimuli. The release of IL-1β ( G ), ( H ) IL-1α, and ( I ) TNFα was determined by ELISA. Bar graphs report the mean ± standard error (SE) for a minimum of 3 experiments, each performed in triplicate. *, p

    Article Snippet: IL-1β, IL-1α, NLRP3, STAT1 and phospho-STAT1(#14994) antibodies were from Cell Signaling (Danvers, MA, USA).

    Techniques: Isolation, Mouse Assay, Enzyme-linked Immunosorbent Assay

    mPPARγKO macrophages produce less IL-1 cytokines in vivo. ( A-C ) WT or mPPARγKO mice were injected with thioglycollate to induce macrophage recruitment to the peritoneal cavity. At day 4, when ~90% of the cells in the peritoneum are macrophages, the mice were given an intraperitoneal injection of LPS (10 μg/200 μl) and 16h later IL-1β ( A ), IL-1α ( B ), and TNFα ( C ) levels in the peritoneal fluid was quantified by ELISA. ( D ) Day 4 peritoneal cell count for WT vs. mPPARγKO mice following thioglycollate injection. Bar graphs report the mean ± standard error (SE) and the individual dots each represent one mouse. The p values for the comparison of WT vs. mPPARγKO mice are shown.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: PPARγ Deficiency Suppresses the Release of IL-1β and α in Macrophages via a Type 1 Interferon Dependent Mechanism

    doi: 10.4049/jimmunol.1800224

    Figure Lengend Snippet: mPPARγKO macrophages produce less IL-1 cytokines in vivo. ( A-C ) WT or mPPARγKO mice were injected with thioglycollate to induce macrophage recruitment to the peritoneal cavity. At day 4, when ~90% of the cells in the peritoneum are macrophages, the mice were given an intraperitoneal injection of LPS (10 μg/200 μl) and 16h later IL-1β ( A ), IL-1α ( B ), and TNFα ( C ) levels in the peritoneal fluid was quantified by ELISA. ( D ) Day 4 peritoneal cell count for WT vs. mPPARγKO mice following thioglycollate injection. Bar graphs report the mean ± standard error (SE) and the individual dots each represent one mouse. The p values for the comparison of WT vs. mPPARγKO mice are shown.

    Article Snippet: IL-1β, IL-1α, NLRP3, STAT1 and phospho-STAT1(#14994) antibodies were from Cell Signaling (Danvers, MA, USA).

    Techniques: In Vivo, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Cell Counting

    PPARγ deficiency leads to decreased levels of IL-1 mRNA and protein levels. ( A ) WT or mPPARγKO (KO) macrophages were stimulated with BSA-PBS (vehicle) or palm-LPS for 16h and the protein level of pro-IL-1β and pro-IL-1α was assessed by western blotting. Tubulin (tub) is shown as a loading control. ( B ) pMACs isolated from WT (open bars) or mPPARγKO mice (filled bars) were treated with vehicle or palm-LPS for 8h and mRNA expression of IL-1β, IL-1α, and NLRP3 was assessed by qRT-PCR. ( C, D ) Kinetic assessment of IL-1β ( C ) and TNFα ( D ) mRNA levels following palm-LPS stimulation in WT and mPPARγKO cells. ( E ) WT or mPPARγ KO cells were treated with palm-LPS for 4h after actinomycin D was added to the culture. The levels of IL-1β mRNA relative to baseline was performed by qRT-PCR. Each genotype was compared to its own baseline which was arbitrarily defined as 1. Bar graphs report the mean ± standard error (SE) for a minimum of 3 experiments, each performed in triplicate. *, p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: PPARγ Deficiency Suppresses the Release of IL-1β and α in Macrophages via a Type 1 Interferon Dependent Mechanism

    doi: 10.4049/jimmunol.1800224

    Figure Lengend Snippet: PPARγ deficiency leads to decreased levels of IL-1 mRNA and protein levels. ( A ) WT or mPPARγKO (KO) macrophages were stimulated with BSA-PBS (vehicle) or palm-LPS for 16h and the protein level of pro-IL-1β and pro-IL-1α was assessed by western blotting. Tubulin (tub) is shown as a loading control. ( B ) pMACs isolated from WT (open bars) or mPPARγKO mice (filled bars) were treated with vehicle or palm-LPS for 8h and mRNA expression of IL-1β, IL-1α, and NLRP3 was assessed by qRT-PCR. ( C, D ) Kinetic assessment of IL-1β ( C ) and TNFα ( D ) mRNA levels following palm-LPS stimulation in WT and mPPARγKO cells. ( E ) WT or mPPARγ KO cells were treated with palm-LPS for 4h after actinomycin D was added to the culture. The levels of IL-1β mRNA relative to baseline was performed by qRT-PCR. Each genotype was compared to its own baseline which was arbitrarily defined as 1. Bar graphs report the mean ± standard error (SE) for a minimum of 3 experiments, each performed in triplicate. *, p

    Article Snippet: IL-1β, IL-1α, NLRP3, STAT1 and phospho-STAT1(#14994) antibodies were from Cell Signaling (Danvers, MA, USA).

    Techniques: Western Blot, Isolation, Mouse Assay, Expressing, Quantitative RT-PCR

    Immunological properties of 3-week mDC cultures in EGM . IL-1α ( A ) and IL-6 ( B ) were detected by ELISA on reseeded cultures after 48 h stimulation with TNFα and LPS. ( A ) ANOVA analysis: p

    Journal: BMC Immunology

    Article Title: The interplay between surfaces and soluble factors define the immunologic and angiogenic properties of myeloid dendritic cells

    doi: 10.1186/1471-2172-12-35

    Figure Lengend Snippet: Immunological properties of 3-week mDC cultures in EGM . IL-1α ( A ) and IL-6 ( B ) were detected by ELISA on reseeded cultures after 48 h stimulation with TNFα and LPS. ( A ) ANOVA analysis: p

    Article Snippet: Standard curves were constructed using recombinant murine IL-1α (220-11), IL-6 (216-16) and VEGF (450-32) (all Peprotech).

    Techniques: Enzyme-linked Immunosorbent Assay

    One-week mDC cultures response to inflammatory stimuli . IL-6 ( A ), IL-1α ( B ) and nitrites ( C ) were detected by ELISA analysis on mDC-cultures after 1 week culture in the presence or absence of 100 ng/ml LPS and 20 ng/ml TNFα. An experiment representative of two independent experiments with n = 2 for each condition is shown. ( A ) IL-6 production by DC cultures. All inflammatory stimuli-treated samples showed significantly higher levels of nitrites when compared to their untreated counterparts as determined by ANOVA analysis followed by Tukey-Kramer Multiple Comparisons post-test. *p

    Journal: BMC Immunology

    Article Title: The interplay between surfaces and soluble factors define the immunologic and angiogenic properties of myeloid dendritic cells

    doi: 10.1186/1471-2172-12-35

    Figure Lengend Snippet: One-week mDC cultures response to inflammatory stimuli . IL-6 ( A ), IL-1α ( B ) and nitrites ( C ) were detected by ELISA analysis on mDC-cultures after 1 week culture in the presence or absence of 100 ng/ml LPS and 20 ng/ml TNFα. An experiment representative of two independent experiments with n = 2 for each condition is shown. ( A ) IL-6 production by DC cultures. All inflammatory stimuli-treated samples showed significantly higher levels of nitrites when compared to their untreated counterparts as determined by ANOVA analysis followed by Tukey-Kramer Multiple Comparisons post-test. *p

    Article Snippet: Standard curves were constructed using recombinant murine IL-1α (220-11), IL-6 (216-16) and VEGF (450-32) (all Peprotech).

    Techniques: Enzyme-linked Immunosorbent Assay

    Tissue expression of IL-1α. a Right lung samples demonstrated higher tissue IL-1α expression compared to the untreated control samples. b Left lung samples revealed upregulated tissue IL-1α expression. Immunohistochemical staining with anti-IL-1α antibody of c normal control and d right lung sample treated with helical tomotherapy with CCRT. * p

    Journal: Radiation Oncology (London, England)

    Article Title: Risk of radiation-induced pneumonitis after helical and static-port tomotherapy in lung cancer patients and experimental rats

    doi: 10.1186/s13014-015-0502-9

    Figure Lengend Snippet: Tissue expression of IL-1α. a Right lung samples demonstrated higher tissue IL-1α expression compared to the untreated control samples. b Left lung samples revealed upregulated tissue IL-1α expression. Immunohistochemical staining with anti-IL-1α antibody of c normal control and d right lung sample treated with helical tomotherapy with CCRT. * p

    Article Snippet: The sections were incubated with primary antibodies, including anti-IL-1α antibody (Abcam) and rabbit anti-IL-1β polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:100, rabbit anti-IL-6 polyclonal antibody (Abcam) at 1:50, and mouse anti-TGF-β monoclonal antibody (Abcam) at 1:25 for 1 h at room temperature.

    Techniques: Expressing, Immunohistochemistry, Staining

    Plasma levels of a IL-1α and b IL-1β according to the experimental rat groups. There were significant differences among the groups at 3 weeks after tomotherapy or CCRT. * p

    Journal: Radiation Oncology (London, England)

    Article Title: Risk of radiation-induced pneumonitis after helical and static-port tomotherapy in lung cancer patients and experimental rats

    doi: 10.1186/s13014-015-0502-9

    Figure Lengend Snippet: Plasma levels of a IL-1α and b IL-1β according to the experimental rat groups. There were significant differences among the groups at 3 weeks after tomotherapy or CCRT. * p

    Article Snippet: The sections were incubated with primary antibodies, including anti-IL-1α antibody (Abcam) and rabbit anti-IL-1β polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:100, rabbit anti-IL-6 polyclonal antibody (Abcam) at 1:50, and mouse anti-TGF-β monoclonal antibody (Abcam) at 1:25 for 1 h at room temperature.

    Techniques:

    The effect of CU-CPT22 on IL-1α expression. hRPE cells were cultured with or without CU-CPT22 or co-cultured with LPS (1000 ng/ml) or Pam3CSK4 (100 ng/ml) for 24 hr (A-C). Unless specified, the concentration of CU-CPT22 was 10 μM, TAK-242 100μM, BAPTA-AM 5 μM, caspase-4 inhibitor, LEVD 4 μM, Ly 294002 75 μM. After incubation, the whole cell lysates were harvested and subjected to ELISA. *p

    Journal: Experimental eye research

    Article Title: Expression and Regulation of Alarmin Cytokine IL-1α in Human Retinal Pigment Epithelial Cells

    doi: 10.1016/j.exer.2018.03.015

    Figure Lengend Snippet: The effect of CU-CPT22 on IL-1α expression. hRPE cells were cultured with or without CU-CPT22 or co-cultured with LPS (1000 ng/ml) or Pam3CSK4 (100 ng/ml) for 24 hr (A-C). Unless specified, the concentration of CU-CPT22 was 10 μM, TAK-242 100μM, BAPTA-AM 5 μM, caspase-4 inhibitor, LEVD 4 μM, Ly 294002 75 μM. After incubation, the whole cell lysates were harvested and subjected to ELISA. *p

    Article Snippet: IL-1α antibody was from Abcam (Cambridge, UK).

    Techniques: Expressing, Cell Culture, Concentration Assay, Incubation, Enzyme-linked Immunosorbent Assay

    Ca 2+ signaling regulates IL-1α expression. hRPE cells were cultured with or without LPS (1000 ng/ml) (A and F), tunicamycin (Tu, 10μM) (B), IL-1β (2 ng/ml) (C), or Pam3CSK4 (Pam, 100 ng/ml) (D) for 6 (F) or 24 hr (A-D) with or without PD150606 (PD, 100 μM) or BAPTA-AM (BAPTA, 5 μM). hRPE cells were incubated with or without ionomycin (3 μM), LPS, and Pam3CSK4 in presence or absence of caspase-4 inhibitor, LEVD (4 μM), and inhabited with ionomycin plus LPS or ionomycin plus Pam3CSK4 for 24 hr (E). After incubation, the whole cell lysates were harvested and subjected to ELISA. Steady-state IL-1α mRNA was analyzed by qPCR (F). *p

    Journal: Experimental eye research

    Article Title: Expression and Regulation of Alarmin Cytokine IL-1α in Human Retinal Pigment Epithelial Cells

    doi: 10.1016/j.exer.2018.03.015

    Figure Lengend Snippet: Ca 2+ signaling regulates IL-1α expression. hRPE cells were cultured with or without LPS (1000 ng/ml) (A and F), tunicamycin (Tu, 10μM) (B), IL-1β (2 ng/ml) (C), or Pam3CSK4 (Pam, 100 ng/ml) (D) for 6 (F) or 24 hr (A-D) with or without PD150606 (PD, 100 μM) or BAPTA-AM (BAPTA, 5 μM). hRPE cells were incubated with or without ionomycin (3 μM), LPS, and Pam3CSK4 in presence or absence of caspase-4 inhibitor, LEVD (4 μM), and inhabited with ionomycin plus LPS or ionomycin plus Pam3CSK4 for 24 hr (E). After incubation, the whole cell lysates were harvested and subjected to ELISA. Steady-state IL-1α mRNA was analyzed by qPCR (F). *p

    Article Snippet: IL-1α antibody was from Abcam (Cambridge, UK).

    Techniques: Expressing, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

    Immunofluorescence analysis of IL-1α expression in hRPE cells. hRPE cells seeded in chamber slides were incubated with LPS (A, 1000 ng/ml), tunicamycin (B, 10 μM) or IL-1β (C, E, F, 2 ng/ml) for 20 hr. Controls include untreated cells (D), IL-1β-treated cells without primary antibody (E), and IL-1β-treated cells with an isotype antibody control (F). The cells were fixed as described in Materials and Methods. The expressed IL-1α is shown in green. The nuclei were stained with 400x bisBenzimide (blue) (A-F, bottom panels). Experiments were repeated twice. Typical images were taken at 400X magnification.

    Journal: Experimental eye research

    Article Title: Expression and Regulation of Alarmin Cytokine IL-1α in Human Retinal Pigment Epithelial Cells

    doi: 10.1016/j.exer.2018.03.015

    Figure Lengend Snippet: Immunofluorescence analysis of IL-1α expression in hRPE cells. hRPE cells seeded in chamber slides were incubated with LPS (A, 1000 ng/ml), tunicamycin (B, 10 μM) or IL-1β (C, E, F, 2 ng/ml) for 20 hr. Controls include untreated cells (D), IL-1β-treated cells without primary antibody (E), and IL-1β-treated cells with an isotype antibody control (F). The cells were fixed as described in Materials and Methods. The expressed IL-1α is shown in green. The nuclei were stained with 400x bisBenzimide (blue) (A-F, bottom panels). Experiments were repeated twice. Typical images were taken at 400X magnification.

    Article Snippet: IL-1α antibody was from Abcam (Cambridge, UK).

    Techniques: Immunofluorescence, Expressing, Incubation, Staining

    IL-1α mRNA synthesis and protein production in hRPE cells. mRNA levels were assessed for cells (A) treated with LPS (1000 ng/ml), tunicamycin (10 μM) or IL-1β (2 ng/ml) for 6 hr. IL-1α protein production was analyzed by ELISA in hRPE cells treated with LPS (B, 1000 ng/ml), tunicamycin (C, 10 μM) or IL-1β (D, 2 ng/ml) for 24 or 48 hr in the presence or absence of caspase-4 inhibitor (LEVD, 4 μM), caspase-1 inhibitor (YVAD, 2 μM), anti-IL-1β antibody (Ab IL-1β) or isotype serium (Ab Ctrl). The p values were calculated by comparing treatment to Ctrl (A), to LPS only (B), to tunicamycin only (C), or to IL-1β only (D). *p

    Journal: Experimental eye research

    Article Title: Expression and Regulation of Alarmin Cytokine IL-1α in Human Retinal Pigment Epithelial Cells

    doi: 10.1016/j.exer.2018.03.015

    Figure Lengend Snippet: IL-1α mRNA synthesis and protein production in hRPE cells. mRNA levels were assessed for cells (A) treated with LPS (1000 ng/ml), tunicamycin (10 μM) or IL-1β (2 ng/ml) for 6 hr. IL-1α protein production was analyzed by ELISA in hRPE cells treated with LPS (B, 1000 ng/ml), tunicamycin (C, 10 μM) or IL-1β (D, 2 ng/ml) for 24 or 48 hr in the presence or absence of caspase-4 inhibitor (LEVD, 4 μM), caspase-1 inhibitor (YVAD, 2 μM), anti-IL-1β antibody (Ab IL-1β) or isotype serium (Ab Ctrl). The p values were calculated by comparing treatment to Ctrl (A), to LPS only (B), to tunicamycin only (C), or to IL-1β only (D). *p

    Article Snippet: IL-1α antibody was from Abcam (Cambridge, UK).

    Techniques: Enzyme-linked Immunosorbent Assay

    Effects of IFNs on caspase-4 expression/activation and LPS-induced IL-1α production. mRNA levels of IL-1α and caspase-4 were analyzed by RT-PCR (A-C) and qPCR (D, G). IL-1α protein was analyzed by ELISA (E and F). Caspase-4 protein cleavage in hRPE cells was shown by Western blot analysis (H). hRPE cells were treated with LPS (1000 ng/ml), IFN-α (1000 U/ml), IFN-β (1000 U/ml), IFN-γ (1000 U/ml), IFN-γ priming or in combination for 6 (A-D, G), 24 (E-F) or 0, 16 and 24 hr (H). In E and F, hRPE cells were pre-incubated with IFN-α, -β and -γ at 1000 U/ml for 16 hr and then switched to fresh growth media with or without LPS (1000 ng/ml) for an additional 24 hr. The p values were calculated by comparing treatment with LPS alone (D, E, F), treatment with LPS plus caspase-4 inhibitor (F), or treatment with control (G). *p

    Journal: Experimental eye research

    Article Title: Expression and Regulation of Alarmin Cytokine IL-1α in Human Retinal Pigment Epithelial Cells

    doi: 10.1016/j.exer.2018.03.015

    Figure Lengend Snippet: Effects of IFNs on caspase-4 expression/activation and LPS-induced IL-1α production. mRNA levels of IL-1α and caspase-4 were analyzed by RT-PCR (A-C) and qPCR (D, G). IL-1α protein was analyzed by ELISA (E and F). Caspase-4 protein cleavage in hRPE cells was shown by Western blot analysis (H). hRPE cells were treated with LPS (1000 ng/ml), IFN-α (1000 U/ml), IFN-β (1000 U/ml), IFN-γ (1000 U/ml), IFN-γ priming or in combination for 6 (A-D, G), 24 (E-F) or 0, 16 and 24 hr (H). In E and F, hRPE cells were pre-incubated with IFN-α, -β and -γ at 1000 U/ml for 16 hr and then switched to fresh growth media with or without LPS (1000 ng/ml) for an additional 24 hr. The p values were calculated by comparing treatment with LPS alone (D, E, F), treatment with LPS plus caspase-4 inhibitor (F), or treatment with control (G). *p

    Article Snippet: IL-1α antibody was from Abcam (Cambridge, UK).

    Techniques: Expressing, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation

    Modulation of IL-1α expression through TLR4 and TLR2 signaling. hRPE cells were cultured with LPS (1000 ng/ml) (A, B) or Pam3CSK4 (Pam 300 ng/ml) or LPS plus 100 ng/ml Pam3CSK4 (B, C) for 24 hr. In some experiments, hRPE cells also were pre-incubated with TAK-242 (75 μM) (A), caspase-4 inhibitor LEVD-CHO (4 μM) (C), TLR2 blocking antibody, or isotype control antibody (C). After stimulations, whole cell lysates were harvested and subjected to ELISA. *p

    Journal: Experimental eye research

    Article Title: Expression and Regulation of Alarmin Cytokine IL-1α in Human Retinal Pigment Epithelial Cells

    doi: 10.1016/j.exer.2018.03.015

    Figure Lengend Snippet: Modulation of IL-1α expression through TLR4 and TLR2 signaling. hRPE cells were cultured with LPS (1000 ng/ml) (A, B) or Pam3CSK4 (Pam 300 ng/ml) or LPS plus 100 ng/ml Pam3CSK4 (B, C) for 24 hr. In some experiments, hRPE cells also were pre-incubated with TAK-242 (75 μM) (A), caspase-4 inhibitor LEVD-CHO (4 μM) (C), TLR2 blocking antibody, or isotype control antibody (C). After stimulations, whole cell lysates were harvested and subjected to ELISA. *p

    Article Snippet: IL-1α antibody was from Abcam (Cambridge, UK).

    Techniques: Expressing, Cell Culture, Incubation, Blocking Assay, Enzyme-linked Immunosorbent Assay

    Effects of PI3K inhibitors on IL-1α expression. hRPE cells were cultured with or without LPS (1000 ng/ml) in the presence or absence of CAL-101, PI-103, Wortmannin and MK 2206 for 6 hr to measure mRNA levels (A, C) or 24 hr to measure protein (B, D). ***p

    Journal: Experimental eye research

    Article Title: Expression and Regulation of Alarmin Cytokine IL-1α in Human Retinal Pigment Epithelial Cells

    doi: 10.1016/j.exer.2018.03.015

    Figure Lengend Snippet: Effects of PI3K inhibitors on IL-1α expression. hRPE cells were cultured with or without LPS (1000 ng/ml) in the presence or absence of CAL-101, PI-103, Wortmannin and MK 2206 for 6 hr to measure mRNA levels (A, C) or 24 hr to measure protein (B, D). ***p

    Article Snippet: IL-1α antibody was from Abcam (Cambridge, UK).

    Techniques: Expressing, Cell Culture

    Synergism and antagonism in growth factor and cytokine-stimulated myoblast proliferation and differentiation. Primary mouse myoblasts were cultured in differentiation medium (DM) with 0.1% DMSO (control) and were stimulated with either FGF2 + EGF + IGF1 (FEI), TNF-α + IL-1α (TI), IL-6 + OSM + LIF (IOL), or their two-way combinations for 72 h. ( a , b ) Proliferative index was determined by nuclei counting from DAPI images. ( a ) Representative DAPI images. Scale bar, 200 μ m. ( c – e ) RT-qPCR analysis of myogenic differentiation genes Myod1, Myogenin , and Myh2 quantified relative to 36b4 (reference control). ( f – h ) Immunoblots for MHC, Myogenin, and Hsp90 (as a loading control). Myogenin ( g ) and MHC ( h ) densitometry, normalized to Hsp90. In ( b – e , g – h ), n = 3 replicates are plotted as log 2 of mean fold change relative to control ± SEM. For co-treatments, an additive model used to identify synergy or antagonism using a modified Bliss independence model with p

    Journal: Cellular and Molecular Bioengineering

    Article Title: Data-Modeling Identifies Conflicting Signaling Axes Governing Myoblast Proliferation and Differentiation Responses to Diverse Ligand Stimuli

    doi: 10.1007/s12195-017-0508-5

    Figure Lengend Snippet: Synergism and antagonism in growth factor and cytokine-stimulated myoblast proliferation and differentiation. Primary mouse myoblasts were cultured in differentiation medium (DM) with 0.1% DMSO (control) and were stimulated with either FGF2 + EGF + IGF1 (FEI), TNF-α + IL-1α (TI), IL-6 + OSM + LIF (IOL), or their two-way combinations for 72 h. ( a , b ) Proliferative index was determined by nuclei counting from DAPI images. ( a ) Representative DAPI images. Scale bar, 200 μ m. ( c – e ) RT-qPCR analysis of myogenic differentiation genes Myod1, Myogenin , and Myh2 quantified relative to 36b4 (reference control). ( f – h ) Immunoblots for MHC, Myogenin, and Hsp90 (as a loading control). Myogenin ( g ) and MHC ( h ) densitometry, normalized to Hsp90. In ( b – e , g – h ), n = 3 replicates are plotted as log 2 of mean fold change relative to control ± SEM. For co-treatments, an additive model used to identify synergy or antagonism using a modified Bliss independence model with p

    Article Snippet: PMBs (passage number 18-20) were seeded at 50,000 cells cm−2 in collagen-coated treated 12-well plates in 1 mL growth media for 2 h, then switched to 1 mL differentiation media (DM: 50% Dulbecco’s Modified Eagle’s Medium, 45% Ham’s F-10, 3% Fetal Bovine Serum, 1% Penicillin–Streptomyocin, 1% l -glutamine) with or without 0.1% DMSO for 3 h. Cells were then stimulated with differentiation media (mock control) or a recombinant mouse protein cocktails consisting of mixtures of the following (final concentrations): 5 ng mL−1 FGF2 (R & D Systems, #3139-FB-025), 10 ng mL−1 EGF (R & D Systems, #2028-EG-200), 10 ng mL−1 IGF1 (R & D Systems, #791-MG-050); 10 ng mL−1 IL6 (R & D Systems, #406-ML-005), 10 ng mL−1 OSM (R & D Systems, #495-MO-025), 10 ng mL−1 LIF (R & D Systems, #8878-LF-025); 10 ng mL−1 TNF-α (R & D Systems, #410-MT-010), 10 ng mL−1 IL-1α (R & D Systems, #400-ML-005).

    Techniques: Cell Culture, Quantitative RT-PCR, Western Blot, Modification

    Cytokine-induced p38 MAPK subtype activation in human CF. (A) Cells stimulated with 10 ng/ml TNFα, 10 ng/ml IL-1α or 25 μg/ml anisomycin for 5–60 min before preparing whole cell homogenates and immunoblotting with phospho-specific and expression antibodies for p38 MAPK, MAPKAPK2 and HSP27. Blots representative of n = 3. (B) CF stimulated with 10 ng/ml TNFα, 10 ng/ml IL-1α or 25 μg/ml anisomycin for 15 min before preparing cell extracts for analysis of p38 subtype phosphorylation by IP/IB method. Samples were immunoprecipitated with pan phospho-p38 antibody then immunoblotted with individual p38 subtype expression antibodies. Blots representative of n = 3. (C) CF stimulated as for (B) and p38 subtype phosphorylation analyzed by ELISA. Bar chart depicts concentration of phosphorylated p38 subtypes (pg/ml). ∗∗∗ P

    Journal: Biochemical and Biophysical Research Communications

    Article Title: p38 MAPK alpha mediates cytokine-induced IL-6 and MMP-3 expression in human cardiac fibroblasts

    doi: 10.1016/j.bbrc.2012.11.071

    Figure Lengend Snippet: Cytokine-induced p38 MAPK subtype activation in human CF. (A) Cells stimulated with 10 ng/ml TNFα, 10 ng/ml IL-1α or 25 μg/ml anisomycin for 5–60 min before preparing whole cell homogenates and immunoblotting with phospho-specific and expression antibodies for p38 MAPK, MAPKAPK2 and HSP27. Blots representative of n = 3. (B) CF stimulated with 10 ng/ml TNFα, 10 ng/ml IL-1α or 25 μg/ml anisomycin for 15 min before preparing cell extracts for analysis of p38 subtype phosphorylation by IP/IB method. Samples were immunoprecipitated with pan phospho-p38 antibody then immunoblotted with individual p38 subtype expression antibodies. Blots representative of n = 3. (C) CF stimulated as for (B) and p38 subtype phosphorylation analyzed by ELISA. Bar chart depicts concentration of phosphorylated p38 subtypes (pg/ml). ∗∗∗ P

    Article Snippet: For signaling experiments, serum-starved CF were stimulated with recombinant human IL-1α (Invitrogen), TNFα (Invitrogen) or anisomycin (Sigma) in serum-free medium before preparing whole cell homogenates.

    Techniques: Activation Assay, Expressing, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Concentration Assay

    Effect of p38α gene silencing on IL-6 and MMP-3 expression. CF were mock-transfected (open bars) or transfected with p38α siRNA (filled bars), cultured for 4 days to promote p38α protein silencing, then stimulated with 10 ng/ml IL-1α for 6–24 h. (A) Real-time RT-PCR data for mRNA levels of IL-6, MMP-3 and p38α, expressed as percentage GAPDH mRNA levels. ∗∗∗ P

    Journal: Biochemical and Biophysical Research Communications

    Article Title: p38 MAPK alpha mediates cytokine-induced IL-6 and MMP-3 expression in human cardiac fibroblasts

    doi: 10.1016/j.bbrc.2012.11.071

    Figure Lengend Snippet: Effect of p38α gene silencing on IL-6 and MMP-3 expression. CF were mock-transfected (open bars) or transfected with p38α siRNA (filled bars), cultured for 4 days to promote p38α protein silencing, then stimulated with 10 ng/ml IL-1α for 6–24 h. (A) Real-time RT-PCR data for mRNA levels of IL-6, MMP-3 and p38α, expressed as percentage GAPDH mRNA levels. ∗∗∗ P

    Article Snippet: For signaling experiments, serum-starved CF were stimulated with recombinant human IL-1α (Invitrogen), TNFα (Invitrogen) or anisomycin (Sigma) in serum-free medium before preparing whole cell homogenates.

    Techniques: Expressing, Transfection, Cell Culture, Quantitative RT-PCR

    Growth and MVD of DA/3 mammary cancer cells in WT BALB/c, IL-1α, or IL-1β KO mice. ( A ) Mice were injected i.f.p. with DA/3 cells (5 × 10 5 cells per mouse). The results represent one of three experiments. Mean ± SEM from each group ( n = 8 per group) are shown. ( B ) MVD ± SEM in DA/3-embedded Matrigel plugs (2 × 10 5 ) in WT, IL-1β, or IL-1α KO mice ( n = 5 in each group).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: IL-1 is required for tumor invasiveness and angiogenesis

    doi: 10.1073/pnas.0437939100

    Figure Lengend Snippet: Growth and MVD of DA/3 mammary cancer cells in WT BALB/c, IL-1α, or IL-1β KO mice. ( A ) Mice were injected i.f.p. with DA/3 cells (5 × 10 5 cells per mouse). The results represent one of three experiments. Mean ± SEM from each group ( n = 8 per group) are shown. ( B ) MVD ± SEM in DA/3-embedded Matrigel plugs (2 × 10 5 ) in WT, IL-1β, or IL-1α KO mice ( n = 5 in each group).

    Article Snippet: Recombinant human IL-1Ra was obtained from Amgen Biologicals; recombinant mouse IL-1α was obtained from R & D Systems.

    Techniques: Mouse Assay, Injection

    MVD in B16 melanoma embedded in Matrigel plugs. ( A ) MVD ± SEM in B16 melanoma-embedded Matrigel plugs in WT or IL-1β KO mice on day 7. IL-1β KO mice were also implanted with Matrigel plugs embedded with a mixture of B16 cells and 100 ng of murine IL-1α per 0.4 ml of Matrigel ( n = 5 in each group). ( B ) Day 7 MVD of Matrigel plugs embedded into WT mice containing a mixture of B16 melanoma cells plus saline, 50 or 100 μg of recombinant human IL-1Ra per 0.4 ml of Matrigel ( n = 5 in each group).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: IL-1 is required for tumor invasiveness and angiogenesis

    doi: 10.1073/pnas.0437939100

    Figure Lengend Snippet: MVD in B16 melanoma embedded in Matrigel plugs. ( A ) MVD ± SEM in B16 melanoma-embedded Matrigel plugs in WT or IL-1β KO mice on day 7. IL-1β KO mice were also implanted with Matrigel plugs embedded with a mixture of B16 cells and 100 ng of murine IL-1α per 0.4 ml of Matrigel ( n = 5 in each group). ( B ) Day 7 MVD of Matrigel plugs embedded into WT mice containing a mixture of B16 melanoma cells plus saline, 50 or 100 μg of recombinant human IL-1Ra per 0.4 ml of Matrigel ( n = 5 in each group).

    Article Snippet: Recombinant human IL-1Ra was obtained from Amgen Biologicals; recombinant mouse IL-1α was obtained from R & D Systems.

    Techniques: Mouse Assay, Recombinant

    VEGF secretion in cocultures of B16 melanoma cells and macrophages from WT, IL-1α, or IL-1β KO mice. PEC were obtained and cocultured with B16 melanoma cells, as indicated in Materials and Methods . Mean ± SEM VEGF levels in 48-h supernatants were measured ( n = 5 experiments).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: IL-1 is required for tumor invasiveness and angiogenesis

    doi: 10.1073/pnas.0437939100

    Figure Lengend Snippet: VEGF secretion in cocultures of B16 melanoma cells and macrophages from WT, IL-1α, or IL-1β KO mice. PEC were obtained and cocultured with B16 melanoma cells, as indicated in Materials and Methods . Mean ± SEM VEGF levels in 48-h supernatants were measured ( n = 5 experiments).

    Article Snippet: Recombinant human IL-1Ra was obtained from Amgen Biologicals; recombinant mouse IL-1α was obtained from R & D Systems.

    Techniques: Mouse Assay

    Effect of combined therapy of methotrexate and coenzyme Q 10 on plasmatic level of IL-1α assessed on day 28. Values are given as arithmetic mean ± S.E.M. Statistical significance was evaluated using unpaired Student′s t-test: ** p

    Journal: Interdisciplinary Toxicology

    Article Title: Utilization of adjuvant arthritis model for evaluation of new approaches in rheumatoid arthritis therapy focused on regulation of immune processes and oxidative stress

    doi: 10.2478/v10102-011-0007-9

    Figure Lengend Snippet: Effect of combined therapy of methotrexate and coenzyme Q 10 on plasmatic level of IL-1α assessed on day 28. Values are given as arithmetic mean ± S.E.M. Statistical significance was evaluated using unpaired Student′s t-test: ** p

    Article Snippet: Rat IL-1α assay in plasma For determination of IL-1α in plasma an ELISA kit from Bender MedSystems was used as described in the product manual Rat IL-1α ELISA BMS627 and BMS627TEN.

    Techniques: