il-1β Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher il 1β
    Effect of RT on LPS stimulated pro-inflammatory cytokines in U-87 cells. LPS pre-treated (500 ng/ml for 20 min) cells were treated with various concentrations of RT before the estimation of cytokines. ( A – D ) Are the graphical representation of optical density of cell supernatants at 405 nm for TNF-α, <t>IL-1β,</t> IL-6 and IL-10. Data represents mean ± S.E.M. of 3 independent experiments. ** p
    Il 1β, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13192 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/il 1β/product/Thermo Fisher
    Average 99 stars, based on 13192 article reviews
    Price from $9.99 to $1999.99
    il 1β - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    95
    Millipore il 1β
    IL7AS and MIR3142HG regulate the <t>IL-1β-stimulated</t> inflammatory response in control fibroblasts. Control fibroblasts were transfected overnight with LNA antisense sequences against IL7AS (A/C/E/G) and MIR3142HG (B/D/F/H) or scrambled (negative) controls. Cell were then stimulated with IL-1β for 24 h prior to isolation of RNA and measurement of IL7AS (A) or MIR3142HG (B) by qRT-PCR or measurement of supernatant IL-6 (C,D) , IL-8 (E,F) , and CCL2 (G,H) by ELISA. Data represents the mean ± SEM of five control individuals. Following normalization against the IL1β-stimulated cells (100%), statistical significance was assessed (vs. IL1β-stimulated cells) using the repeat measures 1-way analysis of variance (ANOVA) with a Dunnett's test where * p
    Il 1β, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 5110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/il 1β/product/Millipore
    Average 95 stars, based on 5110 article reviews
    Price from $9.99 to $1999.99
    il 1β - by Bioz Stars, 2020-10
    95/100 stars
      Buy from Supplier

    94
    Becton Dickinson il 1β
    AMPK pathway activation inhibits the induction of <t>IL-1β</t> and IL-18 production by various inflammasome stimuli in LPS-primed MDMs. Primary MDMs were isolated from healthy controls ( A and B ; n = 5) or type 2 diabetic patients ( C ; n = 11) before and after treatment with metformin for 2 months. A : MDMs were primed with LPS (10 ng/mL) for 4 h in the presence of a high glucose concentration (15 mmol/L) and then treated with compound C (Comp C; 5, 10, or 25 μmol/L) and stimulated with ATP (1 mmol/L for 1 h). Data are expressed as means ± SEM of five independent experiments. B : MDMs were transduced with nonspecific control shRNA lentiviral particles (shNS) or lentiviral shRNA specific for AMPK (shAMPK). Then, the cells were primed with LPS (10 ng/mL) for 4 h in the presence of a high glucose concentration (15 mmol/L) and treated with ATP or MSU (100 μg/mL for 6 h). Data are expressed as means ± SEM of three independent experiments. Representative images of semiquantitative RT-PCR gels run to assess transduction efficiency (top). C : The cells were primed with LPS (10 ng/mL) for 4 h in the presence of autologous sera and then treated with ATP or MSU. A and B : ELISA of IL-1β and IL-18 levels. C : Western blotting analysis of p-AMPKα protein levels. The intensity of each band for each protein was quantified and normalized to the housekeeping protein β-actin ( C , right). Data are expressed as means ± SEM. *** P
    Il 1β, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 6205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/il 1β/product/Becton Dickinson
    Average 94 stars, based on 6205 article reviews
    Price from $9.99 to $1999.99
    il 1β - by Bioz Stars, 2020-10
    94/100 stars
      Buy from Supplier

    99
    Abcam il 1β
    High glucose treatment increases the secretion levels of <t>IL-1β,</t> IL-6 and TNF-α in RPECs. (A-C) Expression levels of (A) IL-1β, (B) IL-6 and (C) TNF-α in the culture medium of cells treated with either 30 mmol/l glucose or 24.4 mmol/l mannitol for 12 h were measured by ELISA. (D and E) Expression levels of p-AKT, total AKT, p-mTOR and total mTOR in cells treated with either 30 mmol/l glucose or 24.4 mmol/l mannitol for 12 h as measured by western blot analysis. Data are presented as the mean ± standard error of the mean; n=3; **P
    Il 1β, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2940 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/il 1β/product/Abcam
    Average 99 stars, based on 2940 article reviews
    Price from $9.99 to $1999.99
    il 1β - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    94
    Santa Cruz Biotechnology il 1β
    Expression of NLRP3 inflammasome components and the mature form of <t>IL-1β.</t> ( A ) LAD2 cells (1 × 10 6 cells per well) were seeded in a 12-well culture plate and were stimulated with SP (1 μΜ), IL-33 (30 ng/mL), or their combination for 24 h. Cell lysates were collected after 24 h, and protein levels of the NLRP3 inflammasome components (NLRP3, ASC, caspase-1), pro-IL-1β, and active IL-1β (p17) were measured by Western blot, using β-actin as loading control (shown in a representative gel of n = 3). SP and IL-33 increase caspase-1 gene expression. ( B ) LAD2 cells (1 × 10 6 cells per well), ( n = 3, ** P
    Il 1β, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 2491 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/il 1β/product/Santa Cruz Biotechnology
    Average 94 stars, based on 2491 article reviews
    Price from $9.99 to $1999.99
    il 1β - by Bioz Stars, 2020-10
    94/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc il 1β
    Recombinant human interleukin-1 receptor antagonist (RhIL-1Ra) ameliorated ConA-induced NOD-like receptor protein 3 (NLRP3) inflammasome activation and pyroptosis. (A) BALB/c mice ( n = 6 for each group) were pretreated with rhIL-1Ra before ConA injection, and samples were extracted at 12 h post ConA treatment. Serum <t>IL-1β</t> and IL-18 were detected by ELISA. (B) Liver homogenates were subjected to caspase-1 activity assay. (C) Western blot of NLRP3, Cleaved caspase-1, and IL-1β in the livers. GAPDH acted as a loading control. Each lane represented a separate animal. Results were obtained from three experiments. Quantification of protein expression were obtained by Image J software. (D) Caspase-1 positive death cells staining were observed by confocal microscopy. FAM-YVAD-FMK (green), propidium iodide (red), Hoechst 33342 (blue). The data were presented as means ± SD (Student’s t -test, * p
    Il 1β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1544 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/il 1β/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1544 article reviews
    Price from $9.99 to $1999.99
    il 1β - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    94
    Bio-Rad il 1β
    Full-length cDNA of largemouth bass Micropterus salmoides , <t>IL-1β.</t> The underlined sequence part is IL-1β signature and grey highlighted sequences are ATTTA motifs, bold letters are N -glycosylation sites and double underlined are AATA motif.
    Il 1β, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 2322 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/il 1β/product/Bio-Rad
    Average 94 stars, based on 2322 article reviews
    Price from $9.99 to $1999.99
    il 1β - by Bioz Stars, 2020-10
    94/100 stars
      Buy from Supplier

    99
    PeproTech il 1β
    Exosome‐delivered mesenchymal‐epithelial transition factor (MET) stimulates macrophages to facilitate tumour growth in vivo. A, The effects of the supernatant from macrophage treated with PBS, MET + exosomes, MET − exosomes, MET − exosomes + recombinant <t>IL‐1β</t> protein, MET + exosomes + IL‐1β neutralizing antibody on tumour growth in a xenograft model. Tumour volume in the xenograft models was measured every 3 d after a 10 d inoculation period. B,C, Final tumour weights were determined and photographed. * P
    Il 1β, supplied by PeproTech, used in various techniques. Bioz Stars score: 99/100, based on 4040 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/il 1β/product/PeproTech
    Average 99 stars, based on 4040 article reviews
    Price from $9.99 to $1999.99
    il 1β - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    94
    Meso Scale Diagnostics LLC il 1β
    (A-B) The levels of cytokines in CSF (pg/mL). The LPS-treated rats exhibited higher levels <t>IL-1β</t> (A) and TNF-α (B), but exendin-4 treatment had no effect on cytokine levels (Student’s t -tests, NS). Non-LPS treated rats were below detection limit. CSF from six animals were not used because of insufficient amounts ( n = 1 [Control], n = 2 [LPS], n = 3 [LPS+Ex4]). (C) The mRNA expression of IL-1β in the striatum. The IL-1β expression was higher in LPS treated rats but was not affected by exendin-4 treatment (two-way ANOVA, LPS effect, F (1,38) = 44.04, p
    Il 1β, supplied by Meso Scale Diagnostics LLC, used in various techniques. Bioz Stars score: 94/100, based on 1124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/il 1β/product/Meso Scale Diagnostics LLC
    Average 94 stars, based on 1124 article reviews
    Price from $9.99 to $1999.99
    il 1β - by Bioz Stars, 2020-10
    94/100 stars
      Buy from Supplier

    99
    R&D Systems mouse il 1 beta il 1f2 antibody
    (A-B) The levels of cytokines in CSF (pg/mL). The LPS-treated rats exhibited higher levels <t>IL-1β</t> (A) and TNF-α (B), but exendin-4 treatment had no effect on cytokine levels (Student’s t -tests, NS). Non-LPS treated rats were below detection limit. CSF from six animals were not used because of insufficient amounts ( n = 1 [Control], n = 2 [LPS], n = 3 [LPS+Ex4]). (C) The mRNA expression of IL-1β in the striatum. The IL-1β expression was higher in LPS treated rats but was not affected by exendin-4 treatment (two-way ANOVA, LPS effect, F (1,38) = 44.04, p
    Mouse Il 1 Beta Il 1f2 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 983 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse il 1 beta il 1f2 antibody/product/R&D Systems
    Average 99 stars, based on 983 article reviews
    Price from $9.99 to $1999.99
    mouse il 1 beta il 1f2 antibody - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    94
    Santa Cruz Biotechnology anti il 1β
    PI3K phosphorylation is involved in APLN-induced <t>IL-1β</t> synthesis. ( A ) OASFs were pretreated with PI3K inhibitors (LY294002, Wortmannin; 10 μM) for 30 min, then incubated with APLN (10 ng/mL) for 24 h. IL-1β mRNA and protein levels were examined by RT-qPCR (n=4) and Western blot (n=3) assays, respectively. ( B ) OASFs were pretreated with PI3K inhibitors (LY294002, Wortmannin; 10 μM) for 30 min, then incubated with APLN (10 ng/mL) for 24 h. Excreted IL-1 β protein levels were examined by ELISA (n=5). ( C ) OASFs were transfected with PI3K siRNA (1 μg) then incubated with APLN (10 ng/mL) for 24 h. IL-1β mRNA levels were examined by ELISA assay (n=5). ( D ) OASFs were transfected with PI3K siRNA (1 μg), then incubated with APLN (10 ng/mL) for 24 h. Excreted IL-1β protein levels were examined by ELISA assay (n=5). ( E ) OASFs were incubated with APLN for the indicated time intervals, and the extent of PI3K phosphorylation was examined by Western blot (n=3). * p
    Anti Il 1β, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 724 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti il 1β/product/Santa Cruz Biotechnology
    Average 94 stars, based on 724 article reviews
    Price from $9.99 to $1999.99
    anti il 1β - by Bioz Stars, 2020-10
    94/100 stars
      Buy from Supplier

    98
    Abcam anti il 1β
    Statistically significant correlations between IL-18 and caspase 1 in LL ( A ) and TT leprosy ( B ), between caspase 1 and <t>IL-1β</t> ( C ), between NLRP1 and IL-18 ( D ), between NLRP3 and IL-18 ( E ), and between NLRP3 and NLRP1 ( F ) in I leprosy. Abbreviations: I, indeterminate; LL, lepromatous leprosy; NLRP1, nucleotide-binding oligomerization domain-like receptor containing pyrin domain 1; NLRP3, nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3; TT, tuberculoid.
    Anti Il 1β, supplied by Abcam, used in various techniques. Bioz Stars score: 98/100, based on 549 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti il 1β/product/Abcam
    Average 98 stars, based on 549 article reviews
    Price from $9.99 to $1999.99
    anti il 1β - by Bioz Stars, 2020-10
    98/100 stars
      Buy from Supplier

    99
    Abcam anti il1 beta antibody
    Statistically significant correlations between IL-18 and caspase 1 in LL ( A ) and TT leprosy ( B ), between caspase 1 and <t>IL-1β</t> ( C ), between NLRP1 and IL-18 ( D ), between NLRP3 and IL-18 ( E ), and between NLRP3 and NLRP1 ( F ) in I leprosy. Abbreviations: I, indeterminate; LL, lepromatous leprosy; NLRP1, nucleotide-binding oligomerization domain-like receptor containing pyrin domain 1; NLRP3, nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3; TT, tuberculoid.
    Anti Il1 Beta Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 313 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti il1 beta antibody/product/Abcam
    Average 99 stars, based on 313 article reviews
    Price from $9.99 to $1999.99
    anti il1 beta antibody - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc anti il 1β
    CBG pre-treatment was able to reduce the levels of pro-inflammatory cytokines in NSC-34 cells treated with the medium of LPS-stimulated RAW 264.7 macrophages. Immunocytochemistry with the quantitative analysis of positive staining showed that the treatment with the medium of LPS-stimulated RAW 264.7 macrophages induced the expression of the pro-inflammatory cytokines <t>IL-1β,</t> TNF-α and IFN-γ. CBG pre-treatment reduced the protein levels of the pro-inflammatory cytokines. The immunocytochemical assays were repeated three times. **** p
    Anti Il 1β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 444 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti il 1β/product/Cell Signaling Technology Inc
    Average 99 stars, based on 444 article reviews
    Price from $9.99 to $1999.99
    anti il 1β - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher interleukin il 1 β
    Effects of HSR on corticosterone-induced proinflammatory cytokine expression. Total RNA was extracted from SK-N-SH cells, and the levels of (a) <t>IL-1</t> β , (b) IL-6, (c) IL-8, and (d) TNF- α mRNA were determined by qRT-PCR. Expression levels of the target genes were normalized to those of β -actin. Data are the mean ± SD ( n = 3). ### p
    Interleukin Il 1 β, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 322 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/interleukin il 1 β/product/Thermo Fisher
    Average 99 stars, based on 322 article reviews
    Price from $9.99 to $1999.99
    interleukin il 1 β - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Abcam rabbit anti il 1β
    Effects of joint IM on serum C5a and pro-inflammatory cytokine expression levels. (A) Expression levels of C5a, <t>IL-1β,</t> TNF-α and IL-17A in the serum; (B) expression levels of C5a, IL-1β, TNF-α and IL-17A in joint cavity fluid. The graphs present the mean ± SEM. *P
    Rabbit Anti Il 1β, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 349 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti il 1β/product/Abcam
    Average 99 stars, based on 349 article reviews
    Price from $9.99 to $1999.99
    rabbit anti il 1β - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    92
    Genzyme il 1β
    Cytokine production (cell-associated or secreted <t>IL-1β,</t> IL-6, and TNF-α) by macrophages exposed to 1 ng of leukotoxin/ml or to 100 ng of lipopolysaccharide/ml from A. actinomycetemcomitans (Aa LPS) or from E. coli (Ec LPS) for 3 h. The
    Il 1β, supplied by Genzyme, used in various techniques. Bioz Stars score: 92/100, based on 364 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/il 1β/product/Genzyme
    Average 92 stars, based on 364 article reviews
    Price from $9.99 to $1999.99
    il 1β - by Bioz Stars, 2020-10
    92/100 stars
      Buy from Supplier

    93
    Bender MedSystems il 1β
    Model representing the induction and inhibition of <t>IL-1β</t> by HPV16. Infection of the basal keratinocytes with HPV16 induces inflammasome dependent IL-1β production sensed by an unknown innate receptor. p53 transcriptional regulation of IRF6 is increased, which we show drives IL-1β transcription. The pro form of IL-1β is cleaved by caspase 1 (red bar). The active form of IL-1β can block the increase in viral copies. However, when the oncoprotein E6 is expressed this drives p53 degradation by E6AP preventing IRF6 and consequently IL-1β transcription. This mechanism of viral inhibition of innate responses may contribute to HPV16 persistence in the host.
    Il 1β, supplied by Bender MedSystems, used in various techniques. Bioz Stars score: 93/100, based on 415 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/il 1β/product/Bender MedSystems
    Average 93 stars, based on 415 article reviews
    Price from $9.99 to $1999.99
    il 1β - by Bioz Stars, 2020-10
    93/100 stars
      Buy from Supplier

    99
    PeproTech recombinant il 1β
    <t>IL-1β</t> impaired cLTP-induced signaling associated with actin dynamics. a Western blots of G-actin and F-actin. b Quantification of the blots shown in a . Data are mean ± SEM from three independent experiments expressed in terms of the ratio of G-actin to F-actin obtained in the control cultures (* p
    Recombinant Il 1β, supplied by PeproTech, used in various techniques. Bioz Stars score: 99/100, based on 270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant il 1β/product/PeproTech
    Average 99 stars, based on 270 article reviews
    Price from $9.99 to $1999.99
    recombinant il 1β - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    97
    Millipore recombinant human il 1β
    ( A ) IP-10, ( B ) RANTES, and ( C ) TNFα secretion levels induced in NHA by <t>IL-1β</t> stimulation (20 ng/ml) are significantly reduced with exposure to Palm Fruit Bioactives at 24 and 96 hours in a dose-dependent manner. The normalized percent change (%) for each PFB treatment condition compared with the positive control, IL-1β stimulation alone, for each time point. Data represent the mean percentage change ± SD of three Luminex replicates (*p
    Recombinant Human Il 1β, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human il 1β/product/Millipore
    Average 97 stars, based on 205 article reviews
    Price from $9.99 to $1999.99
    recombinant human il 1β - by Bioz Stars, 2020-10
    97/100 stars
      Buy from Supplier

    92
    Becton Dickinson human il 1β
    cGAS-dependent inflammasome activation is necessary for the control of MCMV. WT, Aim2 −/− , cGAS −/− , or Ifnar −/− BMDMs were infected with MCMV at multiplicity of infection 1. (a) <t>IL-1β</t> ELISA of supernatants from BMDMs 1 d postinfection. (b and c) WT, Ifnar −/− , or Ifnar −/− cGAS −/− BMDMs were infected with MCMV at multiplicity of infection 1. (b) IL-1β ELISA of supernatants from BMDMs 1–3 d postinfection. (c) Relative MCMV genome copy number from BMDMs 2 d postinfection. (d and e) WT and Ifnar −/− BMDMs were infected in the presence or absence of caspase-1 inhibitor, Z-YVAD, and assayed for IL-1β (d) or relative MCVM genome copy (e). In e, left and right panels are from the same experiment. n = 2 independent experiments. Shown is a representative experiment with technical replicates of six; mean ± SEM. (f and g) WT, Ifnar −/− , or Ifnar −/− cGAS −/− mice were infected with MCMV for 36 h. IL-18 serum levels (f), IFNγ spleen levels (g), and relative MCMV genome copy number from spleens after infection (h). n = 8. Error bars, SEM; *, P
    Human Il 1β, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human il 1β/product/Becton Dickinson
    Average 92 stars, based on 142 article reviews
    Price from $9.99 to $1999.99
    human il 1β - by Bioz Stars, 2020-10
    92/100 stars
      Buy from Supplier

    91
    Santa Cruz Biotechnology rabbit anti il 1β
    HF ameliorates LPS-induced production of <t>IL-1β</t> in macrophages by affecting mRNA stability and processing of mature IL-1β. (A–B) IL-1β production from LPS (500 ng/ml)-primed or -unprimed BMDMs treated with different concentrations of HF or MAZ1310 (control) for 6 h. ATP (5 mM) was added to the LPS-stimulated macrophage cultures for 30 min at the end of time point ( S1 Data ). Statistical significance was determined by student t test. * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005. (C) IL-1β production from LPS-primed macrophages stimulated with MSU (150 ug/ml), or ALU (200 ug/ml) for 6 h, or infected with S . typhimurium (MOI 10) in presence or absence of HF ( S1 Data ). (D) IL-1β and caspase-1 (pro and active) expression by immunoblot analysis from LPS-primed BMDMs treated with HF as indicated; β-actin was used as loading control. (E) ROS levels detected by CM-H2DCFDA staining in macrophages treated with HF or LPS plus HF. (F, G) qRT-PCR analysis of mature IL-1β and pre–IL-1β mRNA levels in J774A.1 cells stimulated with LPS or LPS plus HF ( S1 Data ). (H) Analysis of IL-1β mRNA levels by qRT-PCR in LPS-primed macrophages treated with Act-D for 2 h followed by HF treatment for an additional 2 h ( S1 Data ). * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005 were considered statistically significant. Data are representative of 1 of 3–4 independent experiments. Act-D, actinomycin-D; ALU, aluminum hydroxide; BMDM, bone marrow–derived macrophage; HF, Halofuginone; IL-1β, interleukin 1β; LPS, lipopolysaccharide; Lys., cell lysates; MOI, multiplicity of infection; MSU, monosodium urate; qRT-PCR, quantitative reverse transcription PCR; ROS, reactive oxygen species; Sup., culture supernatant.
    Rabbit Anti Il 1β, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 266 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti il 1β/product/Santa Cruz Biotechnology
    Average 91 stars, based on 266 article reviews
    Price from $9.99 to $1999.99
    rabbit anti il 1β - by Bioz Stars, 2020-10
    91/100 stars
      Buy from Supplier

    90
    Beyotime il 1β
    Regulation of type II collagen and MMP-13 by <t>IL-1β</t> in rat chondrocytes. (A) Reverse transcription-quantitative polymerase chain reaction and (B) western blot analyses demonstrating that IL-1β stimulation decreases type II collagen and increases MMP-13 mRNA and protein expression levels. GAPDH served as an internal control. Data are presented as the mean ± standard deviation. **P
    Il 1β, supplied by Beyotime, used in various techniques. Bioz Stars score: 90/100, based on 222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/il 1β/product/Beyotime
    Average 90 stars, based on 222 article reviews
    Price from $9.99 to $1999.99
    il 1β - by Bioz Stars, 2020-10
    90/100 stars
      Buy from Supplier

    Image Search Results


    Effect of RT on LPS stimulated pro-inflammatory cytokines in U-87 cells. LPS pre-treated (500 ng/ml for 20 min) cells were treated with various concentrations of RT before the estimation of cytokines. ( A – D ) Are the graphical representation of optical density of cell supernatants at 405 nm for TNF-α, IL-1β, IL-6 and IL-10. Data represents mean ± S.E.M. of 3 independent experiments. ** p

    Journal: Scientific Reports

    Article Title: Ultra-diluted Toxicodendron pubescens attenuates pro-inflammatory cytokines and ROS- mediated neuropathic pain in rats

    doi: 10.1038/s41598-018-31971-9

    Figure Lengend Snippet: Effect of RT on LPS stimulated pro-inflammatory cytokines in U-87 cells. LPS pre-treated (500 ng/ml for 20 min) cells were treated with various concentrations of RT before the estimation of cytokines. ( A – D ) Are the graphical representation of optical density of cell supernatants at 405 nm for TNF-α, IL-1β, IL-6 and IL-10. Data represents mean ± S.E.M. of 3 independent experiments. ** p

    Article Snippet: Cytokine ELISA Ready SET-Go kits for TNF-α (Cat: 837324-22: Batch No. E09479-1645), IL-1β (Cat: 887013-22; Batch No. E09323-1645) and IL-6 (Cat: 837064-22: Batch No. E09358-1645) were purchased from e-Biosciences Incorporation, USA.

    Techniques:

    IL7AS and MIR3142HG regulate the IL-1β-stimulated inflammatory response in control fibroblasts. Control fibroblasts were transfected overnight with LNA antisense sequences against IL7AS (A/C/E/G) and MIR3142HG (B/D/F/H) or scrambled (negative) controls. Cell were then stimulated with IL-1β for 24 h prior to isolation of RNA and measurement of IL7AS (A) or MIR3142HG (B) by qRT-PCR or measurement of supernatant IL-6 (C,D) , IL-8 (E,F) , and CCL2 (G,H) by ELISA. Data represents the mean ± SEM of five control individuals. Following normalization against the IL1β-stimulated cells (100%), statistical significance was assessed (vs. IL1β-stimulated cells) using the repeat measures 1-way analysis of variance (ANOVA) with a Dunnett's test where * p

    Journal: Frontiers in Immunology

    Article Title: Long Non-coding RNAs Are Central Regulators of the IL-1β-Induced Inflammatory Response in Normal and Idiopathic Pulmonary Lung Fibroblasts

    doi: 10.3389/fimmu.2018.02906

    Figure Lengend Snippet: IL7AS and MIR3142HG regulate the IL-1β-stimulated inflammatory response in control fibroblasts. Control fibroblasts were transfected overnight with LNA antisense sequences against IL7AS (A/C/E/G) and MIR3142HG (B/D/F/H) or scrambled (negative) controls. Cell were then stimulated with IL-1β for 24 h prior to isolation of RNA and measurement of IL7AS (A) or MIR3142HG (B) by qRT-PCR or measurement of supernatant IL-6 (C,D) , IL-8 (E,F) , and CCL2 (G,H) by ELISA. Data represents the mean ± SEM of five control individuals. Following normalization against the IL1β-stimulated cells (100%), statistical significance was assessed (vs. IL1β-stimulated cells) using the repeat measures 1-way analysis of variance (ANOVA) with a Dunnett's test where * p

    Article Snippet: On day 2, the cells were serum-starved with 2 ml of fresh medium (0.1% FBS) and treated with/without 3 ng/ml IL-1β (recombinant, expressed in E. coli , Sigma-Aldrich, I9401-5UG) for 6 h before all supernatants were collected and cells were harvested for RNA extraction.

    Techniques: Transfection, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    IL-1β-induced expression of IL7AS, MIR3142HG, and the inflammatory mediators is mediated via the NF-κB signaling pathway in control and IPF fibroblasts. Control and IPF fibroblasts were pre-incubated in the stated concentration of TPCA-1 or 0.1% (v/v) DMSO (vehicle) and then incubated in absence or presence of IL-1β for 24 h prior to measurement of IL6, IL8 and CCL2 release (A) and IL7AS and MIR3142HG expression (B) . Data is normalized against IL-1β stimulated cells (100%) and represents the mean ± SEM of five control and IPF patients. The logIC 50 for each individual was determined in GraphPad Prism and comparison between control and IPF groups was performed using an unpaired t -test. The IC 50 was calculated from the mean logIC 50 values.

    Journal: Frontiers in Immunology

    Article Title: Long Non-coding RNAs Are Central Regulators of the IL-1β-Induced Inflammatory Response in Normal and Idiopathic Pulmonary Lung Fibroblasts

    doi: 10.3389/fimmu.2018.02906

    Figure Lengend Snippet: IL-1β-induced expression of IL7AS, MIR3142HG, and the inflammatory mediators is mediated via the NF-κB signaling pathway in control and IPF fibroblasts. Control and IPF fibroblasts were pre-incubated in the stated concentration of TPCA-1 or 0.1% (v/v) DMSO (vehicle) and then incubated in absence or presence of IL-1β for 24 h prior to measurement of IL6, IL8 and CCL2 release (A) and IL7AS and MIR3142HG expression (B) . Data is normalized against IL-1β stimulated cells (100%) and represents the mean ± SEM of five control and IPF patients. The logIC 50 for each individual was determined in GraphPad Prism and comparison between control and IPF groups was performed using an unpaired t -test. The IC 50 was calculated from the mean logIC 50 values.

    Article Snippet: On day 2, the cells were serum-starved with 2 ml of fresh medium (0.1% FBS) and treated with/without 3 ng/ml IL-1β (recombinant, expressed in E. coli , Sigma-Aldrich, I9401-5UG) for 6 h before all supernatants were collected and cells were harvested for RNA extraction.

    Techniques: Expressing, Incubation, Concentration Assay

    Differential expression of mRNAs and lncRNAs following IL-1β-stimulation of control lung fibroblasts . (A) Heat map showing the differential expression of mRNAs in control fibroblasts following IL-1β stimulation for 6 h. (B) Pathway analysis of up-regulated mRNAs. (C) Top 10 most highly expressed lncRNA in non-stimulated control fibroblasts. (D) Heat map showing the differential expression of lncRNAs in control fibroblasts following IL-1β stimulation for 6 h.

    Journal: Frontiers in Immunology

    Article Title: Long Non-coding RNAs Are Central Regulators of the IL-1β-Induced Inflammatory Response in Normal and Idiopathic Pulmonary Lung Fibroblasts

    doi: 10.3389/fimmu.2018.02906

    Figure Lengend Snippet: Differential expression of mRNAs and lncRNAs following IL-1β-stimulation of control lung fibroblasts . (A) Heat map showing the differential expression of mRNAs in control fibroblasts following IL-1β stimulation for 6 h. (B) Pathway analysis of up-regulated mRNAs. (C) Top 10 most highly expressed lncRNA in non-stimulated control fibroblasts. (D) Heat map showing the differential expression of lncRNAs in control fibroblasts following IL-1β stimulation for 6 h.

    Article Snippet: On day 2, the cells were serum-starved with 2 ml of fresh medium (0.1% FBS) and treated with/without 3 ng/ml IL-1β (recombinant, expressed in E. coli , Sigma-Aldrich, I9401-5UG) for 6 h before all supernatants were collected and cells were harvested for RNA extraction.

    Techniques: Expressing

    IL7AS but not MIR3142HG regulates the IL-β-stimulated inflammatory response in IPF fibroblasts. IPF fibroblasts were transfected overnight with LNA antisense sequences against IL7AS (A,C,E,G) and MIR3142HG (B,D,F,H) or scrambled (negative) controls. Cell were then stimulated with IL-1β for 24 h prior to isolation of RNA and measurement of IL7AS (A) or MIR3142HG (B) by qRT-PCR or measurement of supernatant IL-6 (C,D) , IL-8 (E,F) , and CCL2 (G,H) by ELISA. Data represents the mean ± SEM of five IPF individuals. Following normalization against the IL1β-stimulated cells (100%), statistical significance was assessed (vs. IL1β-stimulated cells) using the repeat measures 1-way analysis of variance (ANOVA) with a Dunnett's test where * p

    Journal: Frontiers in Immunology

    Article Title: Long Non-coding RNAs Are Central Regulators of the IL-1β-Induced Inflammatory Response in Normal and Idiopathic Pulmonary Lung Fibroblasts

    doi: 10.3389/fimmu.2018.02906

    Figure Lengend Snippet: IL7AS but not MIR3142HG regulates the IL-β-stimulated inflammatory response in IPF fibroblasts. IPF fibroblasts were transfected overnight with LNA antisense sequences against IL7AS (A,C,E,G) and MIR3142HG (B,D,F,H) or scrambled (negative) controls. Cell were then stimulated with IL-1β for 24 h prior to isolation of RNA and measurement of IL7AS (A) or MIR3142HG (B) by qRT-PCR or measurement of supernatant IL-6 (C,D) , IL-8 (E,F) , and CCL2 (G,H) by ELISA. Data represents the mean ± SEM of five IPF individuals. Following normalization against the IL1β-stimulated cells (100%), statistical significance was assessed (vs. IL1β-stimulated cells) using the repeat measures 1-way analysis of variance (ANOVA) with a Dunnett's test where * p

    Article Snippet: On day 2, the cells were serum-starved with 2 ml of fresh medium (0.1% FBS) and treated with/without 3 ng/ml IL-1β (recombinant, expressed in E. coli , Sigma-Aldrich, I9401-5UG) for 6 h before all supernatants were collected and cells were harvested for RNA extraction.

    Techniques: Transfection, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    IL-1β-induced expression of IL7AS, MIR3142HG, miR-146a, miR-3142 and inflammatory mediators in control and IPF fibroblasts . (A) Control and IPF fibroblasts were incubated in the absence of presence of IL-1β for 24 h and the fold-change in the IL7AS and MIR3142 expression determined by qRT-PCR, (B) ). Control and IPF fibroblasts were incubated in the absence of presence of IL-1β for 24 h before determination of the fold-change in miR-3142 and miR-146a expression by qRT-PCR (C) and the release of IL-6, IL-8, and CCL2 by ELISA (D) . Values are the mean ± SEM of five control and IPF patients and statistical significance was assessed using 1-way analysis of variance (ANOVA) where * p

    Journal: Frontiers in Immunology

    Article Title: Long Non-coding RNAs Are Central Regulators of the IL-1β-Induced Inflammatory Response in Normal and Idiopathic Pulmonary Lung Fibroblasts

    doi: 10.3389/fimmu.2018.02906

    Figure Lengend Snippet: IL-1β-induced expression of IL7AS, MIR3142HG, miR-146a, miR-3142 and inflammatory mediators in control and IPF fibroblasts . (A) Control and IPF fibroblasts were incubated in the absence of presence of IL-1β for 24 h and the fold-change in the IL7AS and MIR3142 expression determined by qRT-PCR, (B) ). Control and IPF fibroblasts were incubated in the absence of presence of IL-1β for 24 h before determination of the fold-change in miR-3142 and miR-146a expression by qRT-PCR (C) and the release of IL-6, IL-8, and CCL2 by ELISA (D) . Values are the mean ± SEM of five control and IPF patients and statistical significance was assessed using 1-way analysis of variance (ANOVA) where * p

    Article Snippet: On day 2, the cells were serum-starved with 2 ml of fresh medium (0.1% FBS) and treated with/without 3 ng/ml IL-1β (recombinant, expressed in E. coli , Sigma-Aldrich, I9401-5UG) for 6 h before all supernatants were collected and cells were harvested for RNA extraction.

    Techniques: Expressing, Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    AMPK pathway activation inhibits the induction of IL-1β and IL-18 production by various inflammasome stimuli in LPS-primed MDMs. Primary MDMs were isolated from healthy controls ( A and B ; n = 5) or type 2 diabetic patients ( C ; n = 11) before and after treatment with metformin for 2 months. A : MDMs were primed with LPS (10 ng/mL) for 4 h in the presence of a high glucose concentration (15 mmol/L) and then treated with compound C (Comp C; 5, 10, or 25 μmol/L) and stimulated with ATP (1 mmol/L for 1 h). Data are expressed as means ± SEM of five independent experiments. B : MDMs were transduced with nonspecific control shRNA lentiviral particles (shNS) or lentiviral shRNA specific for AMPK (shAMPK). Then, the cells were primed with LPS (10 ng/mL) for 4 h in the presence of a high glucose concentration (15 mmol/L) and treated with ATP or MSU (100 μg/mL for 6 h). Data are expressed as means ± SEM of three independent experiments. Representative images of semiquantitative RT-PCR gels run to assess transduction efficiency (top). C : The cells were primed with LPS (10 ng/mL) for 4 h in the presence of autologous sera and then treated with ATP or MSU. A and B : ELISA of IL-1β and IL-18 levels. C : Western blotting analysis of p-AMPKα protein levels. The intensity of each band for each protein was quantified and normalized to the housekeeping protein β-actin ( C , right). Data are expressed as means ± SEM. *** P

    Journal: Diabetes

    Article Title: Upregulated NLRP3 Inflammasome Activation in Patients With Type 2 Diabetes

    doi: 10.2337/db12-0420

    Figure Lengend Snippet: AMPK pathway activation inhibits the induction of IL-1β and IL-18 production by various inflammasome stimuli in LPS-primed MDMs. Primary MDMs were isolated from healthy controls ( A and B ; n = 5) or type 2 diabetic patients ( C ; n = 11) before and after treatment with metformin for 2 months. A : MDMs were primed with LPS (10 ng/mL) for 4 h in the presence of a high glucose concentration (15 mmol/L) and then treated with compound C (Comp C; 5, 10, or 25 μmol/L) and stimulated with ATP (1 mmol/L for 1 h). Data are expressed as means ± SEM of five independent experiments. B : MDMs were transduced with nonspecific control shRNA lentiviral particles (shNS) or lentiviral shRNA specific for AMPK (shAMPK). Then, the cells were primed with LPS (10 ng/mL) for 4 h in the presence of a high glucose concentration (15 mmol/L) and treated with ATP or MSU (100 μg/mL for 6 h). Data are expressed as means ± SEM of three independent experiments. Representative images of semiquantitative RT-PCR gels run to assess transduction efficiency (top). C : The cells were primed with LPS (10 ng/mL) for 4 h in the presence of autologous sera and then treated with ATP or MSU. A and B : ELISA of IL-1β and IL-18 levels. C : Western blotting analysis of p-AMPKα protein levels. The intensity of each band for each protein was quantified and normalized to the housekeeping protein β-actin ( C , right). Data are expressed as means ± SEM. *** P

    Article Snippet: To detect IL-1β and IL-18 production, enzyme-linked immunosorbent assays (ELISAs) were performed for IL-1β (BD Pharmingen, San Diego, CA) and IL-18 (MBL International, Woburn, MA), according to the respective manufacturer’s instructions.

    Techniques: Activation Assay, Isolation, Concentration Assay, Transduction, shRNA, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot

    The antidiabetic drug metformin inhibits IL-1β and IL-18 production induced by various inflammasome stimuli in LPS-primed MDMs. Primary MDMs from healthy controls ( n = 5) were primed with LPS (10 ng/mL for 4 h) in the presence of a high glucose concentration (15 mmol/L). They were then treated with metformin (Met) at the indicated doses ( A , B , and D ; 200 and 500 μmol/L for 60 min) or for the indicated periods of time ( C ; 200 μmol/L for 30, 60, or 120 min), and stimulated with ATP ( A – C ; 1 mmol/L for 1 h), MSU ( C and D ; 100 μg/mL for 6 h), or FFAs ( D ; 200 μmol/L for 16 h). A : Quantitative real-time RT-PCR analysis of Il1β and IL8 mRNA levels. B and D : ELISA analysis of IL-1β and IL-18. C : Western blotting analysis of IL-1β protein levels in cell lysates (Cell, pro-IL-1β [31 kDa]) and supernatants (SN; mature IL-1β [17 kDa]). A , B , and D : Data are expressed as means ± SEM of five independent experiments. *** P

    Journal: Diabetes

    Article Title: Upregulated NLRP3 Inflammasome Activation in Patients With Type 2 Diabetes

    doi: 10.2337/db12-0420

    Figure Lengend Snippet: The antidiabetic drug metformin inhibits IL-1β and IL-18 production induced by various inflammasome stimuli in LPS-primed MDMs. Primary MDMs from healthy controls ( n = 5) were primed with LPS (10 ng/mL for 4 h) in the presence of a high glucose concentration (15 mmol/L). They were then treated with metformin (Met) at the indicated doses ( A , B , and D ; 200 and 500 μmol/L for 60 min) or for the indicated periods of time ( C ; 200 μmol/L for 30, 60, or 120 min), and stimulated with ATP ( A – C ; 1 mmol/L for 1 h), MSU ( C and D ; 100 μg/mL for 6 h), or FFAs ( D ; 200 μmol/L for 16 h). A : Quantitative real-time RT-PCR analysis of Il1β and IL8 mRNA levels. B and D : ELISA analysis of IL-1β and IL-18. C : Western blotting analysis of IL-1β protein levels in cell lysates (Cell, pro-IL-1β [31 kDa]) and supernatants (SN; mature IL-1β [17 kDa]). A , B , and D : Data are expressed as means ± SEM of five independent experiments. *** P

    Article Snippet: To detect IL-1β and IL-18 production, enzyme-linked immunosorbent assays (ELISAs) were performed for IL-1β (BD Pharmingen, San Diego, CA) and IL-18 (MBL International, Woburn, MA), according to the respective manufacturer’s instructions.

    Techniques: Concentration Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot

    Upregulated activation of casapse-1, IL-1β maturation, and production of IL-1β and IL-18 in MDMs from patients with type 2 diabetes (T2D) compared with healthy controls (HCs). MDMs were isolated from T2D patients ( n = 47) and HCs ( n = 57) were primed with LPS (10 ng/mL) for 4 h and stimulated with various ligands: ATP (1 mmol/L for 1 h), MSU (100 μg/mL for 6 h), FFAs (200 μmol/L for 16 h), and IAPP (10 μmol/L for 16 h). A : Western blotting analysis to determine caspase-1 (Casp1) and IL-1β protein levels (cell lysates [Cell], Casp1 p45, and pro-IL-1β [31 kDa]; supernatants [SN], cleaved Casp1 p10, and mature IL-1β [17 kDa]). The intensity of each band for each protein was quantified and normalized to the housekeeping gene β-actin ( A , bottom). Data are expressed as means ± SEM. B : ELISA of IL-1β (top) and IL-18 (bottom) levels in culture supernatants. Cells were left untreated (U; left) or treated with the indicated ligands. Results are expressed as the mean of triplicate samples. Data are representative of two independent experiments. ***P

    Journal: Diabetes

    Article Title: Upregulated NLRP3 Inflammasome Activation in Patients With Type 2 Diabetes

    doi: 10.2337/db12-0420

    Figure Lengend Snippet: Upregulated activation of casapse-1, IL-1β maturation, and production of IL-1β and IL-18 in MDMs from patients with type 2 diabetes (T2D) compared with healthy controls (HCs). MDMs were isolated from T2D patients ( n = 47) and HCs ( n = 57) were primed with LPS (10 ng/mL) for 4 h and stimulated with various ligands: ATP (1 mmol/L for 1 h), MSU (100 μg/mL for 6 h), FFAs (200 μmol/L for 16 h), and IAPP (10 μmol/L for 16 h). A : Western blotting analysis to determine caspase-1 (Casp1) and IL-1β protein levels (cell lysates [Cell], Casp1 p45, and pro-IL-1β [31 kDa]; supernatants [SN], cleaved Casp1 p10, and mature IL-1β [17 kDa]). The intensity of each band for each protein was quantified and normalized to the housekeeping gene β-actin ( A , bottom). Data are expressed as means ± SEM. B : ELISA of IL-1β (top) and IL-18 (bottom) levels in culture supernatants. Cells were left untreated (U; left) or treated with the indicated ligands. Results are expressed as the mean of triplicate samples. Data are representative of two independent experiments. ***P

    Article Snippet: To detect IL-1β and IL-18 production, enzyme-linked immunosorbent assays (ELISAs) were performed for IL-1β (BD Pharmingen, San Diego, CA) and IL-18 (MBL International, Woburn, MA), according to the respective manufacturer’s instructions.

    Techniques: Activation Assay, Isolation, Western Blot, Enzyme-linked Immunosorbent Assay

    Expression profiles of NLRP3, ASC, and inflammatory cytokines in MDMs and sera from patients with type 2 diabetes (T2D) and healthy controls (HCs). A , C–E : MDMs were obtained from 47 patients with T2D and 57 HCs. The MDMs were incubated with media containing autologous sera and stimulated with ultrapure LPS ( A , C, and E; 10 ng/mL) and Pam3CSK4 (Pam, E ; 10 ng/mL) for 4 h. B : MDMs were cultured from T2D patients ( n = 10) and HCs ( n = 10). The protein levels of NLRP3 and ASC were measured by Western blot analysis. The intensity of each band for each protein was quantified and normalized relative to the housekeeping gene β-actin ( B , right). D : Sera collected from the peripheral blood of T2D patients ( n = 47) and HCs ( n = 57). The mRNA expression of Il1β , Nlrp3 , and Asc ( A ) and Il6 , Il8 , Tnfα , and Ccl2 ( E ) was analyzed by quantitative real-time RT-PCR. IL-1β and IL-18 production in culture supernatants ( C ) and sera ( D ) were measured by ELISA. Serum cytokine levels were determined in samples pretreated with protease inhibitors (4% volume/volume). A and E : Results are expressed as the mean concentration of triplicate samples. Data are representative of two independent experiments. B – D : Data are expressed as means ± SEM. *P

    Journal: Diabetes

    Article Title: Upregulated NLRP3 Inflammasome Activation in Patients With Type 2 Diabetes

    doi: 10.2337/db12-0420

    Figure Lengend Snippet: Expression profiles of NLRP3, ASC, and inflammatory cytokines in MDMs and sera from patients with type 2 diabetes (T2D) and healthy controls (HCs). A , C–E : MDMs were obtained from 47 patients with T2D and 57 HCs. The MDMs were incubated with media containing autologous sera and stimulated with ultrapure LPS ( A , C, and E; 10 ng/mL) and Pam3CSK4 (Pam, E ; 10 ng/mL) for 4 h. B : MDMs were cultured from T2D patients ( n = 10) and HCs ( n = 10). The protein levels of NLRP3 and ASC were measured by Western blot analysis. The intensity of each band for each protein was quantified and normalized relative to the housekeeping gene β-actin ( B , right). D : Sera collected from the peripheral blood of T2D patients ( n = 47) and HCs ( n = 57). The mRNA expression of Il1β , Nlrp3 , and Asc ( A ) and Il6 , Il8 , Tnfα , and Ccl2 ( E ) was analyzed by quantitative real-time RT-PCR. IL-1β and IL-18 production in culture supernatants ( C ) and sera ( D ) were measured by ELISA. Serum cytokine levels were determined in samples pretreated with protease inhibitors (4% volume/volume). A and E : Results are expressed as the mean concentration of triplicate samples. Data are representative of two independent experiments. B – D : Data are expressed as means ± SEM. *P

    Article Snippet: To detect IL-1β and IL-18 production, enzyme-linked immunosorbent assays (ELISAs) were performed for IL-1β (BD Pharmingen, San Diego, CA) and IL-18 (MBL International, Woburn, MA), according to the respective manufacturer’s instructions.

    Techniques: Expressing, Incubation, Cell Culture, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Concentration Assay

    Upregulated NLRP3 inflammasome activation in patients with type 2 diabetes (T2D) is mediated by mitochondrial ROS. A : PBMCs isolated from T2D patients ( n = 9) and healthy controls (HCs; n = 9) were primed with LPS (10 ng/mL) for 4 h and then stimulated with ATP (1 mmol/L) or HMGB1 (10 ng/mL) for 1 h in the absence or presence of Mito-TEMPO (mit; 200 μmol/L). Then, the cells were stained with MitoSOX, gated for the CD14 + population, and analyzed by flow cytometry. Representative images (left) and quantitative analysis of mean fluorescence intensities (right) are shown ( A , right). Data are expressed as means ± SEM. B : MDMs from T2D patients ( n = 5) and HCs ( n = 5) were primed with LPS (10 ng/mL, for 4 h), and then stimulated with ATP (1 mmol/L) or HMGB1 (10 ng/mL) for 1 h in the absence or presence of Mito-TEMPO (mit; 200 μmol/L). C : MDMs from T2D patients ( n = 5) and HCs ( n = 5) were transduced with nonspecific control shRNA lentiviral particles (shNS) or lentiviral shRNA specific for NLRP3 (shNLRP3) or ASC (shASC), primed with LPS, and stimulated with ATP (1 mmol/L for 1 h) or MSU (100 μg/mL for 6 h). ELISA analysis of IL-1β ( B and C ) and IL-18 ( B ). Data are expressed as means ± SEM. C : Representative images of gels run to assess transduction efficiency by semiquantitative RT-PCR analysis (top). ***P

    Journal: Diabetes

    Article Title: Upregulated NLRP3 Inflammasome Activation in Patients With Type 2 Diabetes

    doi: 10.2337/db12-0420

    Figure Lengend Snippet: Upregulated NLRP3 inflammasome activation in patients with type 2 diabetes (T2D) is mediated by mitochondrial ROS. A : PBMCs isolated from T2D patients ( n = 9) and healthy controls (HCs; n = 9) were primed with LPS (10 ng/mL) for 4 h and then stimulated with ATP (1 mmol/L) or HMGB1 (10 ng/mL) for 1 h in the absence or presence of Mito-TEMPO (mit; 200 μmol/L). Then, the cells were stained with MitoSOX, gated for the CD14 + population, and analyzed by flow cytometry. Representative images (left) and quantitative analysis of mean fluorescence intensities (right) are shown ( A , right). Data are expressed as means ± SEM. B : MDMs from T2D patients ( n = 5) and HCs ( n = 5) were primed with LPS (10 ng/mL, for 4 h), and then stimulated with ATP (1 mmol/L) or HMGB1 (10 ng/mL) for 1 h in the absence or presence of Mito-TEMPO (mit; 200 μmol/L). C : MDMs from T2D patients ( n = 5) and HCs ( n = 5) were transduced with nonspecific control shRNA lentiviral particles (shNS) or lentiviral shRNA specific for NLRP3 (shNLRP3) or ASC (shASC), primed with LPS, and stimulated with ATP (1 mmol/L for 1 h) or MSU (100 μg/mL for 6 h). ELISA analysis of IL-1β ( B and C ) and IL-18 ( B ). Data are expressed as means ± SEM. C : Representative images of gels run to assess transduction efficiency by semiquantitative RT-PCR analysis (top). ***P

    Article Snippet: To detect IL-1β and IL-18 production, enzyme-linked immunosorbent assays (ELISAs) were performed for IL-1β (BD Pharmingen, San Diego, CA) and IL-18 (MBL International, Woburn, MA), according to the respective manufacturer’s instructions.

    Techniques: Activation Assay, Isolation, Staining, Flow Cytometry, Cytometry, Fluorescence, Transduction, shRNA, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

    Metformin treatment inhibits the secretion of mature IL-1β and caspase-1 activation in MDMs from patients with type 2 diabetes (T2D). MDMs were isolated from T2D patients ( n = 11) before (Before) and after (After) treatment with metformin for 2 months. MDMs were primed with LPS (10 ng/mL) for 4 h, and then stimulated with various ligands: ATP (1 mmol/L for 1 h), MSU (100 μg/mL for 6 h), and FFAs (200 μmol/L for 16 h). A : Western blotting analysis to determine caspase-1 (Casp1) and IL-1β protein levels (cell lysates [Cell], Casp1 p45, and pro-IL-1β [31 kDa]; supernatants [SN], cleaved Casp1 p10, and mature IL-1β [17 kDa]) ( A , right). The intensity of each band for each protein was quantified and normalized to the housekeeping gene β-actin. Data are expressed as means ± SEM. ***P

    Journal: Diabetes

    Article Title: Upregulated NLRP3 Inflammasome Activation in Patients With Type 2 Diabetes

    doi: 10.2337/db12-0420

    Figure Lengend Snippet: Metformin treatment inhibits the secretion of mature IL-1β and caspase-1 activation in MDMs from patients with type 2 diabetes (T2D). MDMs were isolated from T2D patients ( n = 11) before (Before) and after (After) treatment with metformin for 2 months. MDMs were primed with LPS (10 ng/mL) for 4 h, and then stimulated with various ligands: ATP (1 mmol/L for 1 h), MSU (100 μg/mL for 6 h), and FFAs (200 μmol/L for 16 h). A : Western blotting analysis to determine caspase-1 (Casp1) and IL-1β protein levels (cell lysates [Cell], Casp1 p45, and pro-IL-1β [31 kDa]; supernatants [SN], cleaved Casp1 p10, and mature IL-1β [17 kDa]) ( A , right). The intensity of each band for each protein was quantified and normalized to the housekeeping gene β-actin. Data are expressed as means ± SEM. ***P

    Article Snippet: To detect IL-1β and IL-18 production, enzyme-linked immunosorbent assays (ELISAs) were performed for IL-1β (BD Pharmingen, San Diego, CA) and IL-18 (MBL International, Woburn, MA), according to the respective manufacturer’s instructions.

    Techniques: Activation Assay, Isolation, Western Blot

    High glucose treatment increases the secretion levels of IL-1β, IL-6 and TNF-α in RPECs. (A-C) Expression levels of (A) IL-1β, (B) IL-6 and (C) TNF-α in the culture medium of cells treated with either 30 mmol/l glucose or 24.4 mmol/l mannitol for 12 h were measured by ELISA. (D and E) Expression levels of p-AKT, total AKT, p-mTOR and total mTOR in cells treated with either 30 mmol/l glucose or 24.4 mmol/l mannitol for 12 h as measured by western blot analysis. Data are presented as the mean ± standard error of the mean; n=3; **P

    Journal: Molecular Medicine Reports

    Article Title: Curcumin inhibits high glucose-induced inflammatory injury in human retinal pigment epithelial cells through the ROS-PI3K/AKT/mTOR signaling pathway

    doi: 10.3892/mmr.2018.9749

    Figure Lengend Snippet: High glucose treatment increases the secretion levels of IL-1β, IL-6 and TNF-α in RPECs. (A-C) Expression levels of (A) IL-1β, (B) IL-6 and (C) TNF-α in the culture medium of cells treated with either 30 mmol/l glucose or 24.4 mmol/l mannitol for 12 h were measured by ELISA. (D and E) Expression levels of p-AKT, total AKT, p-mTOR and total mTOR in cells treated with either 30 mmol/l glucose or 24.4 mmol/l mannitol for 12 h as measured by western blot analysis. Data are presented as the mean ± standard error of the mean; n=3; **P

    Article Snippet: Following incubation, the medium was collected and TNF-α (catalog no. ab181421), IL-1β (catalog no. ab100562) and IL-6 (catalog no. ab46027) content were measured by ELISA kits (Abcam, Cambridge, UK), according to the manufacturer's protocols.

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

    CUR treatment reduces the high glucose-induced secretion of IL-1β, IL-6 and TNF-α via the ROS/PI3K/AKT/mTOR signaling pathway in RPECs. (A-C) Expression levels of (A) IL-6, (B) IL-1β and (C) TNF-α in cells treated with 30 mmol/l glucose, 10 µmol/l CUR or their combination were measured by ELISA. (D) ROS formation in cells treated with high glucose, 10 µmol/l CUR or their combination for 12 h was measured by 2′,7′-dichlorodihydrofluororescein diacetate staining (green); nuclei were stained with DAPI (blue); magnification, ×600. (E and F) Expression levels of (E) p-AKT and AKT, and (F) p-mTOR and mTOR in cells treated with 30 mmol/l glucose, 10 µmol/l CUR or their combination were measured by western blot analysis. **P

    Journal: Molecular Medicine Reports

    Article Title: Curcumin inhibits high glucose-induced inflammatory injury in human retinal pigment epithelial cells through the ROS-PI3K/AKT/mTOR signaling pathway

    doi: 10.3892/mmr.2018.9749

    Figure Lengend Snippet: CUR treatment reduces the high glucose-induced secretion of IL-1β, IL-6 and TNF-α via the ROS/PI3K/AKT/mTOR signaling pathway in RPECs. (A-C) Expression levels of (A) IL-6, (B) IL-1β and (C) TNF-α in cells treated with 30 mmol/l glucose, 10 µmol/l CUR or their combination were measured by ELISA. (D) ROS formation in cells treated with high glucose, 10 µmol/l CUR or their combination for 12 h was measured by 2′,7′-dichlorodihydrofluororescein diacetate staining (green); nuclei were stained with DAPI (blue); magnification, ×600. (E and F) Expression levels of (E) p-AKT and AKT, and (F) p-mTOR and mTOR in cells treated with 30 mmol/l glucose, 10 µmol/l CUR or their combination were measured by western blot analysis. **P

    Article Snippet: Following incubation, the medium was collected and TNF-α (catalog no. ab181421), IL-1β (catalog no. ab100562) and IL-6 (catalog no. ab46027) content were measured by ELISA kits (Abcam, Cambridge, UK), according to the manufacturer's protocols.

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

    HG treatment increases cytokine secretion RPECs via the ROS/PI3K/AKT/mTOR signaling pathway. (A) ROS formation in cells treated with high glucose or mannitol for 12 h was measured by 2′,7′-dichlorodihydrofluororescein diacetate staining (green); nuclei were stained with DAPI (blue); magnification, ×600. (B and C) Expression levels of (B) p-AKT and total AKT, and (C) p-mTOR and total mTOR in cells treated with 30 mmol/l glucose and 1 mmol/l NAC, either alone or in combination, were measured by western blot analysis. (D-F) Expression levels of (D) IL-1β, (E) IL-6 and (F) TNF-α in the culture medium of cells treated with 30 mmol/l glucose alone or in combination with 1 mmol/l NAC, 1 µmol/l LY294002 or 10 µmol/l rapamycin were measured by ELISA. Data are presented as the mean ± standard error of the mean; **P

    Journal: Molecular Medicine Reports

    Article Title: Curcumin inhibits high glucose-induced inflammatory injury in human retinal pigment epithelial cells through the ROS-PI3K/AKT/mTOR signaling pathway

    doi: 10.3892/mmr.2018.9749

    Figure Lengend Snippet: HG treatment increases cytokine secretion RPECs via the ROS/PI3K/AKT/mTOR signaling pathway. (A) ROS formation in cells treated with high glucose or mannitol for 12 h was measured by 2′,7′-dichlorodihydrofluororescein diacetate staining (green); nuclei were stained with DAPI (blue); magnification, ×600. (B and C) Expression levels of (B) p-AKT and total AKT, and (C) p-mTOR and total mTOR in cells treated with 30 mmol/l glucose and 1 mmol/l NAC, either alone or in combination, were measured by western blot analysis. (D-F) Expression levels of (D) IL-1β, (E) IL-6 and (F) TNF-α in the culture medium of cells treated with 30 mmol/l glucose alone or in combination with 1 mmol/l NAC, 1 µmol/l LY294002 or 10 µmol/l rapamycin were measured by ELISA. Data are presented as the mean ± standard error of the mean; **P

    Article Snippet: Following incubation, the medium was collected and TNF-α (catalog no. ab181421), IL-1β (catalog no. ab100562) and IL-6 (catalog no. ab46027) content were measured by ELISA kits (Abcam, Cambridge, UK), according to the manufacturer's protocols.

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

    Expression of NLRP3 inflammasome components and the mature form of IL-1β. ( A ) LAD2 cells (1 × 10 6 cells per well) were seeded in a 12-well culture plate and were stimulated with SP (1 μΜ), IL-33 (30 ng/mL), or their combination for 24 h. Cell lysates were collected after 24 h, and protein levels of the NLRP3 inflammasome components (NLRP3, ASC, caspase-1), pro-IL-1β, and active IL-1β (p17) were measured by Western blot, using β-actin as loading control (shown in a representative gel of n = 3). SP and IL-33 increase caspase-1 gene expression. ( B ) LAD2 cells (1 × 10 6 cells per well), ( n = 3, ** P

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

    Article Title: Substance P and IL-33 administered together stimulate a marked secretion of IL-1β from human mast cells, inhibited by methoxyluteolin

    doi: 10.1073/pnas.1810133115

    Figure Lengend Snippet: Expression of NLRP3 inflammasome components and the mature form of IL-1β. ( A ) LAD2 cells (1 × 10 6 cells per well) were seeded in a 12-well culture plate and were stimulated with SP (1 μΜ), IL-33 (30 ng/mL), or their combination for 24 h. Cell lysates were collected after 24 h, and protein levels of the NLRP3 inflammasome components (NLRP3, ASC, caspase-1), pro-IL-1β, and active IL-1β (p17) were measured by Western blot, using β-actin as loading control (shown in a representative gel of n = 3). SP and IL-33 increase caspase-1 gene expression. ( B ) LAD2 cells (1 × 10 6 cells per well), ( n = 3, ** P

    Article Snippet: The membranes were probed with the following primary antibodies at 1:1,000 dilutions: NLRP3, pro-IL-1β, caspase-1, β-actin (Cell Signaling Technology), ASC, and cleaved IL-1β (Santa Cruz Biotechnology).

    Techniques: Expressing, Western Blot

    Methoxyluteolin inhibits the secretion of IL-1β. LAD2 cells (1 × 10 5 cells per well) were seeded in a 96-well culture plate, were pretreated with methoxyluteolin (MET, 1–100 μM) for 2 h, and then were stimulated with the combination of SP (1 μM) and IL-33 (30 ng/mL) for 24 h. LAD2 cells were also pretreated with AC-YVAD-CMK (YVAD 25–100 μM) or glybenclamide (GLY 25–100 μM). Control cells were treated with 0.1% DMSO, the highest concentration corresponding to that of 100 μM methoxyluteolin. Collected lysates ( A ) and supernatant fluids ( B and C ) were assayed for IL-1β using ELISA ( n = 3, * P

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

    Article Title: Substance P and IL-33 administered together stimulate a marked secretion of IL-1β from human mast cells, inhibited by methoxyluteolin

    doi: 10.1073/pnas.1810133115

    Figure Lengend Snippet: Methoxyluteolin inhibits the secretion of IL-1β. LAD2 cells (1 × 10 5 cells per well) were seeded in a 96-well culture plate, were pretreated with methoxyluteolin (MET, 1–100 μM) for 2 h, and then were stimulated with the combination of SP (1 μM) and IL-33 (30 ng/mL) for 24 h. LAD2 cells were also pretreated with AC-YVAD-CMK (YVAD 25–100 μM) or glybenclamide (GLY 25–100 μM). Control cells were treated with 0.1% DMSO, the highest concentration corresponding to that of 100 μM methoxyluteolin. Collected lysates ( A ) and supernatant fluids ( B and C ) were assayed for IL-1β using ELISA ( n = 3, * P

    Article Snippet: The membranes were probed with the following primary antibodies at 1:1,000 dilutions: NLRP3, pro-IL-1β, caspase-1, β-actin (Cell Signaling Technology), ASC, and cleaved IL-1β (Santa Cruz Biotechnology).

    Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

    Selection of the optimal doses to study IL-1β secretion stimulated by SP and IL-33 when administered in combination. ( A and B ) LAD2 cells (1 × 10 5 cells per well) were stimulated with SP (0.01–1 μΜ) ( n = 3, * P

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

    Article Title: Substance P and IL-33 administered together stimulate a marked secretion of IL-1β from human mast cells, inhibited by methoxyluteolin

    doi: 10.1073/pnas.1810133115

    Figure Lengend Snippet: Selection of the optimal doses to study IL-1β secretion stimulated by SP and IL-33 when administered in combination. ( A and B ) LAD2 cells (1 × 10 5 cells per well) were stimulated with SP (0.01–1 μΜ) ( n = 3, * P

    Article Snippet: The membranes were probed with the following primary antibodies at 1:1,000 dilutions: NLRP3, pro-IL-1β, caspase-1, β-actin (Cell Signaling Technology), ASC, and cleaved IL-1β (Santa Cruz Biotechnology).

    Techniques: Selection

    Diagrammatic representation of the stimulatory effect of SP and IL-33 on IL-1β synthesis and secretion and the proposed point of inhibition of methoxyluteolin. Our evidence indicates that (1) SP and IL-33 activate their respective receptors and stimulate synthesis of procaspase-1 and pro-IL-1β, possibly via NF-κB, activation, which is inhibited by methoxyluteolin. (2) Procaspase-1 and pro-IL-1β are released from the nucleus, a process that could also be inhibited by methoxyluteolin. (3) In the cytoplasm, caspase-1, which is already active, converts pro-IL-1β to active IL-1β. (4) Some of the procaspase-1 is converted to caspase-1, but this may be a minor contribution since the NLRP3 inflammasome does not seem to be involved. (5) IL-1β and active caspase-1 are then secreted extracellularly, a process that is also inhibited by methoxyluteolin. Orange boxes and ovals indicate facts supported by our findings. Open boxes and ovals indicate pathways not supported by our data. The (T) attached to methoxyluteolin indicates possible points of inhibition.

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

    Article Title: Substance P and IL-33 administered together stimulate a marked secretion of IL-1β from human mast cells, inhibited by methoxyluteolin

    doi: 10.1073/pnas.1810133115

    Figure Lengend Snippet: Diagrammatic representation of the stimulatory effect of SP and IL-33 on IL-1β synthesis and secretion and the proposed point of inhibition of methoxyluteolin. Our evidence indicates that (1) SP and IL-33 activate their respective receptors and stimulate synthesis of procaspase-1 and pro-IL-1β, possibly via NF-κB, activation, which is inhibited by methoxyluteolin. (2) Procaspase-1 and pro-IL-1β are released from the nucleus, a process that could also be inhibited by methoxyluteolin. (3) In the cytoplasm, caspase-1, which is already active, converts pro-IL-1β to active IL-1β. (4) Some of the procaspase-1 is converted to caspase-1, but this may be a minor contribution since the NLRP3 inflammasome does not seem to be involved. (5) IL-1β and active caspase-1 are then secreted extracellularly, a process that is also inhibited by methoxyluteolin. Orange boxes and ovals indicate facts supported by our findings. Open boxes and ovals indicate pathways not supported by our data. The (T) attached to methoxyluteolin indicates possible points of inhibition.

    Article Snippet: The membranes were probed with the following primary antibodies at 1:1,000 dilutions: NLRP3, pro-IL-1β, caspase-1, β-actin (Cell Signaling Technology), ASC, and cleaved IL-1β (Santa Cruz Biotechnology).

    Techniques: Inhibition, Activation Assay

    ( A ) SP and IL-33 stimulate IL-1β secretion. LAD2 cells (1 × 10 5 cells per well) were seeded in a 96-well culture plate and stimulated with LPS (100 ng/mL), ATP (5 μM), SP (1 μΜ), IL-33 (30 ng/mL), nigericin (10 µM), TNF (50 ng/mL), IFN-γ (100 U), IgE (1 µg/mL)/anti-IgE (5 µg/mL), or their combinations as shown for 24 h. Control cells were treated with culture medium only ( n = 3, ** P

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

    Article Title: Substance P and IL-33 administered together stimulate a marked secretion of IL-1β from human mast cells, inhibited by methoxyluteolin

    doi: 10.1073/pnas.1810133115

    Figure Lengend Snippet: ( A ) SP and IL-33 stimulate IL-1β secretion. LAD2 cells (1 × 10 5 cells per well) were seeded in a 96-well culture plate and stimulated with LPS (100 ng/mL), ATP (5 μM), SP (1 μΜ), IL-33 (30 ng/mL), nigericin (10 µM), TNF (50 ng/mL), IFN-γ (100 U), IgE (1 µg/mL)/anti-IgE (5 µg/mL), or their combinations as shown for 24 h. Control cells were treated with culture medium only ( n = 3, ** P

    Article Snippet: The membranes were probed with the following primary antibodies at 1:1,000 dilutions: NLRP3, pro-IL-1β, caspase-1, β-actin (Cell Signaling Technology), ASC, and cleaved IL-1β (Santa Cruz Biotechnology).

    Techniques:

    NK-1 receptor antagonists inhibit IL-1β secretion. ( A ) LAD2 cells were pretreated with NK-1R antagonists L-733,060 (10 μM) ( n = 3, ** P

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

    Article Title: Substance P and IL-33 administered together stimulate a marked secretion of IL-1β from human mast cells, inhibited by methoxyluteolin

    doi: 10.1073/pnas.1810133115

    Figure Lengend Snippet: NK-1 receptor antagonists inhibit IL-1β secretion. ( A ) LAD2 cells were pretreated with NK-1R antagonists L-733,060 (10 μM) ( n = 3, ** P

    Article Snippet: The membranes were probed with the following primary antibodies at 1:1,000 dilutions: NLRP3, pro-IL-1β, caspase-1, β-actin (Cell Signaling Technology), ASC, and cleaved IL-1β (Santa Cruz Biotechnology).

    Techniques:

    SP and IL-33 markedly enhance IL-1β gene expression and secretion. LAD2 cells (1 × 10 6 cells per well) ( A ) and hCBMCs (0.3 × 10 6 cell per well) ( B ) were seeded in a 12-well culture plate and were stimulated with SP (1 μM), IL-33 (30 ng/mL), or their combination for 6 h. IL-1β mRNA expression levels were measured by qRT-PCR and were normalized to human GAPDH endogenous control ( n = 3, **** P

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

    Article Title: Substance P and IL-33 administered together stimulate a marked secretion of IL-1β from human mast cells, inhibited by methoxyluteolin

    doi: 10.1073/pnas.1810133115

    Figure Lengend Snippet: SP and IL-33 markedly enhance IL-1β gene expression and secretion. LAD2 cells (1 × 10 6 cells per well) ( A ) and hCBMCs (0.3 × 10 6 cell per well) ( B ) were seeded in a 12-well culture plate and were stimulated with SP (1 μM), IL-33 (30 ng/mL), or their combination for 6 h. IL-1β mRNA expression levels were measured by qRT-PCR and were normalized to human GAPDH endogenous control ( n = 3, **** P

    Article Snippet: The membranes were probed with the following primary antibodies at 1:1,000 dilutions: NLRP3, pro-IL-1β, caspase-1, β-actin (Cell Signaling Technology), ASC, and cleaved IL-1β (Santa Cruz Biotechnology).

    Techniques: Expressing, Quantitative RT-PCR

    Methoxyluteolin inhibits gene and protein expression of IL-1β as well as protein expression of procaspase I and pro-IL-1β. LAD2 cells (1 × 10 6 cells per well) were seeded in a 12-well culture plate, were preincubated with methoxyluteolin (MET, 50 μM), AC-YVAD-CMK (YVAD, 50 μM), or glybenclamide (GLY, 50 μM), and then were stimulated with the combination of SP (1 μM) and IL-33 (30 ng/mL) for 6 h. ( A ) Gene expression of IL-1β was measured by qRT-PCR and normalized to human GAPDH endogenous control. ( n = 3, * P

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

    Article Title: Substance P and IL-33 administered together stimulate a marked secretion of IL-1β from human mast cells, inhibited by methoxyluteolin

    doi: 10.1073/pnas.1810133115

    Figure Lengend Snippet: Methoxyluteolin inhibits gene and protein expression of IL-1β as well as protein expression of procaspase I and pro-IL-1β. LAD2 cells (1 × 10 6 cells per well) were seeded in a 12-well culture plate, were preincubated with methoxyluteolin (MET, 50 μM), AC-YVAD-CMK (YVAD, 50 μM), or glybenclamide (GLY, 50 μM), and then were stimulated with the combination of SP (1 μM) and IL-33 (30 ng/mL) for 6 h. ( A ) Gene expression of IL-1β was measured by qRT-PCR and normalized to human GAPDH endogenous control. ( n = 3, * P

    Article Snippet: The membranes were probed with the following primary antibodies at 1:1,000 dilutions: NLRP3, pro-IL-1β, caspase-1, β-actin (Cell Signaling Technology), ASC, and cleaved IL-1β (Santa Cruz Biotechnology).

    Techniques: Expressing, Quantitative RT-PCR

    Recombinant human interleukin-1 receptor antagonist (RhIL-1Ra) ameliorated ConA-induced NOD-like receptor protein 3 (NLRP3) inflammasome activation and pyroptosis. (A) BALB/c mice ( n = 6 for each group) were pretreated with rhIL-1Ra before ConA injection, and samples were extracted at 12 h post ConA treatment. Serum IL-1β and IL-18 were detected by ELISA. (B) Liver homogenates were subjected to caspase-1 activity assay. (C) Western blot of NLRP3, Cleaved caspase-1, and IL-1β in the livers. GAPDH acted as a loading control. Each lane represented a separate animal. Results were obtained from three experiments. Quantification of protein expression were obtained by Image J software. (D) Caspase-1 positive death cells staining were observed by confocal microscopy. FAM-YVAD-FMK (green), propidium iodide (red), Hoechst 33342 (blue). The data were presented as means ± SD (Student’s t -test, * p

    Journal: Frontiers in Immunology

    Article Title: NOD-Like Receptor Protein 3 Inflammasome-Dependent IL-1β Accelerated ConA-Induced Hepatitis

    doi: 10.3389/fimmu.2018.00758

    Figure Lengend Snippet: Recombinant human interleukin-1 receptor antagonist (RhIL-1Ra) ameliorated ConA-induced NOD-like receptor protein 3 (NLRP3) inflammasome activation and pyroptosis. (A) BALB/c mice ( n = 6 for each group) were pretreated with rhIL-1Ra before ConA injection, and samples were extracted at 12 h post ConA treatment. Serum IL-1β and IL-18 were detected by ELISA. (B) Liver homogenates were subjected to caspase-1 activity assay. (C) Western blot of NLRP3, Cleaved caspase-1, and IL-1β in the livers. GAPDH acted as a loading control. Each lane represented a separate animal. Results were obtained from three experiments. Quantification of protein expression were obtained by Image J software. (D) Caspase-1 positive death cells staining were observed by confocal microscopy. FAM-YVAD-FMK (green), propidium iodide (red), Hoechst 33342 (blue). The data were presented as means ± SD (Student’s t -test, * p

    Article Snippet: The primary antibodies used for western blot, including antibodies to NLRP3 (15101), Cleaved caspase-1 (67314), IL-1β (12242), GAPDH (51332), JAK2 (3230), p-JAK2 (Tyr1007/1008) (3776), STAT3 (4904), and p-STAT3 (Tyr705) (91315), were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Recombinant, Activation Assay, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot, Expressing, Software, Staining, Confocal Microscopy

    Administration of ConA-induced inflammatory cells infiltration into liver tissues followed by reactive oxygen species (ROS) generation. ROS contributed to NOD-like receptor protein 3 inflammasome activation and caspase-1 cleavage, which elicited IL-1β production and pyroptosis, thus accelerating liver inflammation and injury.

    Journal: Frontiers in Immunology

    Article Title: NOD-Like Receptor Protein 3 Inflammasome-Dependent IL-1β Accelerated ConA-Induced Hepatitis

    doi: 10.3389/fimmu.2018.00758

    Figure Lengend Snippet: Administration of ConA-induced inflammatory cells infiltration into liver tissues followed by reactive oxygen species (ROS) generation. ROS contributed to NOD-like receptor protein 3 inflammasome activation and caspase-1 cleavage, which elicited IL-1β production and pyroptosis, thus accelerating liver inflammation and injury.

    Article Snippet: The primary antibodies used for western blot, including antibodies to NLRP3 (15101), Cleaved caspase-1 (67314), IL-1β (12242), GAPDH (51332), JAK2 (3230), p-JAK2 (Tyr1007/1008) (3776), STAT3 (4904), and p-STAT3 (Tyr705) (91315), were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Activation Assay

    N -acetyl-cysteine (NAC) attenuated ConA-induced hepatitis via suppressing NOD-like receptor protein 3 (NLRP3) inflammasome activation. (A) BALB/c mice ( n = 6 for each group) were pretreated with NAC and subsequent exposed to ConA challenge for 12 h, then sacrificed for collecting samples. Representative hematoxylin and eosin images of liver tissues were shown (100×, magnification: 400×). (B) Serum ALT and aspartate transaminase (AST) activity were assessed. (C) Western blot was performed to detect NLRP3 inflammasome-associated protein NLRP3, Cleaved caspase-1 and IL-1β in the livers. Each lane represented a separate animal. Results shown represented three independent experiments. Quantification of protein expression with Image J software. (D) The concentrations of IL-1β and IL-18 in serum were measured by ELISA. (E) Caspase-1 enzymatic activity in liver homogenates. The data were presented as means ± SD (Student’s t -test, * p

    Journal: Frontiers in Immunology

    Article Title: NOD-Like Receptor Protein 3 Inflammasome-Dependent IL-1β Accelerated ConA-Induced Hepatitis

    doi: 10.3389/fimmu.2018.00758

    Figure Lengend Snippet: N -acetyl-cysteine (NAC) attenuated ConA-induced hepatitis via suppressing NOD-like receptor protein 3 (NLRP3) inflammasome activation. (A) BALB/c mice ( n = 6 for each group) were pretreated with NAC and subsequent exposed to ConA challenge for 12 h, then sacrificed for collecting samples. Representative hematoxylin and eosin images of liver tissues were shown (100×, magnification: 400×). (B) Serum ALT and aspartate transaminase (AST) activity were assessed. (C) Western blot was performed to detect NLRP3 inflammasome-associated protein NLRP3, Cleaved caspase-1 and IL-1β in the livers. Each lane represented a separate animal. Results shown represented three independent experiments. Quantification of protein expression with Image J software. (D) The concentrations of IL-1β and IL-18 in serum were measured by ELISA. (E) Caspase-1 enzymatic activity in liver homogenates. The data were presented as means ± SD (Student’s t -test, * p

    Article Snippet: The primary antibodies used for western blot, including antibodies to NLRP3 (15101), Cleaved caspase-1 (67314), IL-1β (12242), GAPDH (51332), JAK2 (3230), p-JAK2 (Tyr1007/1008) (3776), STAT3 (4904), and p-STAT3 (Tyr705) (91315), were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Activation Assay, Mouse Assay, AST Assay, Activity Assay, Western Blot, Expressing, Software, Enzyme-linked Immunosorbent Assay

    NOD-like receptor protein 3 (NLRP3) deficiency alleviated ConA-induced liver injury. (A) NLRP3 −/− mice and WT mice ( n = 6 for each group) were treated with ConA (20 mg/kg), and corresponding samples were isolated from mice at 0 and 12 h post ConA challenge. Hepatocellular damage was detected by hematoxylin and eosin (H E) and representative images of liver tissues were shown (100×, magnification: 400×). (B) Serum ALT and aspartate transaminase levels in WT and mice were detected. (C) The protein levels of NLRP3, cleaved caspase-1 and IL-1β in the livers of mice with autoimmune hepatitis (AIH) were analyzed by western blot. GAPDH was used as a loading control. Each lane represented a separate animal. Results shown were obtained from three experiments. Quantification was presented in bar graphs. (D) Pyroptosis was detected by serum lactate dehydrogenase (LDH). (E) Representative fluorescence images of liver tissues co-stained with FAM-YVAD-FMK and propidium iodide (PI). FAM-YVAD-FMK (green), PI (red), Hoechst 33342 (blue). The data were presented as means ± SD (Student’s t -test, * p

    Journal: Frontiers in Immunology

    Article Title: NOD-Like Receptor Protein 3 Inflammasome-Dependent IL-1β Accelerated ConA-Induced Hepatitis

    doi: 10.3389/fimmu.2018.00758

    Figure Lengend Snippet: NOD-like receptor protein 3 (NLRP3) deficiency alleviated ConA-induced liver injury. (A) NLRP3 −/− mice and WT mice ( n = 6 for each group) were treated with ConA (20 mg/kg), and corresponding samples were isolated from mice at 0 and 12 h post ConA challenge. Hepatocellular damage was detected by hematoxylin and eosin (H E) and representative images of liver tissues were shown (100×, magnification: 400×). (B) Serum ALT and aspartate transaminase levels in WT and mice were detected. (C) The protein levels of NLRP3, cleaved caspase-1 and IL-1β in the livers of mice with autoimmune hepatitis (AIH) were analyzed by western blot. GAPDH was used as a loading control. Each lane represented a separate animal. Results shown were obtained from three experiments. Quantification was presented in bar graphs. (D) Pyroptosis was detected by serum lactate dehydrogenase (LDH). (E) Representative fluorescence images of liver tissues co-stained with FAM-YVAD-FMK and propidium iodide (PI). FAM-YVAD-FMK (green), PI (red), Hoechst 33342 (blue). The data were presented as means ± SD (Student’s t -test, * p

    Article Snippet: The primary antibodies used for western blot, including antibodies to NLRP3 (15101), Cleaved caspase-1 (67314), IL-1β (12242), GAPDH (51332), JAK2 (3230), p-JAK2 (Tyr1007/1008) (3776), STAT3 (4904), and p-STAT3 (Tyr705) (91315), were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Mouse Assay, Isolation, Western Blot, Fluorescence, Staining

    Caspase-1 deficiency suppressed ConA-induced hepatitis. (A) Caspase-1 −/− mice and WT mice ( n = 6 for each group) were subjected to ConA (20 mg/kg) treatment, and samples were collected after exposure to ConA for 12 h. Representative hematoxylin and eosin images of liver tissues were shown (100×, magnification: 400×). (B) Serum ALT and aspartate transaminase (AST) levels were analyzed. (C) Western blot analysis of NOD-like receptor protein 3, Cleaved caspase-1 and IL-1β in the livers. Densitometric quantification were normalized to GAPDH. Each lane represented a separate animal. Data shown represented three independent experiments. (D) Lactate dehydrogenase (LDH) release into serum was measured as signs of pyroptosis. (E) Pyroptosis in the livers was observed by confocal fluorescence microscopy. FAM-YVAD-FMK (green), propidium iodide (red), Hoechst 33342 (blue). The data were presented as means ± SD (Student’s t -test, * p

    Journal: Frontiers in Immunology

    Article Title: NOD-Like Receptor Protein 3 Inflammasome-Dependent IL-1β Accelerated ConA-Induced Hepatitis

    doi: 10.3389/fimmu.2018.00758

    Figure Lengend Snippet: Caspase-1 deficiency suppressed ConA-induced hepatitis. (A) Caspase-1 −/− mice and WT mice ( n = 6 for each group) were subjected to ConA (20 mg/kg) treatment, and samples were collected after exposure to ConA for 12 h. Representative hematoxylin and eosin images of liver tissues were shown (100×, magnification: 400×). (B) Serum ALT and aspartate transaminase (AST) levels were analyzed. (C) Western blot analysis of NOD-like receptor protein 3, Cleaved caspase-1 and IL-1β in the livers. Densitometric quantification were normalized to GAPDH. Each lane represented a separate animal. Data shown represented three independent experiments. (D) Lactate dehydrogenase (LDH) release into serum was measured as signs of pyroptosis. (E) Pyroptosis in the livers was observed by confocal fluorescence microscopy. FAM-YVAD-FMK (green), propidium iodide (red), Hoechst 33342 (blue). The data were presented as means ± SD (Student’s t -test, * p

    Article Snippet: The primary antibodies used for western blot, including antibodies to NLRP3 (15101), Cleaved caspase-1 (67314), IL-1β (12242), GAPDH (51332), JAK2 (3230), p-JAK2 (Tyr1007/1008) (3776), STAT3 (4904), and p-STAT3 (Tyr705) (91315), were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Mouse Assay, AST Assay, Western Blot, Fluorescence, Microscopy

    Blocking IL-1β protected ConA-treated mice from acute hepatitis. (A) BALB/c mice ( n = 6 for each group) were pretreated with recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) and subsequently exposed to ConA challenge for 12 h, then sacrificed for collecting samples. Representative H E images of liver tissues were shown (100×, magnification: 400×). (B) Serum ALT and aspartate transaminase (AST) activity were assessed. (C) Serum TNF-α was analyzed by enzyme-linked immune sorbent assay (ELISA). (D) Representative immunohistochemical images of CD68 or CD11b staining in the liver tissue of mice. Quantification of the number of CD11b-positive cells and CD68-positive cells were obtained in four visual fields (×100) in each group. (E) Level of IL-17 in the serum and livers were assessed by ELISA. (F) Primary splenocytes were treated with 10 µg/ml rhIL-1Ra in the presence of 1 µg/ml ConA. The concentration of IL-17 in the supernatants was measured by ELISA. (G) Western blot was performed to detect JAK2, p-JAK2 (Tyr1007/1008), STAT3, and p-STAT3 (Tyr705) in the livers. Each lane represented a separate animal. The blots were representative of three experiments. Quantification of protein expression with Image J software. The data were presented as means ± SD (Student’s t -test, * p

    Journal: Frontiers in Immunology

    Article Title: NOD-Like Receptor Protein 3 Inflammasome-Dependent IL-1β Accelerated ConA-Induced Hepatitis

    doi: 10.3389/fimmu.2018.00758

    Figure Lengend Snippet: Blocking IL-1β protected ConA-treated mice from acute hepatitis. (A) BALB/c mice ( n = 6 for each group) were pretreated with recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) and subsequently exposed to ConA challenge for 12 h, then sacrificed for collecting samples. Representative H E images of liver tissues were shown (100×, magnification: 400×). (B) Serum ALT and aspartate transaminase (AST) activity were assessed. (C) Serum TNF-α was analyzed by enzyme-linked immune sorbent assay (ELISA). (D) Representative immunohistochemical images of CD68 or CD11b staining in the liver tissue of mice. Quantification of the number of CD11b-positive cells and CD68-positive cells were obtained in four visual fields (×100) in each group. (E) Level of IL-17 in the serum and livers were assessed by ELISA. (F) Primary splenocytes were treated with 10 µg/ml rhIL-1Ra in the presence of 1 µg/ml ConA. The concentration of IL-17 in the supernatants was measured by ELISA. (G) Western blot was performed to detect JAK2, p-JAK2 (Tyr1007/1008), STAT3, and p-STAT3 (Tyr705) in the livers. Each lane represented a separate animal. The blots were representative of three experiments. Quantification of protein expression with Image J software. The data were presented as means ± SD (Student’s t -test, * p

    Article Snippet: The primary antibodies used for western blot, including antibodies to NLRP3 (15101), Cleaved caspase-1 (67314), IL-1β (12242), GAPDH (51332), JAK2 (3230), p-JAK2 (Tyr1007/1008) (3776), STAT3 (4904), and p-STAT3 (Tyr705) (91315), were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Blocking Assay, Mouse Assay, Recombinant, AST Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Concentration Assay, Western Blot, Expressing, Software

    NOD-like receptor protein 3 (NLRP3) inflammasome activation and IL-1β production in ConA-induced hepatitis. (A) BALB/c mice ( n = 6 for each group) were intravenous administrated with ConA (20 mg/kg), and sera and liver tissues were obtained following ConA injection at 0, 3, 6, and 12 h. The expression of NLRP3, Cleaved caspase-1, and IL-1β in the livers were detected by western blot analysis. GAPDH was provided as a loading control. Each lane represented a separate animal. The blots were representative of three experiments. Densitometric values of these proteins were quantified using the Image J software. (B) Serum concentrations of IL-1β and IL-18 were analyzed by enzyme-linked immune sorbent assay. (C) Caspase-1 enzymatic activity in the liver homogenates was measured. (D) Lactate dehydrogenase (LDH) release into serum. (E) FAM-YVAD-FMK and propidium iodide (PI) double staining of liver tissues were applied to detect pyroptosis. FAM-YVAD-FMK (green), PI (red), Hoechst 33342 (blue), Scale bars = 50 µm. (F) The protein levels of NLRP3 pathways in the primary hepatocytes and nonparenchymal liver cells isolated from ConA-treated mice were detected by western blot analysis. GAPDH was used as a loading control. Each lane represented a separate animal. Results represented three independent experiments. Quantification was presented. (G) The expression of NLRP3 in primary hepatocytes was displayed by immunofluorescence. NLRP3 (Red), Hoechst 33342 (blue), scale bars = 10 µm. The data were presented as means ± SD (Student’s t -test, * p

    Journal: Frontiers in Immunology

    Article Title: NOD-Like Receptor Protein 3 Inflammasome-Dependent IL-1β Accelerated ConA-Induced Hepatitis

    doi: 10.3389/fimmu.2018.00758

    Figure Lengend Snippet: NOD-like receptor protein 3 (NLRP3) inflammasome activation and IL-1β production in ConA-induced hepatitis. (A) BALB/c mice ( n = 6 for each group) were intravenous administrated with ConA (20 mg/kg), and sera and liver tissues were obtained following ConA injection at 0, 3, 6, and 12 h. The expression of NLRP3, Cleaved caspase-1, and IL-1β in the livers were detected by western blot analysis. GAPDH was provided as a loading control. Each lane represented a separate animal. The blots were representative of three experiments. Densitometric values of these proteins were quantified using the Image J software. (B) Serum concentrations of IL-1β and IL-18 were analyzed by enzyme-linked immune sorbent assay. (C) Caspase-1 enzymatic activity in the liver homogenates was measured. (D) Lactate dehydrogenase (LDH) release into serum. (E) FAM-YVAD-FMK and propidium iodide (PI) double staining of liver tissues were applied to detect pyroptosis. FAM-YVAD-FMK (green), PI (red), Hoechst 33342 (blue), Scale bars = 50 µm. (F) The protein levels of NLRP3 pathways in the primary hepatocytes and nonparenchymal liver cells isolated from ConA-treated mice were detected by western blot analysis. GAPDH was used as a loading control. Each lane represented a separate animal. Results represented three independent experiments. Quantification was presented. (G) The expression of NLRP3 in primary hepatocytes was displayed by immunofluorescence. NLRP3 (Red), Hoechst 33342 (blue), scale bars = 10 µm. The data were presented as means ± SD (Student’s t -test, * p

    Article Snippet: The primary antibodies used for western blot, including antibodies to NLRP3 (15101), Cleaved caspase-1 (67314), IL-1β (12242), GAPDH (51332), JAK2 (3230), p-JAK2 (Tyr1007/1008) (3776), STAT3 (4904), and p-STAT3 (Tyr705) (91315), were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Activation Assay, Mouse Assay, Injection, Expressing, Western Blot, Software, Activity Assay, Double Staining, Isolation, Immunofluorescence

    Full-length cDNA of largemouth bass Micropterus salmoides , IL-1β. The underlined sequence part is IL-1β signature and grey highlighted sequences are ATTTA motifs, bold letters are N -glycosylation sites and double underlined are AATA motif.

    Journal: International Journal of Molecular Sciences

    Article Title: Identification, Molecular Cloning of IL-1β and Its Expression Profile during Nocardia seriolae Infection in Largemouth Bass, Micropterus salmoides

    doi: 10.3390/ijms17101670

    Figure Lengend Snippet: Full-length cDNA of largemouth bass Micropterus salmoides , IL-1β. The underlined sequence part is IL-1β signature and grey highlighted sequences are ATTTA motifs, bold letters are N -glycosylation sites and double underlined are AATA motif.

    Article Snippet: The real-time PCR program was 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 58 °C (IL-1β), 60 °C (TNF-α) for 15 s and 72 °C for 35 s. The reaction (n = 3) was carried out using iQSYBR Green Supermix (Bio-Rad Laboratories).

    Techniques: Sequencing

    Analysis and amplification of putative largemouth bass Micropterus salmoides in head kidney ( a ) and spleen ( b ), IL-1β fragments resolved by RT-PCR and gel electrophoresis.

    Journal: International Journal of Molecular Sciences

    Article Title: Identification, Molecular Cloning of IL-1β and Its Expression Profile during Nocardia seriolae Infection in Largemouth Bass, Micropterus salmoides

    doi: 10.3390/ijms17101670

    Figure Lengend Snippet: Analysis and amplification of putative largemouth bass Micropterus salmoides in head kidney ( a ) and spleen ( b ), IL-1β fragments resolved by RT-PCR and gel electrophoresis.

    Article Snippet: The real-time PCR program was 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 58 °C (IL-1β), 60 °C (TNF-α) for 15 s and 72 °C for 35 s. The reaction (n = 3) was carried out using iQSYBR Green Supermix (Bio-Rad Laboratories).

    Techniques: Amplification, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis

    The amino acid sequences of known teleost IL-1βs were aligned with largemouth bass, Micropterus salmoides , IL-1β and a phylogenetic tree was constructed using the neighbor-joining method.

    Journal: International Journal of Molecular Sciences

    Article Title: Identification, Molecular Cloning of IL-1β and Its Expression Profile during Nocardia seriolae Infection in Largemouth Bass, Micropterus salmoides

    doi: 10.3390/ijms17101670

    Figure Lengend Snippet: The amino acid sequences of known teleost IL-1βs were aligned with largemouth bass, Micropterus salmoides , IL-1β and a phylogenetic tree was constructed using the neighbor-joining method.

    Article Snippet: The real-time PCR program was 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 58 °C (IL-1β), 60 °C (TNF-α) for 15 s and 72 °C for 35 s. The reaction (n = 3) was carried out using iQSYBR Green Supermix (Bio-Rad Laboratories).

    Techniques: Construct

    Amino acid sequence alignment of largemouth bass ( Micropterus salmoides ) IL-1β deduced protein to Mandarin perch (AAV6501.1), Striped trumpeter (ACQ99510.1), European seabass (CAC41006.1), cobia (AFV60967.1), Nile Tilapia (XP_003460673.3), Olive flounder (BAM66988.1), Fugu (NP_001267019.1), Grouper (ABV02593.1), Atlantic salmon (CAC83518.1), Common carp (BAA24538.1), mouse (AAA39276.1) and human (AAA59135.1). Arrow-aspartic region, the IL-1 signature is indicated by a black box; - - - - Sequence gaps, “*” identical residues, “:” conserved substitution, and “.” semi conserved substitution.

    Journal: International Journal of Molecular Sciences

    Article Title: Identification, Molecular Cloning of IL-1β and Its Expression Profile during Nocardia seriolae Infection in Largemouth Bass, Micropterus salmoides

    doi: 10.3390/ijms17101670

    Figure Lengend Snippet: Amino acid sequence alignment of largemouth bass ( Micropterus salmoides ) IL-1β deduced protein to Mandarin perch (AAV6501.1), Striped trumpeter (ACQ99510.1), European seabass (CAC41006.1), cobia (AFV60967.1), Nile Tilapia (XP_003460673.3), Olive flounder (BAM66988.1), Fugu (NP_001267019.1), Grouper (ABV02593.1), Atlantic salmon (CAC83518.1), Common carp (BAA24538.1), mouse (AAA39276.1) and human (AAA59135.1). Arrow-aspartic region, the IL-1 signature is indicated by a black box; - - - - Sequence gaps, “*” identical residues, “:” conserved substitution, and “.” semi conserved substitution.

    Article Snippet: The real-time PCR program was 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 58 °C (IL-1β), 60 °C (TNF-α) for 15 s and 72 °C for 35 s. The reaction (n = 3) was carried out using iQSYBR Green Supermix (Bio-Rad Laboratories).

    Techniques: Sequencing

    Predicted secondary structure of IL-1β protein containing one helix shown as a cylindrical structure.

    Journal: International Journal of Molecular Sciences

    Article Title: Identification, Molecular Cloning of IL-1β and Its Expression Profile during Nocardia seriolae Infection in Largemouth Bass, Micropterus salmoides

    doi: 10.3390/ijms17101670

    Figure Lengend Snippet: Predicted secondary structure of IL-1β protein containing one helix shown as a cylindrical structure.

    Article Snippet: The real-time PCR program was 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 58 °C (IL-1β), 60 °C (TNF-α) for 15 s and 72 °C for 35 s. The reaction (n = 3) was carried out using iQSYBR Green Supermix (Bio-Rad Laboratories).

    Techniques:

    Analysis of largemouth bass, Micropterus salmoides , IL-1β expression in the spleen ( A ) and head kidney ( B ). The results are presented as the mean ± SD ( n = 3) and mean values with different alphabetical letters are significantly different ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Identification, Molecular Cloning of IL-1β and Its Expression Profile during Nocardia seriolae Infection in Largemouth Bass, Micropterus salmoides

    doi: 10.3390/ijms17101670

    Figure Lengend Snippet: Analysis of largemouth bass, Micropterus salmoides , IL-1β expression in the spleen ( A ) and head kidney ( B ). The results are presented as the mean ± SD ( n = 3) and mean values with different alphabetical letters are significantly different ( p

    Article Snippet: The real-time PCR program was 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 58 °C (IL-1β), 60 °C (TNF-α) for 15 s and 72 °C for 35 s. The reaction (n = 3) was carried out using iQSYBR Green Supermix (Bio-Rad Laboratories).

    Techniques: Expressing

    IL-1β domains in various fish species, mouse and human. The IL-1β comparison of structure were predicted by SMART analysis of the amino acid sequence from Mandarin perch (AAV6501.1), Striped trumpeter (ACQ99510.1), European seabass (CAC41006.1), Nile Tilapia (XP_003460673.3), Common carp (BAA24538.1), mouse (AAA39276.1) and human (AAA59135.1).

    Journal: International Journal of Molecular Sciences

    Article Title: Identification, Molecular Cloning of IL-1β and Its Expression Profile during Nocardia seriolae Infection in Largemouth Bass, Micropterus salmoides

    doi: 10.3390/ijms17101670

    Figure Lengend Snippet: IL-1β domains in various fish species, mouse and human. The IL-1β comparison of structure were predicted by SMART analysis of the amino acid sequence from Mandarin perch (AAV6501.1), Striped trumpeter (ACQ99510.1), European seabass (CAC41006.1), Nile Tilapia (XP_003460673.3), Common carp (BAA24538.1), mouse (AAA39276.1) and human (AAA59135.1).

    Article Snippet: The real-time PCR program was 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 58 °C (IL-1β), 60 °C (TNF-α) for 15 s and 72 °C for 35 s. The reaction (n = 3) was carried out using iQSYBR Green Supermix (Bio-Rad Laboratories).

    Techniques: Fluorescence In Situ Hybridization, Sequencing

    Predicted 3D structure of largemouth bass IL-1β protein.

    Journal: International Journal of Molecular Sciences

    Article Title: Identification, Molecular Cloning of IL-1β and Its Expression Profile during Nocardia seriolae Infection in Largemouth Bass, Micropterus salmoides

    doi: 10.3390/ijms17101670

    Figure Lengend Snippet: Predicted 3D structure of largemouth bass IL-1β protein.

    Article Snippet: The real-time PCR program was 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 58 °C (IL-1β), 60 °C (TNF-α) for 15 s and 72 °C for 35 s. The reaction (n = 3) was carried out using iQSYBR Green Supermix (Bio-Rad Laboratories).

    Techniques:

    Exosome‐delivered mesenchymal‐epithelial transition factor (MET) stimulates macrophages to facilitate tumour growth in vivo. A, The effects of the supernatant from macrophage treated with PBS, MET + exosomes, MET − exosomes, MET − exosomes + recombinant IL‐1β protein, MET + exosomes + IL‐1β neutralizing antibody on tumour growth in a xenograft model. Tumour volume in the xenograft models was measured every 3 d after a 10 d inoculation period. B,C, Final tumour weights were determined and photographed. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Helicobacter pylori‐induced exosomal MET educates tumour‐associated macrophages to promote gastric cancer progression, et al. Helicobacter pylori‐induced exosomal MET educates tumour‐associated macrophages to promote gastric cancer progression

    doi: 10.1111/jcmm.13847

    Figure Lengend Snippet: Exosome‐delivered mesenchymal‐epithelial transition factor (MET) stimulates macrophages to facilitate tumour growth in vivo. A, The effects of the supernatant from macrophage treated with PBS, MET + exosomes, MET − exosomes, MET − exosomes + recombinant IL‐1β protein, MET + exosomes + IL‐1β neutralizing antibody on tumour growth in a xenograft model. Tumour volume in the xenograft models was measured every 3 d after a 10 d inoculation period. B,C, Final tumour weights were determined and photographed. * P

    Article Snippet: MGC‐803 cells were incubated in the supernatant of THP‐1‐derived macrophages, which were stimulated with PBS, MET+ exosomes, MET− exosomes, MET− exosomes + IL‐1β (PeproTech, Rocky Hill, NJ, USA, #AF‐200‐01B, 1 ng/mL), or MET+ exosomes + IL‐1β neutralizing antibody (Abcam, #ab9722, 3 μg/mL).

    Techniques: In Vivo, Recombinant

    Improved inhibition of NF-κB pathway by circRNA aptamers. a , Design of circular RNAs that contain NF-κB pathway-inhibiting aptamers. Circular RNAs are designed to contain a F30 3-way junction (black) with Broccoli on one arm, and an NF-κB aptamer on the other arm, while the circularizing stem forms at the base of the F30 3-way junction. This design allows for functional investigation of pathway-inhibiting aptamers while also probing for abundance of this circular RNA using Broccoli fluorescence. b , Tornado efficiently expresses the NF-κB aptamer as a racRNA in HEK293 cells. HEK293 cells were transfected with a plasmid expressing the NF-κB aptamer as a linear RNA or a a racRNA for two days. Cells were then treated with actinomycin D (ActD) for 6 h and RNA was harvested to detect aptamer expression levels before and after actinomycin D treatment. Aptamer levels were detected based on in-gel staining using DFHBI-1T to detect the Broccoli incorporated into the RNA. The Tornado-expressed racRNA generates a single bright Broccoli-fluorescent bands that is resistant to actinomycin D treatment, while the linear band is barely detected (outlined in white and shown in a brightness-adjusted image on the right). c , The NF-κB aptamer is an effective pathway inhibitor when expressed using the Tornado expression system. IL-1β-induced NF-κB pathway activation was detected by luminescence in cells encoding luciferase driven by a NF-κB-promoter. Cells expressed either the circular or linear NF-κB aptamer as indicated. IL-1β-induced luminescence is 8% inhibited by linear expression and 70% inhibited by the circular expression. Circular RNA-expressing cells that were pre-sorted for aptamer expression based on their green fluorescence shows more efficient pathway inhibition (~85%). Luminescence was normalized according to the number of cells present during the assay. All signals were normalized to that of the cells expressing RNA without the NF-κB aptamer after activation. Data in this panel represent the mean (n = 3 or 4 stimulation and assay independent samples).

    Journal: Nature biotechnology

    Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts

    doi: 10.1038/s41587-019-0090-6

    Figure Lengend Snippet: Improved inhibition of NF-κB pathway by circRNA aptamers. a , Design of circular RNAs that contain NF-κB pathway-inhibiting aptamers. Circular RNAs are designed to contain a F30 3-way junction (black) with Broccoli on one arm, and an NF-κB aptamer on the other arm, while the circularizing stem forms at the base of the F30 3-way junction. This design allows for functional investigation of pathway-inhibiting aptamers while also probing for abundance of this circular RNA using Broccoli fluorescence. b , Tornado efficiently expresses the NF-κB aptamer as a racRNA in HEK293 cells. HEK293 cells were transfected with a plasmid expressing the NF-κB aptamer as a linear RNA or a a racRNA for two days. Cells were then treated with actinomycin D (ActD) for 6 h and RNA was harvested to detect aptamer expression levels before and after actinomycin D treatment. Aptamer levels were detected based on in-gel staining using DFHBI-1T to detect the Broccoli incorporated into the RNA. The Tornado-expressed racRNA generates a single bright Broccoli-fluorescent bands that is resistant to actinomycin D treatment, while the linear band is barely detected (outlined in white and shown in a brightness-adjusted image on the right). c , The NF-κB aptamer is an effective pathway inhibitor when expressed using the Tornado expression system. IL-1β-induced NF-κB pathway activation was detected by luminescence in cells encoding luciferase driven by a NF-κB-promoter. Cells expressed either the circular or linear NF-κB aptamer as indicated. IL-1β-induced luminescence is 8% inhibited by linear expression and 70% inhibited by the circular expression. Circular RNA-expressing cells that were pre-sorted for aptamer expression based on their green fluorescence shows more efficient pathway inhibition (~85%). Luminescence was normalized according to the number of cells present during the assay. All signals were normalized to that of the cells expressing RNA without the NF-κB aptamer after activation. Data in this panel represent the mean (n = 3 or 4 stimulation and assay independent samples).

    Article Snippet: Cells were stimulated with 50 ng/mL Recombinant Human IL-1β (Peprotech 200–01b) for 2.5 h. Luminescence was generated with the One-Glo™ Luciferase Assay System (Promega E6110) as recommended by the manufacturer and detected at 570 (Molecular Devices SpectraMax® L Microplate Reader).

    Techniques: Inhibition, Functional Assay, Fluorescence, Transfection, Plasmid Preparation, Expressing, Staining, Activation Assay, Luciferase

    Interleukin 1β (IL-1β) induces resistance to cisplatin and upregulates the expression of TP63 . ( A ) Non-invasive MCF-7 cells and invasive 6D cells were treated with 100 µM cisplatin (MCF-7 + cisplatin) and (6D + cisplatin). MCF-7 cells without any treatment were utilized as the control of live cells in the assay (100% viability). Cells were harvested at 48 h and quantification of viable cells performed using the WST-1 reagent. Data are presented as percentage ± SD of viable cells relative to untreated cells from three independent experiments. ( B ) Relative expression of the TP63 gene was determined by qPCR in MCF-7 and 6D cells. Results represent the average of three independent experiments ± SD. ( C ) (a,b) Representative Western blot and densitometry analysis of total extracts from MCF-7 and 6D cells. The membranes were challenged with anti-TP63 antibody and anti-β-actin for protein load control. The densitometric analysis shows data in three blots from independent experiments. In all of them, ΔNp63α levels were normalized relative to the protein levels in MCF-7 cells. Asterisks indicate significance at p = 0.001.

    Journal: International Journal of Molecular Sciences

    Article Title: IL-1β Inflammatory Cytokine-Induced TP63 Isoform ∆NP63α Signaling Cascade Contributes to Cisplatin Resistance in Human Breast Cancer Cells

    doi: 10.3390/ijms20020270

    Figure Lengend Snippet: Interleukin 1β (IL-1β) induces resistance to cisplatin and upregulates the expression of TP63 . ( A ) Non-invasive MCF-7 cells and invasive 6D cells were treated with 100 µM cisplatin (MCF-7 + cisplatin) and (6D + cisplatin). MCF-7 cells without any treatment were utilized as the control of live cells in the assay (100% viability). Cells were harvested at 48 h and quantification of viable cells performed using the WST-1 reagent. Data are presented as percentage ± SD of viable cells relative to untreated cells from three independent experiments. ( B ) Relative expression of the TP63 gene was determined by qPCR in MCF-7 and 6D cells. Results represent the average of three independent experiments ± SD. ( C ) (a,b) Representative Western blot and densitometry analysis of total extracts from MCF-7 and 6D cells. The membranes were challenged with anti-TP63 antibody and anti-β-actin for protein load control. The densitometric analysis shows data in three blots from independent experiments. In all of them, ΔNp63α levels were normalized relative to the protein levels in MCF-7 cells. Asterisks indicate significance at p = 0.001.

    Article Snippet: For all the experiments, the 6D cells were re-stimulated with 20 ng/mL human recombinant IL-1β (Peprotech, Rocky Hill, NJ, USA) for 48 h to ensure their homogeneous response to IL-1β [ ].

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

    ΔNp63α plays a role in the IL-1β induction of cisplatin resistance in 6D cells. Cells were transfected with empty vector (Mock), non-specific short hairpin RNA (Scramble), and the specific silencing RNA (shRNAp63α). ( A ) TP63 expression was evaluated by qPCR. Results represent the average of three independent experiments ± SD. Asterisks correspond to p = 0.001 relative to the controls, Mock and Scramble. ( B (a)) Representative Western blot of ΔNp63α protein levels in the 6D cells. ( B (b)) Densitometric values corresponding to ΔNp63α levels in ( B (a)) were normalized to those of β-actin. ( C ) Cell viability levels determined in ΔNp63α-silenced and non-silenced cells in the absence or presence of cisplatin. Data represent the average of four independent experiments ± SD. Asterisks indicate significance relative to 6D cells at p = 0.001. ( D ) Comet assay to evaluate DNA integrity and damage by cisplatin in MCF-7, non-silenced, and shRNAp63α-silenced 6D cells. Cells not treated and treated with cisplatin were mixed with low-melting point agarose, lysed, and subjected to electrophoresis, followed by staining with ethidium bromide. DNA was visualized by fluorescence microscopy; scale bar = 100 μm. The insets in the right panel show magnified images taken from each condition; scale bar = 25 μm.

    Journal: International Journal of Molecular Sciences

    Article Title: IL-1β Inflammatory Cytokine-Induced TP63 Isoform ∆NP63α Signaling Cascade Contributes to Cisplatin Resistance in Human Breast Cancer Cells

    doi: 10.3390/ijms20020270

    Figure Lengend Snippet: ΔNp63α plays a role in the IL-1β induction of cisplatin resistance in 6D cells. Cells were transfected with empty vector (Mock), non-specific short hairpin RNA (Scramble), and the specific silencing RNA (shRNAp63α). ( A ) TP63 expression was evaluated by qPCR. Results represent the average of three independent experiments ± SD. Asterisks correspond to p = 0.001 relative to the controls, Mock and Scramble. ( B (a)) Representative Western blot of ΔNp63α protein levels in the 6D cells. ( B (b)) Densitometric values corresponding to ΔNp63α levels in ( B (a)) were normalized to those of β-actin. ( C ) Cell viability levels determined in ΔNp63α-silenced and non-silenced cells in the absence or presence of cisplatin. Data represent the average of four independent experiments ± SD. Asterisks indicate significance relative to 6D cells at p = 0.001. ( D ) Comet assay to evaluate DNA integrity and damage by cisplatin in MCF-7, non-silenced, and shRNAp63α-silenced 6D cells. Cells not treated and treated with cisplatin were mixed with low-melting point agarose, lysed, and subjected to electrophoresis, followed by staining with ethidium bromide. DNA was visualized by fluorescence microscopy; scale bar = 100 μm. The insets in the right panel show magnified images taken from each condition; scale bar = 25 μm.

    Article Snippet: For all the experiments, the 6D cells were re-stimulated with 20 ng/mL human recombinant IL-1β (Peprotech, Rocky Hill, NJ, USA) for 48 h to ensure their homogeneous response to IL-1β [ ].

    Techniques: Transfection, Plasmid Preparation, shRNA, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Single Cell Gel Electrophoresis, Electrophoresis, Staining, Fluorescence, Microscopy

    (A-B) The levels of cytokines in CSF (pg/mL). The LPS-treated rats exhibited higher levels IL-1β (A) and TNF-α (B), but exendin-4 treatment had no effect on cytokine levels (Student’s t -tests, NS). Non-LPS treated rats were below detection limit. CSF from six animals were not used because of insufficient amounts ( n = 1 [Control], n = 2 [LPS], n = 3 [LPS+Ex4]). (C) The mRNA expression of IL-1β in the striatum. The IL-1β expression was higher in LPS treated rats but was not affected by exendin-4 treatment (two-way ANOVA, LPS effect, F (1,38) = 44.04, p

    Journal: Journal of Parkinson's Disease

    Article Title: Exendin-4 Treatment Improves LPS-Induced Depressive-Like Behavior Without Affecting Pro-Inflammatory Cytokines

    doi: 10.3233/JPD-171068

    Figure Lengend Snippet: (A-B) The levels of cytokines in CSF (pg/mL). The LPS-treated rats exhibited higher levels IL-1β (A) and TNF-α (B), but exendin-4 treatment had no effect on cytokine levels (Student’s t -tests, NS). Non-LPS treated rats were below detection limit. CSF from six animals were not used because of insufficient amounts ( n = 1 [Control], n = 2 [LPS], n = 3 [LPS+Ex4]). (C) The mRNA expression of IL-1β in the striatum. The IL-1β expression was higher in LPS treated rats but was not affected by exendin-4 treatment (two-way ANOVA, LPS effect, F (1,38) = 44.04, p

    Article Snippet: Cytokine protein level assay We quantified the CSF levels of IL-1β and TNF-α, serum levels of IL-1β, and supernatant (cell media) levels of IL-1β and TNF-α from cell cultures by electrochemiluminescence in a SECTOR SI6000 imager (Meso-Scale Discovery, USA) following the manufacturer’s instructions.

    Techniques: Expressing

    (A-B) The levels of cytokines in supernatants from adult primary microglia cell cultures. The cell cultures were treated with LPS (2 μg/mL) and exendin-4 (2 μM and 12 μM) and incubated for 24 hours ( n = 3). No significant difference in the levels of IL-1β and TNF-α could be observed between the groups, (one-way ANOVA, NS). The IL-1β levels in the control group were below detection range. Bars represent the mean±SEM.

    Journal: Journal of Parkinson's Disease

    Article Title: Exendin-4 Treatment Improves LPS-Induced Depressive-Like Behavior Without Affecting Pro-Inflammatory Cytokines

    doi: 10.3233/JPD-171068

    Figure Lengend Snippet: (A-B) The levels of cytokines in supernatants from adult primary microglia cell cultures. The cell cultures were treated with LPS (2 μg/mL) and exendin-4 (2 μM and 12 μM) and incubated for 24 hours ( n = 3). No significant difference in the levels of IL-1β and TNF-α could be observed between the groups, (one-way ANOVA, NS). The IL-1β levels in the control group were below detection range. Bars represent the mean±SEM.

    Article Snippet: Cytokine protein level assay We quantified the CSF levels of IL-1β and TNF-α, serum levels of IL-1β, and supernatant (cell media) levels of IL-1β and TNF-α from cell cultures by electrochemiluminescence in a SECTOR SI6000 imager (Meso-Scale Discovery, USA) following the manufacturer’s instructions.

    Techniques: Incubation

    PI3K phosphorylation is involved in APLN-induced IL-1β synthesis. ( A ) OASFs were pretreated with PI3K inhibitors (LY294002, Wortmannin; 10 μM) for 30 min, then incubated with APLN (10 ng/mL) for 24 h. IL-1β mRNA and protein levels were examined by RT-qPCR (n=4) and Western blot (n=3) assays, respectively. ( B ) OASFs were pretreated with PI3K inhibitors (LY294002, Wortmannin; 10 μM) for 30 min, then incubated with APLN (10 ng/mL) for 24 h. Excreted IL-1 β protein levels were examined by ELISA (n=5). ( C ) OASFs were transfected with PI3K siRNA (1 μg) then incubated with APLN (10 ng/mL) for 24 h. IL-1β mRNA levels were examined by ELISA assay (n=5). ( D ) OASFs were transfected with PI3K siRNA (1 μg), then incubated with APLN (10 ng/mL) for 24 h. Excreted IL-1β protein levels were examined by ELISA assay (n=5). ( E ) OASFs were incubated with APLN for the indicated time intervals, and the extent of PI3K phosphorylation was examined by Western blot (n=3). * p

    Journal: Aging (Albany NY)

    Article Title: Apelin enhances IL-1β expression in human synovial fibroblasts by inhibiting miR-144-3p through the PI3K and ERK pathways

    doi: 10.18632/aging.103195

    Figure Lengend Snippet: PI3K phosphorylation is involved in APLN-induced IL-1β synthesis. ( A ) OASFs were pretreated with PI3K inhibitors (LY294002, Wortmannin; 10 μM) for 30 min, then incubated with APLN (10 ng/mL) for 24 h. IL-1β mRNA and protein levels were examined by RT-qPCR (n=4) and Western blot (n=3) assays, respectively. ( B ) OASFs were pretreated with PI3K inhibitors (LY294002, Wortmannin; 10 μM) for 30 min, then incubated with APLN (10 ng/mL) for 24 h. Excreted IL-1 β protein levels were examined by ELISA (n=5). ( C ) OASFs were transfected with PI3K siRNA (1 μg) then incubated with APLN (10 ng/mL) for 24 h. IL-1β mRNA levels were examined by ELISA assay (n=5). ( D ) OASFs were transfected with PI3K siRNA (1 μg), then incubated with APLN (10 ng/mL) for 24 h. Excreted IL-1β protein levels were examined by ELISA assay (n=5). ( E ) OASFs were incubated with APLN for the indicated time intervals, and the extent of PI3K phosphorylation was examined by Western blot (n=3). * p

    Article Snippet: All sections were stained with primary anti-IL-1β (1:200) (Santa Cruz Biotechnology).

    Techniques: Incubation, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection

    APLN stimulates IL-1β expression in OASFs in concentration- and time-dependent manners. ( A ) Human OASFs were incubated with 0, 1, 3, and 10 ng/mL of APLN for 24 h, and IL-1β mRNA expression levels were examined by RT-qPCR (n=4). ( B ) OASFs were incubated under various concentrations of APLN for 24 h, and IL-1β expression levels were examined by Western blot (n=3). ( C ) OASFs were cultured under various concentrations of APLN for 24 h, and excreted IL-1β were examined by ELISA assay (n=5). ( D ) OASFs were incubated with 10 ng/mL of APLN for 0, 6, 12, and 24 h. IL-1β mRNA levels were examined by RT-qPCR (n=4). ( E ) IL-1β protein synthesis levels were examined by Western blot (n=3). ( F ) Excretion of IL-1β protein levels in human OASFs was examined by ELISA (n=5). * p

    Journal: Aging (Albany NY)

    Article Title: Apelin enhances IL-1β expression in human synovial fibroblasts by inhibiting miR-144-3p through the PI3K and ERK pathways

    doi: 10.18632/aging.103195

    Figure Lengend Snippet: APLN stimulates IL-1β expression in OASFs in concentration- and time-dependent manners. ( A ) Human OASFs were incubated with 0, 1, 3, and 10 ng/mL of APLN for 24 h, and IL-1β mRNA expression levels were examined by RT-qPCR (n=4). ( B ) OASFs were incubated under various concentrations of APLN for 24 h, and IL-1β expression levels were examined by Western blot (n=3). ( C ) OASFs were cultured under various concentrations of APLN for 24 h, and excreted IL-1β were examined by ELISA assay (n=5). ( D ) OASFs were incubated with 10 ng/mL of APLN for 0, 6, 12, and 24 h. IL-1β mRNA levels were examined by RT-qPCR (n=4). ( E ) IL-1β protein synthesis levels were examined by Western blot (n=3). ( F ) Excretion of IL-1β protein levels in human OASFs was examined by ELISA (n=5). * p

    Article Snippet: All sections were stained with primary anti-IL-1β (1:200) (Santa Cruz Biotechnology).

    Techniques: Expressing, Concentration Assay, Incubation, Quantitative RT-PCR, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

    APLN-induced suppression of miRNA-144-3p enhances IL-1β production. ( A ) Open-source software (TargetScan, miRMap, RNAhybrid, and miRWalk) was used to identify which miRNAs could possibly interfere with IL-1β transcription. ( B ) OASFs were incubated with APLN (0, 1, 3, and 10 ng/mL). Levels of miR-144-3p expression were examined by RT-qPCR assay (n=4). ( C , D ) OASFs were transfected with miR-144-3p mimic and then stimulated with APLN (10 ng/mL). mRNA and excreted protein levels were examined by RT-qPCR (n=4) and ELISA assays (n=5). ( E ) OASFs were transfected with the mut-IL-1β-3′UTR plasmid with or without miRNA-144-3p mimic, then stimulated with APLN (10 ng/mL). Relative luciferase activity reflected IL-1β promoter activity (n=6). ( F ) OASFs were treated with PI3K or ERK inhibitor then incubated with APLN. miR-144-3p expression levels were examined by RT-qPCR assay (n=4). Results are expressed as the mean ± S.E.M. * p

    Journal: Aging (Albany NY)

    Article Title: Apelin enhances IL-1β expression in human synovial fibroblasts by inhibiting miR-144-3p through the PI3K and ERK pathways

    doi: 10.18632/aging.103195

    Figure Lengend Snippet: APLN-induced suppression of miRNA-144-3p enhances IL-1β production. ( A ) Open-source software (TargetScan, miRMap, RNAhybrid, and miRWalk) was used to identify which miRNAs could possibly interfere with IL-1β transcription. ( B ) OASFs were incubated with APLN (0, 1, 3, and 10 ng/mL). Levels of miR-144-3p expression were examined by RT-qPCR assay (n=4). ( C , D ) OASFs were transfected with miR-144-3p mimic and then stimulated with APLN (10 ng/mL). mRNA and excreted protein levels were examined by RT-qPCR (n=4) and ELISA assays (n=5). ( E ) OASFs were transfected with the mut-IL-1β-3′UTR plasmid with or without miRNA-144-3p mimic, then stimulated with APLN (10 ng/mL). Relative luciferase activity reflected IL-1β promoter activity (n=6). ( F ) OASFs were treated with PI3K or ERK inhibitor then incubated with APLN. miR-144-3p expression levels were examined by RT-qPCR assay (n=4). Results are expressed as the mean ± S.E.M. * p

    Article Snippet: All sections were stained with primary anti-IL-1β (1:200) (Santa Cruz Biotechnology).

    Techniques: Software, Incubation, Expressing, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Luciferase, Activity Assay

    ERK phosphorylation is involved in APLN-induced IL-1β synthesis. ( A ) OASFs were pretreated with ERK inhibitors (PD98059, U0126; 10 μM) for 30 min, then incubated with APLN (10 ng/mL) for 24 h. IL-1β mRNA and protein levels were examined by RT-qPCR (n=4) and Western blot (n=3) assays, respectively. ( B ) OASFs were pretreated with ERK inhibitors (PD98059, U0126; 10 μM) for 30 min, then incubated with APLN (10 ng/mL) for 24 h. Excreted IL-1β protein levels were examined by ELISA (n=5). ( C ) OASFs were transfected with ERK siRNA (1 μg), then incubated with APLN (10 ng/mL) for 24 h. IL-1β mRNA levels were examined by ELISA assay (n=5). ( D ) OASFs were transfected with ERK siRNA (1 μg), then incubated with APLN (10 ng/mL) for 24 h. Excreted IL-1β protein levels were examined by ELISA assay (n=5). ( E ) OASFs were incubated with APLN (10 ng/mL) for the indicated time intervals, and the extent of ERK phosphorylation was examined by Western blot (n=3). ( F ) OASFs were pretreated with LY294002 and Wortmannin (10 μM) for 30 min, then incubated with APLN (10 ng/mL) for 24 h. The extent of ERK phosphorylation was examined by Western blot (n=3). * p

    Journal: Aging (Albany NY)

    Article Title: Apelin enhances IL-1β expression in human synovial fibroblasts by inhibiting miR-144-3p through the PI3K and ERK pathways

    doi: 10.18632/aging.103195

    Figure Lengend Snippet: ERK phosphorylation is involved in APLN-induced IL-1β synthesis. ( A ) OASFs were pretreated with ERK inhibitors (PD98059, U0126; 10 μM) for 30 min, then incubated with APLN (10 ng/mL) for 24 h. IL-1β mRNA and protein levels were examined by RT-qPCR (n=4) and Western blot (n=3) assays, respectively. ( B ) OASFs were pretreated with ERK inhibitors (PD98059, U0126; 10 μM) for 30 min, then incubated with APLN (10 ng/mL) for 24 h. Excreted IL-1β protein levels were examined by ELISA (n=5). ( C ) OASFs were transfected with ERK siRNA (1 μg), then incubated with APLN (10 ng/mL) for 24 h. IL-1β mRNA levels were examined by ELISA assay (n=5). ( D ) OASFs were transfected with ERK siRNA (1 μg), then incubated with APLN (10 ng/mL) for 24 h. Excreted IL-1β protein levels were examined by ELISA assay (n=5). ( E ) OASFs were incubated with APLN (10 ng/mL) for the indicated time intervals, and the extent of ERK phosphorylation was examined by Western blot (n=3). ( F ) OASFs were pretreated with LY294002 and Wortmannin (10 μM) for 30 min, then incubated with APLN (10 ng/mL) for 24 h. The extent of ERK phosphorylation was examined by Western blot (n=3). * p

    Article Snippet: All sections were stained with primary anti-IL-1β (1:200) (Santa Cruz Biotechnology).

    Techniques: Incubation, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection

    shAPLN administration mitigates the histologic severity of OA. ( A ) Staining of specimens with H E, Safranin-O, IL-1β and APLN from the control knee (n=6), ACLT knee (n=6), and shAPLN-transfected ACLT knee (n=6). ( B ) Cartilage degeneration scores were calculated for articular cartilage sections stained with Safranin-O. ( C ) Synovial membrane inflammation score. Magnified area of synovium used to generate synovial inflammation score in all samples. Scoring was performed in H E-stained slides. ( D ) IHC analysis of proportions of IL-1β-positive cells (red arrows) and APLN-positive cells in synovial lining tissues in specimens from control knees (n=6), ACLT knees (n=6), and shAPLN-transfected ACLT knees (n=6). * p

    Journal: Aging (Albany NY)

    Article Title: Apelin enhances IL-1β expression in human synovial fibroblasts by inhibiting miR-144-3p through the PI3K and ERK pathways

    doi: 10.18632/aging.103195

    Figure Lengend Snippet: shAPLN administration mitigates the histologic severity of OA. ( A ) Staining of specimens with H E, Safranin-O, IL-1β and APLN from the control knee (n=6), ACLT knee (n=6), and shAPLN-transfected ACLT knee (n=6). ( B ) Cartilage degeneration scores were calculated for articular cartilage sections stained with Safranin-O. ( C ) Synovial membrane inflammation score. Magnified area of synovium used to generate synovial inflammation score in all samples. Scoring was performed in H E-stained slides. ( D ) IHC analysis of proportions of IL-1β-positive cells (red arrows) and APLN-positive cells in synovial lining tissues in specimens from control knees (n=6), ACLT knees (n=6), and shAPLN-transfected ACLT knees (n=6). * p

    Article Snippet: All sections were stained with primary anti-IL-1β (1:200) (Santa Cruz Biotechnology).

    Techniques: Staining, Transfection, Immunohistochemistry

    APLN expression is positively correlated with IL-1β expression in OA patients. ( A ) IHC staining showing increased levels of APLN and IL-1β expression in OA synovial tissue (n=8) compared to normal healthy tissue (n=5). ( B , C ) The IHC score of APLN and IL-1β are presented. ( D ) Correlation between levels of APLN and IL-1β expression in synovial tissues retrieved from OA patients.

    Journal: Aging (Albany NY)

    Article Title: Apelin enhances IL-1β expression in human synovial fibroblasts by inhibiting miR-144-3p through the PI3K and ERK pathways

    doi: 10.18632/aging.103195

    Figure Lengend Snippet: APLN expression is positively correlated with IL-1β expression in OA patients. ( A ) IHC staining showing increased levels of APLN and IL-1β expression in OA synovial tissue (n=8) compared to normal healthy tissue (n=5). ( B , C ) The IHC score of APLN and IL-1β are presented. ( D ) Correlation between levels of APLN and IL-1β expression in synovial tissues retrieved from OA patients.

    Article Snippet: All sections were stained with primary anti-IL-1β (1:200) (Santa Cruz Biotechnology).

    Techniques: Expressing, Immunohistochemistry, Staining

    Schematic diagram summarizes the mechanism whereby APLN promotes IL-1β production in OASFs. APLN induces inflammatory IL-1β production in OASFs by downregulating miR-144-3p through the PI3K and ERK signaling pathways.

    Journal: Aging (Albany NY)

    Article Title: Apelin enhances IL-1β expression in human synovial fibroblasts by inhibiting miR-144-3p through the PI3K and ERK pathways

    doi: 10.18632/aging.103195

    Figure Lengend Snippet: Schematic diagram summarizes the mechanism whereby APLN promotes IL-1β production in OASFs. APLN induces inflammatory IL-1β production in OASFs by downregulating miR-144-3p through the PI3K and ERK signaling pathways.

    Article Snippet: All sections were stained with primary anti-IL-1β (1:200) (Santa Cruz Biotechnology).

    Techniques:

    Statistically significant correlations between IL-18 and caspase 1 in LL ( A ) and TT leprosy ( B ), between caspase 1 and IL-1β ( C ), between NLRP1 and IL-18 ( D ), between NLRP3 and IL-18 ( E ), and between NLRP3 and NLRP1 ( F ) in I leprosy. Abbreviations: I, indeterminate; LL, lepromatous leprosy; NLRP1, nucleotide-binding oligomerization domain-like receptor containing pyrin domain 1; NLRP3, nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3; TT, tuberculoid.

    Journal: Infection and Drug Resistance

    Article Title: The inflammasome in leprosy skin lesions: an immunohistochemical evaluation

    doi: 10.2147/IDR.S172806

    Figure Lengend Snippet: Statistically significant correlations between IL-18 and caspase 1 in LL ( A ) and TT leprosy ( B ), between caspase 1 and IL-1β ( C ), between NLRP1 and IL-18 ( D ), between NLRP3 and IL-18 ( E ), and between NLRP3 and NLRP1 ( F ) in I leprosy. Abbreviations: I, indeterminate; LL, lepromatous leprosy; NLRP1, nucleotide-binding oligomerization domain-like receptor containing pyrin domain 1; NLRP3, nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3; TT, tuberculoid.

    Article Snippet: Immunohistochemical analysis with monoclonal anti-NLRP1 (Abcam Cambridge, Cambridgeshire, UK; ab 98181), anti-NLRP3 (Abcam; ab 214185), anti-caspase 1 (Abcam; ab 18503), anti-IL-1β (Abcam; ab 14367), and anti-IL-18 (Abcam; ab 9722) antibodies followed the method involving formation of a biotin–streptavidin peroxidase complex according to the protocol described previously.

    Techniques: Binding Assay

    Immunohistochemical patterns of the inflammasome in leprosy skin lesions. Notes: Note that immunostaining occurs predominantly in immune cells of the granulomatous inflammatory infiltrate representative of the clinical forms LL, I leprosy, and TT leprosy. Immunostaining for NLRP1 in LL ( A ), I leprosy ( B ), and TT leprosy ( C ); for NLRP3 in LL ( D ), I leprosy ( E ), and TT leprosy ( F ); for caspase 1 in LL ( G ), I leprosy ( H ), and TT leprosy ( I ); for IL-1β in LL ( J ), I leprosy ( K ), and TT leprosy ( L ); and for IL-18 in LL ( M ), I leprosy ( N ), and TT leprosy ( O ). The arrows indicate the stained areas bounded by brownish color coincident with the cell bodies in the granulomatous inflammatory infiltrate (magnification, 200×). Abbreviations: I, indeterminate; LL, lepromatous leprosy; NLRP1, nucleotide-binding oligomerization domain-like receptor containing pyrin domain 1; NLRP3, nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3; TT, tuberculoid.

    Journal: Infection and Drug Resistance

    Article Title: The inflammasome in leprosy skin lesions: an immunohistochemical evaluation

    doi: 10.2147/IDR.S172806

    Figure Lengend Snippet: Immunohistochemical patterns of the inflammasome in leprosy skin lesions. Notes: Note that immunostaining occurs predominantly in immune cells of the granulomatous inflammatory infiltrate representative of the clinical forms LL, I leprosy, and TT leprosy. Immunostaining for NLRP1 in LL ( A ), I leprosy ( B ), and TT leprosy ( C ); for NLRP3 in LL ( D ), I leprosy ( E ), and TT leprosy ( F ); for caspase 1 in LL ( G ), I leprosy ( H ), and TT leprosy ( I ); for IL-1β in LL ( J ), I leprosy ( K ), and TT leprosy ( L ); and for IL-18 in LL ( M ), I leprosy ( N ), and TT leprosy ( O ). The arrows indicate the stained areas bounded by brownish color coincident with the cell bodies in the granulomatous inflammatory infiltrate (magnification, 200×). Abbreviations: I, indeterminate; LL, lepromatous leprosy; NLRP1, nucleotide-binding oligomerization domain-like receptor containing pyrin domain 1; NLRP3, nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3; TT, tuberculoid.

    Article Snippet: Immunohistochemical analysis with monoclonal anti-NLRP1 (Abcam Cambridge, Cambridgeshire, UK; ab 98181), anti-NLRP3 (Abcam; ab 214185), anti-caspase 1 (Abcam; ab 18503), anti-IL-1β (Abcam; ab 14367), and anti-IL-18 (Abcam; ab 9722) antibodies followed the method involving formation of a biotin–streptavidin peroxidase complex according to the protocol described previously.

    Techniques: Immunohistochemistry, Immunostaining, Staining, Binding Assay

    CBG pre-treatment was able to reduce the levels of pro-inflammatory cytokines in NSC-34 cells treated with the medium of LPS-stimulated RAW 264.7 macrophages. Immunocytochemistry with the quantitative analysis of positive staining showed that the treatment with the medium of LPS-stimulated RAW 264.7 macrophages induced the expression of the pro-inflammatory cytokines IL-1β, TNF-α and IFN-γ. CBG pre-treatment reduced the protein levels of the pro-inflammatory cytokines. The immunocytochemical assays were repeated three times. **** p

    Journal: International Journal of Molecular Sciences

    Article Title: In Vitro Model of Neuroinflammation: Efficacy of Cannabigerol, a Non-Psychoactive Cannabinoid

    doi: 10.3390/ijms19071992

    Figure Lengend Snippet: CBG pre-treatment was able to reduce the levels of pro-inflammatory cytokines in NSC-34 cells treated with the medium of LPS-stimulated RAW 264.7 macrophages. Immunocytochemistry with the quantitative analysis of positive staining showed that the treatment with the medium of LPS-stimulated RAW 264.7 macrophages induced the expression of the pro-inflammatory cytokines IL-1β, TNF-α and IFN-γ. CBG pre-treatment reduced the protein levels of the pro-inflammatory cytokines. The immunocytochemical assays were repeated three times. **** p

    Article Snippet: After three washes with PBS, cells were blocked with horse serum +0.1% Triton X-100 for 20 min and incubated overnight at 4°C with the following primary antibodies: - anti Bax (1:50; Santa Cruz Biotechnology); - anti Bcl-2 (1:50; Santa Cruz Biotechnology); - anti IL-1β (1:250; Cell Signaling Technology); - anti IFN-γ (1:50; Santa Cruz Biotechnology); - anti TNF-α (1:250; Cell Signaling Technology); - anti SOD1 (1:100; Abcam, Cambridge, UK); - anti iNOS (1:50; Santa Cruz Biotechnology); - anti nitrotyrosine (1:1000; Millipore); - anti Nrf-2 (1:50; Santa Cruz Biotechnology); - anti PPARγ (1:50; Santa Cruz Biotechnology).

    Techniques: Immunocytochemistry, Staining, Expressing

    Effects of HSR on corticosterone-induced proinflammatory cytokine expression. Total RNA was extracted from SK-N-SH cells, and the levels of (a) IL-1 β , (b) IL-6, (c) IL-8, and (d) TNF- α mRNA were determined by qRT-PCR. Expression levels of the target genes were normalized to those of β -actin. Data are the mean ± SD ( n = 3). ### p

    Journal: BioMed Research International

    Article Title: Antidepressant-Like and Neuroprotective Effects of Ethanol Extract from the Root Bark of Hibiscus syriacus L.

    doi: 10.1155/2018/7383869

    Figure Lengend Snippet: Effects of HSR on corticosterone-induced proinflammatory cytokine expression. Total RNA was extracted from SK-N-SH cells, and the levels of (a) IL-1 β , (b) IL-6, (c) IL-8, and (d) TNF- α mRNA were determined by qRT-PCR. Expression levels of the target genes were normalized to those of β -actin. Data are the mean ± SD ( n = 3). ### p

    Article Snippet: Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using TaqMan assays (Applied Biosystems, Foster City, CA, USA) specific for interleukin (IL)-1 β , IL-6, IL-8, and tumor necrosis factor (TNF)- α on a QuantStudio™ 6 Flex RT-PCR system (Applied Biosystems).

    Techniques: Expressing, Quantitative RT-PCR

    Effects of joint IM on serum C5a and pro-inflammatory cytokine expression levels. (A) Expression levels of C5a, IL-1β, TNF-α and IL-17A in the serum; (B) expression levels of C5a, IL-1β, TNF-α and IL-17A in joint cavity fluid. The graphs present the mean ± SEM. *P

    Journal: Molecular Medicine Reports

    Article Title: C5a aggravates dysfunction of the articular cartilage and synovial fluid in rats with knee joint immobilization

    doi: 10.3892/mmr.2018.9208

    Figure Lengend Snippet: Effects of joint IM on serum C5a and pro-inflammatory cytokine expression levels. (A) Expression levels of C5a, IL-1β, TNF-α and IL-17A in the serum; (B) expression levels of C5a, IL-1β, TNF-α and IL-17A in joint cavity fluid. The graphs present the mean ± SEM. *P

    Article Snippet: Membranes were incubated in blocking buffer (cat. no. P0023B; Beyotime Institute of Biotechnology) for 8 h at 4°C, and subsequently incubated with rabbit anti-C5a (cat. no. ab11876; 1:2,000), rabbit anti-IL-1β (cat. no. ab200478; 1:2,000), rabbit anti-TNF-α (cat. no. ab9755; 1:2,000; all Abcam, Cambridge, UK;), rabbit anti-IL-17A (cat. no. SAB3701458-100UG; 1:2,000; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), and rabbit anti-GAPDH (cat. no. KC-5G5; 1:5,000; Kangchen BioTech Co., Ltd., Shanghai, China) for 8 h at 4°C.

    Techniques: Expressing

    Cytokine production (cell-associated or secreted IL-1β, IL-6, and TNF-α) by macrophages exposed to 1 ng of leukotoxin/ml or to 100 ng of lipopolysaccharide/ml from A. actinomycetemcomitans (Aa LPS) or from E. coli (Ec LPS) for 3 h. The

    Journal: Infection and Immunity

    Article Title: Abundant Secretion of Bioactive Interleukin-1? by Human Macrophages Induced by Actinobacillus actinomycetemcomitans Leukotoxin

    doi: 10.1128/IAI.73.1.453-458.2005

    Figure Lengend Snippet: Cytokine production (cell-associated or secreted IL-1β, IL-6, and TNF-α) by macrophages exposed to 1 ng of leukotoxin/ml or to 100 ng of lipopolysaccharide/ml from A. actinomycetemcomitans (Aa LPS) or from E. coli (Ec LPS) for 3 h. The

    Article Snippet: To inhibit autocrine activation by secreted IL-1β, antibodies to IL-1β (polyclonal rabbit anti-human; Genzyme Corp., Cambridge, Mass.) were added (10 μg/ml of medium).

    Techniques:

    Induction of bone resorption by supernatants from macrophages (MΦ-sup) incubated for 3 h with 1 ng of leukotoxin (Ltx)/ml. Monoclonal antibodies against IL-1α, IL-1β, or IRAP were added to some supernatants. The resorption is determined

    Journal: Infection and Immunity

    Article Title: Abundant Secretion of Bioactive Interleukin-1? by Human Macrophages Induced by Actinobacillus actinomycetemcomitans Leukotoxin

    doi: 10.1128/IAI.73.1.453-458.2005

    Figure Lengend Snippet: Induction of bone resorption by supernatants from macrophages (MΦ-sup) incubated for 3 h with 1 ng of leukotoxin (Ltx)/ml. Monoclonal antibodies against IL-1α, IL-1β, or IRAP were added to some supernatants. The resorption is determined

    Article Snippet: To inhibit autocrine activation by secreted IL-1β, antibodies to IL-1β (polyclonal rabbit anti-human; Genzyme Corp., Cambridge, Mass.) were added (10 μg/ml of medium).

    Techniques: Incubation

    IL-1β production (cell associated or secreted) by macrophages exposed to 1 ng of leukotoxin/ml for 3 h in the presence of the caspase 1 inhibitor (inh.) Ac-YVAD-CMK or monoclonal antibodies to IL-1β. The cell-associated and secreted amounts

    Journal: Infection and Immunity

    Article Title: Abundant Secretion of Bioactive Interleukin-1? by Human Macrophages Induced by Actinobacillus actinomycetemcomitans Leukotoxin

    doi: 10.1128/IAI.73.1.453-458.2005

    Figure Lengend Snippet: IL-1β production (cell associated or secreted) by macrophages exposed to 1 ng of leukotoxin/ml for 3 h in the presence of the caspase 1 inhibitor (inh.) Ac-YVAD-CMK or monoclonal antibodies to IL-1β. The cell-associated and secreted amounts

    Article Snippet: To inhibit autocrine activation by secreted IL-1β, antibodies to IL-1β (polyclonal rabbit anti-human; Genzyme Corp., Cambridge, Mass.) were added (10 μg/ml of medium).

    Techniques:

    Levels of mRNA for the cytokines IL-1β, IL-6, and TNF-α and for the constitutive enzyme GAPDH in macrophages exposed to 1 ng of leukotoxin/ml from A. actinomycetemcomitans or to 100 ng of E. coli LPS (Ec LPS)/ml for 3 h. The corresponding

    Journal: Infection and Immunity

    Article Title: Abundant Secretion of Bioactive Interleukin-1? by Human Macrophages Induced by Actinobacillus actinomycetemcomitans Leukotoxin

    doi: 10.1128/IAI.73.1.453-458.2005

    Figure Lengend Snippet: Levels of mRNA for the cytokines IL-1β, IL-6, and TNF-α and for the constitutive enzyme GAPDH in macrophages exposed to 1 ng of leukotoxin/ml from A. actinomycetemcomitans or to 100 ng of E. coli LPS (Ec LPS)/ml for 3 h. The corresponding

    Article Snippet: To inhibit autocrine activation by secreted IL-1β, antibodies to IL-1β (polyclonal rabbit anti-human; Genzyme Corp., Cambridge, Mass.) were added (10 μg/ml of medium).

    Techniques:

    Model representing the induction and inhibition of IL-1β by HPV16. Infection of the basal keratinocytes with HPV16 induces inflammasome dependent IL-1β production sensed by an unknown innate receptor. p53 transcriptional regulation of IRF6 is increased, which we show drives IL-1β transcription. The pro form of IL-1β is cleaved by caspase 1 (red bar). The active form of IL-1β can block the increase in viral copies. However, when the oncoprotein E6 is expressed this drives p53 degradation by E6AP preventing IRF6 and consequently IL-1β transcription. This mechanism of viral inhibition of innate responses may contribute to HPV16 persistence in the host.

    Journal: PLoS Pathogens

    Article Title: Human papillomavirus type 16 antagonizes IRF6 regulation of IL-1β

    doi: 10.1371/journal.ppat.1007158

    Figure Lengend Snippet: Model representing the induction and inhibition of IL-1β by HPV16. Infection of the basal keratinocytes with HPV16 induces inflammasome dependent IL-1β production sensed by an unknown innate receptor. p53 transcriptional regulation of IRF6 is increased, which we show drives IL-1β transcription. The pro form of IL-1β is cleaved by caspase 1 (red bar). The active form of IL-1β can block the increase in viral copies. However, when the oncoprotein E6 is expressed this drives p53 degradation by E6AP preventing IRF6 and consequently IL-1β transcription. This mechanism of viral inhibition of innate responses may contribute to HPV16 persistence in the host.

    Article Snippet: After the indicated period (see figure legend), the supernatant was harvested and quantified for IL-1β by ELISA (Bender Med System) or IL-18 [ ].

    Techniques: Inhibition, Infection, Blocking Assay

    HPV16 oncoproteins inhibit pro-IL-1β levels. (A) Human keratinocytes transduced with pLXSN or 16E6E7 were stimulated with AIM2 and (B) NLPR3 ligands and both pro-IL-1β and IL-1β from cell lysates or supernatants were analysed by immunoblotting. β-actin was used as a loading control. Densitometry analysis was performed n = 3. (C) Immunoblotting of pro-IL-1β in human keratinocytes transduced with pLXSN or 16E6E7. (D) Cervical cancer cell lines were lysed and immunoblotting for pro-IL-1β was performed. n = 4 (E) Human keratinocytes transduced with pLXSN or 16E6E7 were treated for 24 h with N-CBZ-Leu-Leu-Leu-al. Cells were harvested and p53, E6 as well as pro-IL-1β levels were determined by immunoblotting. Right, p53 densitometry levels were normalized to β-actin. Below, immunoblot analysis of the 16E6 protein. n = 3. (F) RNA was extracted from Human keratinocytes transduced with pLXSN or 16E6E7 and IL-1β transcripts relative expression was determined by RT-qPCR. n = 5. (G) RNA was extracted from patient derived cervical cancer cell lines and IL-1β transcripts were determined by RT-qPCR. n = 6. Panels A-E. Data are representative of n independent experiments performed in triplicate. Shown are the mean ± SEM with ***, P

    Journal: PLoS Pathogens

    Article Title: Human papillomavirus type 16 antagonizes IRF6 regulation of IL-1β

    doi: 10.1371/journal.ppat.1007158

    Figure Lengend Snippet: HPV16 oncoproteins inhibit pro-IL-1β levels. (A) Human keratinocytes transduced with pLXSN or 16E6E7 were stimulated with AIM2 and (B) NLPR3 ligands and both pro-IL-1β and IL-1β from cell lysates or supernatants were analysed by immunoblotting. β-actin was used as a loading control. Densitometry analysis was performed n = 3. (C) Immunoblotting of pro-IL-1β in human keratinocytes transduced with pLXSN or 16E6E7. (D) Cervical cancer cell lines were lysed and immunoblotting for pro-IL-1β was performed. n = 4 (E) Human keratinocytes transduced with pLXSN or 16E6E7 were treated for 24 h with N-CBZ-Leu-Leu-Leu-al. Cells were harvested and p53, E6 as well as pro-IL-1β levels were determined by immunoblotting. Right, p53 densitometry levels were normalized to β-actin. Below, immunoblot analysis of the 16E6 protein. n = 3. (F) RNA was extracted from Human keratinocytes transduced with pLXSN or 16E6E7 and IL-1β transcripts relative expression was determined by RT-qPCR. n = 5. (G) RNA was extracted from patient derived cervical cancer cell lines and IL-1β transcripts were determined by RT-qPCR. n = 6. Panels A-E. Data are representative of n independent experiments performed in triplicate. Shown are the mean ± SEM with ***, P

    Article Snippet: After the indicated period (see figure legend), the supernatant was harvested and quantified for IL-1β by ELISA (Bender Med System) or IL-18 [ ].

    Techniques: Transduction, Expressing, Quantitative RT-PCR, Derivative Assay

    16E6E7 block IL-1β production in primary human keratinocytes and in cervical cancer derived cells lines. (A) Analysis of the IL-1β production by ELISA in human keratinocytes transduced with pLXSN or 16E6E7 stimulated with nigericin or poly dA:dT. n = 10. (B) Human keratinocytes transduced with pLXSN or 16E6E7 transfected with a siRNA targeting 16E6E7 (+) or the scramble control (-). Cells were stimulated with the NLRP3 ligand nigericin and IL-1β secretion was measured by ELISA. Middle, western blot of E6 or E7 siRNA efficacy on 16E6E7 or PLXSN transduced cells. n = 4. Left SiHa cell were treated with a siRNA targeting 16E6E7 (+) or the scramble control (-). Cells were stimulated with the NLRP3 ligand nigericin and IL-1β secretion was measured by ELISA. n = 4. (C) IL-8 bioassay: HEK293T cells transiently expressing the IL-8 promoter linked to luciferase gene were treated with increasing concentrations of recombinant IL-1β ± Anakinra. Twenty four h post treatment cells were harvested and luciferase activity was measured. n = 4. (D) IL-8 bioassay using supernatants from human keratinocytes transduced with pLXSN or 16E6E7± AIM2 ligand poly dA:dT. n = 4. (E) Cervical cancer cells (SiHa) were transfected with a siRNA targeting 16E6E7 or the scramble control. The cells were stimulated with the NLRP3 ligand nigericin, AIM2 ligand poly dA:dT or 16QsV (200 v.g.e per cell) and IL-1β was measured by ELISA. n = 4. (F) IL-8 bioassay using supernatants from cervical cancer cell lines ± nigericin. n = 6. Data are representative of n independent experiments performed in triplicate. Shown are the mean ± SEM with ***, P

    Journal: PLoS Pathogens

    Article Title: Human papillomavirus type 16 antagonizes IRF6 regulation of IL-1β

    doi: 10.1371/journal.ppat.1007158

    Figure Lengend Snippet: 16E6E7 block IL-1β production in primary human keratinocytes and in cervical cancer derived cells lines. (A) Analysis of the IL-1β production by ELISA in human keratinocytes transduced with pLXSN or 16E6E7 stimulated with nigericin or poly dA:dT. n = 10. (B) Human keratinocytes transduced with pLXSN or 16E6E7 transfected with a siRNA targeting 16E6E7 (+) or the scramble control (-). Cells were stimulated with the NLRP3 ligand nigericin and IL-1β secretion was measured by ELISA. Middle, western blot of E6 or E7 siRNA efficacy on 16E6E7 or PLXSN transduced cells. n = 4. Left SiHa cell were treated with a siRNA targeting 16E6E7 (+) or the scramble control (-). Cells were stimulated with the NLRP3 ligand nigericin and IL-1β secretion was measured by ELISA. n = 4. (C) IL-8 bioassay: HEK293T cells transiently expressing the IL-8 promoter linked to luciferase gene were treated with increasing concentrations of recombinant IL-1β ± Anakinra. Twenty four h post treatment cells were harvested and luciferase activity was measured. n = 4. (D) IL-8 bioassay using supernatants from human keratinocytes transduced with pLXSN or 16E6E7± AIM2 ligand poly dA:dT. n = 4. (E) Cervical cancer cells (SiHa) were transfected with a siRNA targeting 16E6E7 or the scramble control. The cells were stimulated with the NLRP3 ligand nigericin, AIM2 ligand poly dA:dT or 16QsV (200 v.g.e per cell) and IL-1β was measured by ELISA. n = 4. (F) IL-8 bioassay using supernatants from cervical cancer cell lines ± nigericin. n = 6. Data are representative of n independent experiments performed in triplicate. Shown are the mean ± SEM with ***, P

    Article Snippet: After the indicated period (see figure legend), the supernatant was harvested and quantified for IL-1β by ELISA (Bender Med System) or IL-18 [ ].

    Techniques: Blocking Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Transduction, Transfection, Western Blot, Expressing, Luciferase, Recombinant, Activity Assay

    HPV16 E6 down-regulates the IL-1β promoter in cervical cells via the ISRE site. (A) NIKs were co-transfected with the IL-1β promoter with increasing concentrations of pLXSN HPV16E6E7 or 16E6 or 16E7 as indicated. After 48 h, cells were harvested and luciferase activity was measured. n = 5. (B) Relative expression of 16E6E7, 16E6 or 16E7 were measured by RT-qPCR. n = 5. (C) Primary human keratinocytes transduced with 16E6 and treated with a scramble or siRNA against 16E6. Protein levels of pro-IL-1β and loading control β-tubulin were evaluated by immunoblotting. n = 4. (D) Schematic representation of IL-1β promoter luciferase deletion mutations. (E) WT and deleted IL-1β promoter constructs were transiently transfected into NIKs expressing pLXSN or 16E6. After 48 h, cells were harvested and luciferase activity was measured. n = 4. (F) Schematic representation of the IL-1β LILRE site. (G) WT and deleted or mutated IL-1β promoter constructs were transiently transfected into NIKs expressing pLXSN or 16E6. After 48 h, cells were harvested and luciferase activity was measured. n = 4. Data are representative of n independent experiments performed in triplicate. Panel A and B shown are the mean ± SEM with ***, P

    Journal: PLoS Pathogens

    Article Title: Human papillomavirus type 16 antagonizes IRF6 regulation of IL-1β

    doi: 10.1371/journal.ppat.1007158

    Figure Lengend Snippet: HPV16 E6 down-regulates the IL-1β promoter in cervical cells via the ISRE site. (A) NIKs were co-transfected with the IL-1β promoter with increasing concentrations of pLXSN HPV16E6E7 or 16E6 or 16E7 as indicated. After 48 h, cells were harvested and luciferase activity was measured. n = 5. (B) Relative expression of 16E6E7, 16E6 or 16E7 were measured by RT-qPCR. n = 5. (C) Primary human keratinocytes transduced with 16E6 and treated with a scramble or siRNA against 16E6. Protein levels of pro-IL-1β and loading control β-tubulin were evaluated by immunoblotting. n = 4. (D) Schematic representation of IL-1β promoter luciferase deletion mutations. (E) WT and deleted IL-1β promoter constructs were transiently transfected into NIKs expressing pLXSN or 16E6. After 48 h, cells were harvested and luciferase activity was measured. n = 4. (F) Schematic representation of the IL-1β LILRE site. (G) WT and deleted or mutated IL-1β promoter constructs were transiently transfected into NIKs expressing pLXSN or 16E6. After 48 h, cells were harvested and luciferase activity was measured. n = 4. Data are representative of n independent experiments performed in triplicate. Panel A and B shown are the mean ± SEM with ***, P

    Article Snippet: After the indicated period (see figure legend), the supernatant was harvested and quantified for IL-1β by ELISA (Bender Med System) or IL-18 [ ].

    Techniques: Transfection, Luciferase, Activity Assay, Expressing, Quantitative RT-PCR, Transduction, Construct

    IRF6 and not IRF8 is recruited to the IL-1β promoter which is blocked by HPV16E6. (A) HEK293 cells were co-transfected with IL-1β promoter luciferase construct along with the empty vector pUNO, IRF8 or IRF6 plasmid at the indicated concentration. Post 48 h cells were lysed and luciferase activity measured. n = 4. (B) Oligo pulldown assay for WT or the mutated ISRE site using protein lysates from HEK293 cells transfected with IRF6 or IRF8. Bound proteins were assessed by immunoblotting for IRF8 or IRF6. Input controls (10%). n = 4. (C) Immunoblot analysis of IRF6 protein levels in in pLXSN and 16E6 transduced human primary keratinocytes. n = 4. (D) IRF6 relative levels were measured in pLXSN, 16E6 and 16E7 transduced human primary keratinocytes by RT-qPCR. n = 4. (E) Immunofluorescent staining of IRF6 in human keratinocytes transduced with pLXSN or HPV16E6. Left, semi-quantative analysis of IRF6 was examined by calculating immunofluorescent intensity. The mean and S.E.M of five fields were plotted. n = 4. (F) Immunoblot analysis of IRF6 protein levels in C33A and NIKs. n = 4. (G) IRF6 mRNA levels detected by RT-qPCR in NIKs and CaSki cells. n = 4. (H) NIKs and CaSki cells were co-transfected with IL-1β promoter luciferase construct ± siRNA for 16E6. Post 48 h cells were lysed and luciferase activity measured. (I) C33A cells were treated with control PsV or 16QsV at different v.g.e per cell for 24 h and IRF6 protein levels were examined by immunoblot. n = 3. (J) C33A cells were treated with control PsV or HPV16 at different v.g.e for 24h and IRF6 mRNA levels were examined by RT-qPCR and (below) viral DNA expression of E7 vs β2-microgloubulin. n = 3. (K), ChIP using IgG, IRF6 or IRF8 antibodies was performed for the ISRE site on C33A cells infected with HPV16 or PsV for 24 h. n = 3. Data are representative of n independent experiments performed in triplicate. Panels A, E, J and K are shown as the mean ± SEM with ***, P

    Journal: PLoS Pathogens

    Article Title: Human papillomavirus type 16 antagonizes IRF6 regulation of IL-1β

    doi: 10.1371/journal.ppat.1007158

    Figure Lengend Snippet: IRF6 and not IRF8 is recruited to the IL-1β promoter which is blocked by HPV16E6. (A) HEK293 cells were co-transfected with IL-1β promoter luciferase construct along with the empty vector pUNO, IRF8 or IRF6 plasmid at the indicated concentration. Post 48 h cells were lysed and luciferase activity measured. n = 4. (B) Oligo pulldown assay for WT or the mutated ISRE site using protein lysates from HEK293 cells transfected with IRF6 or IRF8. Bound proteins were assessed by immunoblotting for IRF8 or IRF6. Input controls (10%). n = 4. (C) Immunoblot analysis of IRF6 protein levels in in pLXSN and 16E6 transduced human primary keratinocytes. n = 4. (D) IRF6 relative levels were measured in pLXSN, 16E6 and 16E7 transduced human primary keratinocytes by RT-qPCR. n = 4. (E) Immunofluorescent staining of IRF6 in human keratinocytes transduced with pLXSN or HPV16E6. Left, semi-quantative analysis of IRF6 was examined by calculating immunofluorescent intensity. The mean and S.E.M of five fields were plotted. n = 4. (F) Immunoblot analysis of IRF6 protein levels in C33A and NIKs. n = 4. (G) IRF6 mRNA levels detected by RT-qPCR in NIKs and CaSki cells. n = 4. (H) NIKs and CaSki cells were co-transfected with IL-1β promoter luciferase construct ± siRNA for 16E6. Post 48 h cells were lysed and luciferase activity measured. (I) C33A cells were treated with control PsV or 16QsV at different v.g.e per cell for 24 h and IRF6 protein levels were examined by immunoblot. n = 3. (J) C33A cells were treated with control PsV or HPV16 at different v.g.e for 24h and IRF6 mRNA levels were examined by RT-qPCR and (below) viral DNA expression of E7 vs β2-microgloubulin. n = 3. (K), ChIP using IgG, IRF6 or IRF8 antibodies was performed for the ISRE site on C33A cells infected with HPV16 or PsV for 24 h. n = 3. Data are representative of n independent experiments performed in triplicate. Panels A, E, J and K are shown as the mean ± SEM with ***, P

    Article Snippet: After the indicated period (see figure legend), the supernatant was harvested and quantified for IL-1β by ELISA (Bender Med System) or IL-18 [ ].

    Techniques: Transfection, Luciferase, Construct, Plasmid Preparation, Concentration Assay, Activity Assay, Quantitative RT-PCR, Staining, Transduction, Expressing, Chromatin Immunoprecipitation, Infection

    HPV16-positive cervical cancer lesions contain less IRF6 and IL-1β. (A) Immunofluorescence of normal cervical issue and HPV16+ cervical cancer biopsies. IL-1β (green), p53 (red) with trace indicating the basal layer and nucleus (white). Normal (HPV−) and a neoplastic biopsy (HPV16+) from one representative patient out of six with similar results are shown. Bars represent a scale of 10 μm. For each stained biopsy, six fields were examined IL-1β staining was counted manually and the percentage scored out of 100 cells. n = 4. (B) RNA was extracted from normal (29) and cervical cancer biopsies (29). IL-1β relative and IRF6 mRNA levels were measured by RT-qPCR. n = 4. (C) Table of immunohistochemical scoring IRF6 in patients at different stages of cervical neoplasia. Scoring, *** strong, ** medium, * low and—no staining (4 normal, 8 CINI, 8 CINII, 5 CINIII). n = 2. All tissue staining data were examined by two pathologists. (D), Immunohistochemical staining of IRF6 in cervical tissue in patients with CINI, II or III. n = 2. (E) RT-qPCR of IL-1β and IRF6 mRNA expression levels in normal vs neoplastic cervical tissue. n = 3. A p53 site is required to bind to the IRF6 promoter but is lost in cervical cancer tissues . (F) ChIP analysis was performed on normal and cervical neoplastic tissue for IRF6 binding on ISRE site on the IL-1β promoter. n = 3.

    Journal: PLoS Pathogens

    Article Title: Human papillomavirus type 16 antagonizes IRF6 regulation of IL-1β

    doi: 10.1371/journal.ppat.1007158

    Figure Lengend Snippet: HPV16-positive cervical cancer lesions contain less IRF6 and IL-1β. (A) Immunofluorescence of normal cervical issue and HPV16+ cervical cancer biopsies. IL-1β (green), p53 (red) with trace indicating the basal layer and nucleus (white). Normal (HPV−) and a neoplastic biopsy (HPV16+) from one representative patient out of six with similar results are shown. Bars represent a scale of 10 μm. For each stained biopsy, six fields were examined IL-1β staining was counted manually and the percentage scored out of 100 cells. n = 4. (B) RNA was extracted from normal (29) and cervical cancer biopsies (29). IL-1β relative and IRF6 mRNA levels were measured by RT-qPCR. n = 4. (C) Table of immunohistochemical scoring IRF6 in patients at different stages of cervical neoplasia. Scoring, *** strong, ** medium, * low and—no staining (4 normal, 8 CINI, 8 CINII, 5 CINIII). n = 2. All tissue staining data were examined by two pathologists. (D), Immunohistochemical staining of IRF6 in cervical tissue in patients with CINI, II or III. n = 2. (E) RT-qPCR of IL-1β and IRF6 mRNA expression levels in normal vs neoplastic cervical tissue. n = 3. A p53 site is required to bind to the IRF6 promoter but is lost in cervical cancer tissues . (F) ChIP analysis was performed on normal and cervical neoplastic tissue for IRF6 binding on ISRE site on the IL-1β promoter. n = 3.

    Article Snippet: After the indicated period (see figure legend), the supernatant was harvested and quantified for IL-1β by ELISA (Bender Med System) or IL-18 [ ].

    Techniques: Immunofluorescence, Staining, Quantitative RT-PCR, Immunohistochemistry, Expressing, Chromatin Immunoprecipitation, Binding Assay

    HPV16 induces transient IL-1β secretion by keratinocytes. (A) Human primary keratinocytes were treated as indicated with 16QsV or PsV (at 200 viral genome equivalents (v.g.e) per cell). IL-1β transcripts were determined by RT-qPCR. n = 5. (B) As in A, E1, E6 and E7 mRNA relative levels were determined by RT-qPCR. n = 4. (C) Human keratinocytes were treated at 4, 8 and 24 h with 16QsV at different v.g.e per cell. Supernatants were harvested and IL-1β or IL-18 production was measured by ELISA. PsV or L1/L2 fractions were added as controls. n = 5. (D) Human keratinocytes were treated with 16QsV or PsV at decreasing v.g.e per cell for 24 h ± recombinant IL-1β (200pg/ml). Cells were harvested and E7 mRNA levels were measured by RT-qPCR. n = 5. (E) Human keratinocytes were treated with 16QsV or PsV (200 v.g.e) for 24 h ± IL-1R inhibitor (Anakinra). Cells were harvested and E1 mRNA levels were measured by RTqPCR. qPCR. n = 5. Data are representative of n independent experiments performed. Shown are the mean ± SEM with ***, P

    Journal: PLoS Pathogens

    Article Title: Human papillomavirus type 16 antagonizes IRF6 regulation of IL-1β

    doi: 10.1371/journal.ppat.1007158

    Figure Lengend Snippet: HPV16 induces transient IL-1β secretion by keratinocytes. (A) Human primary keratinocytes were treated as indicated with 16QsV or PsV (at 200 viral genome equivalents (v.g.e) per cell). IL-1β transcripts were determined by RT-qPCR. n = 5. (B) As in A, E1, E6 and E7 mRNA relative levels were determined by RT-qPCR. n = 4. (C) Human keratinocytes were treated at 4, 8 and 24 h with 16QsV at different v.g.e per cell. Supernatants were harvested and IL-1β or IL-18 production was measured by ELISA. PsV or L1/L2 fractions were added as controls. n = 5. (D) Human keratinocytes were treated with 16QsV or PsV at decreasing v.g.e per cell for 24 h ± recombinant IL-1β (200pg/ml). Cells were harvested and E7 mRNA levels were measured by RT-qPCR. n = 5. (E) Human keratinocytes were treated with 16QsV or PsV (200 v.g.e) for 24 h ± IL-1R inhibitor (Anakinra). Cells were harvested and E1 mRNA levels were measured by RTqPCR. qPCR. n = 5. Data are representative of n independent experiments performed. Shown are the mean ± SEM with ***, P

    Article Snippet: After the indicated period (see figure legend), the supernatant was harvested and quantified for IL-1β by ELISA (Bender Med System) or IL-18 [ ].

    Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Recombinant, Real-time Polymerase Chain Reaction

    Loss of p53 inhibition by 16E6 restores IRF6 activity. (A) Table defining the 16E6 mutations that alter p53, E6AP or PDZ binding sites. (B) NIKs were co-transfected with IL-1β promoter luciferase construct along with the HPV16E6 WT and mutated constructs at the indicated concentration. n = 5. (C) Human primary keratinocytes were transfected with cas9/sgRNA for p53 or control and 36h later cells were examined for mRNA levels for p53, IL-1β or IRF6. n = 4. (D) siRNAE6AP (+) or siRNA scramble control (-) was transfected into PLXSN or 16E6 transduced human keratinocytes for 48 h, cells were harvested for protein and RNA. Western blot analysis for p53 and β-actin. Left top, RT-qPCR for IL-1β and, left below IRF6. n = 3, (E) NIKs were co-transfected with the IL-1β promoter and pLXSN, E6, p53 or E6 with p53. Luciferase activity was measured 48 h post infection. n = 4. (F) 16E6 transduced primary keratinocytes were transfected with vector control (-), p53 or IRF6 expression vectors. Twenty-four hours later cells were harvested and pro-IL-1β, p53 or IRF6 levels were examined by immunoblotting. n = 4. Data are representative of n independent experiments performed in triplicate. Shown are the mean ± SEM with ***, P

    Journal: PLoS Pathogens

    Article Title: Human papillomavirus type 16 antagonizes IRF6 regulation of IL-1β

    doi: 10.1371/journal.ppat.1007158

    Figure Lengend Snippet: Loss of p53 inhibition by 16E6 restores IRF6 activity. (A) Table defining the 16E6 mutations that alter p53, E6AP or PDZ binding sites. (B) NIKs were co-transfected with IL-1β promoter luciferase construct along with the HPV16E6 WT and mutated constructs at the indicated concentration. n = 5. (C) Human primary keratinocytes were transfected with cas9/sgRNA for p53 or control and 36h later cells were examined for mRNA levels for p53, IL-1β or IRF6. n = 4. (D) siRNAE6AP (+) or siRNA scramble control (-) was transfected into PLXSN or 16E6 transduced human keratinocytes for 48 h, cells were harvested for protein and RNA. Western blot analysis for p53 and β-actin. Left top, RT-qPCR for IL-1β and, left below IRF6. n = 3, (E) NIKs were co-transfected with the IL-1β promoter and pLXSN, E6, p53 or E6 with p53. Luciferase activity was measured 48 h post infection. n = 4. (F) 16E6 transduced primary keratinocytes were transfected with vector control (-), p53 or IRF6 expression vectors. Twenty-four hours later cells were harvested and pro-IL-1β, p53 or IRF6 levels were examined by immunoblotting. n = 4. Data are representative of n independent experiments performed in triplicate. Shown are the mean ± SEM with ***, P

    Article Snippet: After the indicated period (see figure legend), the supernatant was harvested and quantified for IL-1β by ELISA (Bender Med System) or IL-18 [ ].

    Techniques: Inhibition, Activity Assay, Binding Assay, Transfection, Luciferase, Construct, Concentration Assay, Western Blot, Quantitative RT-PCR, Infection, Plasmid Preparation, Expressing

    IL-1β impaired cLTP-induced signaling associated with actin dynamics. a Western blots of G-actin and F-actin. b Quantification of the blots shown in a . Data are mean ± SEM from three independent experiments expressed in terms of the ratio of G-actin to F-actin obtained in the control cultures (* p

    Journal: Journal of Neuroinflammation

    Article Title: IL-1β suppresses cLTP-induced surface expression of GluA1 and actin polymerization via ceramide-mediated Src activation

    doi: 10.1186/s12974-018-1158-9

    Figure Lengend Snippet: IL-1β impaired cLTP-induced signaling associated with actin dynamics. a Western blots of G-actin and F-actin. b Quantification of the blots shown in a . Data are mean ± SEM from three independent experiments expressed in terms of the ratio of G-actin to F-actin obtained in the control cultures (* p

    Article Snippet: Recombinant IL-1β (PeproTech) was dissolved in DMEM and used after one freeze–thaw cycle at 50 ng/ml.

    Techniques: Western Blot

    Neutral sphingomyelinase inhibitor GW4869 attenuated IL-1β-mediated suppression of cLTP-induced synaptic plasticity. a Representative fluorescence images show F-actin, which was stained by phallodin, in unstimulated control cells and cells treated with cLTP in the presence or absence of IL-1β with or without GW4869 (5 μM). Scale bar, 10 μm. b Quantification of the images shown in a . The intensity of phallodin staining was normalized by MAP2 staining. Data are mean ± SEM from three independent experiments expressed in terms of control (* p

    Journal: Journal of Neuroinflammation

    Article Title: IL-1β suppresses cLTP-induced surface expression of GluA1 and actin polymerization via ceramide-mediated Src activation

    doi: 10.1186/s12974-018-1158-9

    Figure Lengend Snippet: Neutral sphingomyelinase inhibitor GW4869 attenuated IL-1β-mediated suppression of cLTP-induced synaptic plasticity. a Representative fluorescence images show F-actin, which was stained by phallodin, in unstimulated control cells and cells treated with cLTP in the presence or absence of IL-1β with or without GW4869 (5 μM). Scale bar, 10 μm. b Quantification of the images shown in a . The intensity of phallodin staining was normalized by MAP2 staining. Data are mean ± SEM from three independent experiments expressed in terms of control (* p

    Article Snippet: Recombinant IL-1β (PeproTech) was dissolved in DMEM and used after one freeze–thaw cycle at 50 ng/ml.

    Techniques: Fluorescence, Staining

    Src inhibitor PP2 attenuated IL-1β-mediated suppression of cLTP-induced synaptic plasticity. a Gel image shows Western blots of phosphorylated Src (P-Src). Hippocampal neurons were exposed to IL-1β for 20 min. b Quantification of the blots shown in a. Data are mean ± SEM from three independent experiments expressed in terms of P-Src obtained in the control cultures (* p

    Journal: Journal of Neuroinflammation

    Article Title: IL-1β suppresses cLTP-induced surface expression of GluA1 and actin polymerization via ceramide-mediated Src activation

    doi: 10.1186/s12974-018-1158-9

    Figure Lengend Snippet: Src inhibitor PP2 attenuated IL-1β-mediated suppression of cLTP-induced synaptic plasticity. a Gel image shows Western blots of phosphorylated Src (P-Src). Hippocampal neurons were exposed to IL-1β for 20 min. b Quantification of the blots shown in a. Data are mean ± SEM from three independent experiments expressed in terms of P-Src obtained in the control cultures (* p

    Article Snippet: Recombinant IL-1β (PeproTech) was dissolved in DMEM and used after one freeze–thaw cycle at 50 ng/ml.

    Techniques: Western Blot

    IL-1β impaired cLTP-induced GluA1 insertion and spine formation. a ” section. Scale bar: top panel, 10 μm; bottom panel, 5 μm. b Quantification of the images shown in a . Data are mean ± SEM from three independent experiments expressed in terms of control (* p

    Journal: Journal of Neuroinflammation

    Article Title: IL-1β suppresses cLTP-induced surface expression of GluA1 and actin polymerization via ceramide-mediated Src activation

    doi: 10.1186/s12974-018-1158-9

    Figure Lengend Snippet: IL-1β impaired cLTP-induced GluA1 insertion and spine formation. a ” section. Scale bar: top panel, 10 μm; bottom panel, 5 μm. b Quantification of the images shown in a . Data are mean ± SEM from three independent experiments expressed in terms of control (* p

    Article Snippet: Recombinant IL-1β (PeproTech) was dissolved in DMEM and used after one freeze–thaw cycle at 50 ng/ml.

    Techniques:

    IL-1β impaired cLTP-induced actin polymerization. a Cells were fixed following cLTP (30 min) and F-actin staining by phallodin. Scale bar, 5 μm. b Quantification of the images shown in a . Data are mean ± SEM from three independent experiments expressed in terms of control (* p

    Journal: Journal of Neuroinflammation

    Article Title: IL-1β suppresses cLTP-induced surface expression of GluA1 and actin polymerization via ceramide-mediated Src activation

    doi: 10.1186/s12974-018-1158-9

    Figure Lengend Snippet: IL-1β impaired cLTP-induced actin polymerization. a Cells were fixed following cLTP (30 min) and F-actin staining by phallodin. Scale bar, 5 μm. b Quantification of the images shown in a . Data are mean ± SEM from three independent experiments expressed in terms of control (* p

    Article Snippet: Recombinant IL-1β (PeproTech) was dissolved in DMEM and used after one freeze–thaw cycle at 50 ng/ml.

    Techniques: Staining

    IL-1β treatment decreased the cLTP-induced F-actin stabilization. FRAP analyses were performed using hippocampal neurons at 16–18 DIV that were transfected with Lifeact-GFP. a Illustration of FRAP setting. b Representative fluorescence images show spines of control group (-cLTP, top panel), cLTP group (middle panel), and cLTP+IL-1β group (bottom) during FRAP. Scale bar, 2 μm c Analysis of immobile fractions from data obtained in b ” section. Data are mean ± SEM ( n = 4, (* p

    Journal: Journal of Neuroinflammation

    Article Title: IL-1β suppresses cLTP-induced surface expression of GluA1 and actin polymerization via ceramide-mediated Src activation

    doi: 10.1186/s12974-018-1158-9

    Figure Lengend Snippet: IL-1β treatment decreased the cLTP-induced F-actin stabilization. FRAP analyses were performed using hippocampal neurons at 16–18 DIV that were transfected with Lifeact-GFP. a Illustration of FRAP setting. b Representative fluorescence images show spines of control group (-cLTP, top panel), cLTP group (middle panel), and cLTP+IL-1β group (bottom) during FRAP. Scale bar, 2 μm c Analysis of immobile fractions from data obtained in b ” section. Data are mean ± SEM ( n = 4, (* p

    Article Snippet: Recombinant IL-1β (PeproTech) was dissolved in DMEM and used after one freeze–thaw cycle at 50 ng/ml.

    Techniques: Transfection, Fluorescence

    The effect of IL-1β on cLTP-induced GluA1 insertion was mediated by the regulation of actin dynamics. a The expression of sGluA1 in unstimulated control cells and cells treated with cLTP in the presence or absence of IL-1β with or without jasplakinolide (200 nM). b Quantification of the images shown in a . Data are mean ± SEM from three independent experiments expressed in terms of control (* p

    Journal: Journal of Neuroinflammation

    Article Title: IL-1β suppresses cLTP-induced surface expression of GluA1 and actin polymerization via ceramide-mediated Src activation

    doi: 10.1186/s12974-018-1158-9

    Figure Lengend Snippet: The effect of IL-1β on cLTP-induced GluA1 insertion was mediated by the regulation of actin dynamics. a The expression of sGluA1 in unstimulated control cells and cells treated with cLTP in the presence or absence of IL-1β with or without jasplakinolide (200 nM). b Quantification of the images shown in a . Data are mean ± SEM from three independent experiments expressed in terms of control (* p

    Article Snippet: Recombinant IL-1β (PeproTech) was dissolved in DMEM and used after one freeze–thaw cycle at 50 ng/ml.

    Techniques: Expressing

    ( A ) IP-10, ( B ) RANTES, and ( C ) TNFα secretion levels induced in NHA by IL-1β stimulation (20 ng/ml) are significantly reduced with exposure to Palm Fruit Bioactives at 24 and 96 hours in a dose-dependent manner. The normalized percent change (%) for each PFB treatment condition compared with the positive control, IL-1β stimulation alone, for each time point. Data represent the mean percentage change ± SD of three Luminex replicates (*p

    Journal: Scientific Reports

    Article Title: Palm Fruit Bioactives modulate human astrocyte activity in vitro altering the cytokine secretome reducing levels of TNFα, RANTES and IP-10

    doi: 10.1038/s41598-018-34763-3

    Figure Lengend Snippet: ( A ) IP-10, ( B ) RANTES, and ( C ) TNFα secretion levels induced in NHA by IL-1β stimulation (20 ng/ml) are significantly reduced with exposure to Palm Fruit Bioactives at 24 and 96 hours in a dose-dependent manner. The normalized percent change (%) for each PFB treatment condition compared with the positive control, IL-1β stimulation alone, for each time point. Data represent the mean percentage change ± SD of three Luminex replicates (*p

    Article Snippet: At this time, to induce an inflammatory condition, cells were treated with recombinant human IL-1β (Sigma-Aldrich, St Louis, Missouri, USA) at a concentration of 20 ng/mL as commonly used for the activation of astrocytes , , .

    Techniques: Positive Control, Luminex

    Profiles of cytokines/chemokines produced by human astrocytes with/without IL-1β stimulation reveal 2 profiles: normal (basal) and inflammatory (post-IL-1β) The inflammatory profile consists of those secreted cytokines/chemokines which were significantly altered with IL-1β stimulation compared to their basal expression levels at 24 h or 96 h, while the normal profile consists of those cytokines/chemokines detectable in the basal state.

    Journal: Scientific Reports

    Article Title: Palm Fruit Bioactives modulate human astrocyte activity in vitro altering the cytokine secretome reducing levels of TNFα, RANTES and IP-10

    doi: 10.1038/s41598-018-34763-3

    Figure Lengend Snippet: Profiles of cytokines/chemokines produced by human astrocytes with/without IL-1β stimulation reveal 2 profiles: normal (basal) and inflammatory (post-IL-1β) The inflammatory profile consists of those secreted cytokines/chemokines which were significantly altered with IL-1β stimulation compared to their basal expression levels at 24 h or 96 h, while the normal profile consists of those cytokines/chemokines detectable in the basal state.

    Article Snippet: At this time, to induce an inflammatory condition, cells were treated with recombinant human IL-1β (Sigma-Aldrich, St Louis, Missouri, USA) at a concentration of 20 ng/mL as commonly used for the activation of astrocytes , , .

    Techniques: Produced, Expressing

    Time-dependent differential expression of cytokines/chemokines induced by IL-1β stimulation in human astrocytes. Cytokines induced within 24 hours are designated “early short response.” Cytokines produced after 96 hours of induction are designated “delayed response.” Cytokines induced within 24 h which remain elevated at 96 hours are designated “early-prolonged response”.

    Journal: Scientific Reports

    Article Title: Palm Fruit Bioactives modulate human astrocyte activity in vitro altering the cytokine secretome reducing levels of TNFα, RANTES and IP-10

    doi: 10.1038/s41598-018-34763-3

    Figure Lengend Snippet: Time-dependent differential expression of cytokines/chemokines induced by IL-1β stimulation in human astrocytes. Cytokines induced within 24 hours are designated “early short response.” Cytokines produced after 96 hours of induction are designated “delayed response.” Cytokines induced within 24 h which remain elevated at 96 hours are designated “early-prolonged response”.

    Article Snippet: At this time, to induce an inflammatory condition, cells were treated with recombinant human IL-1β (Sigma-Aldrich, St Louis, Missouri, USA) at a concentration of 20 ng/mL as commonly used for the activation of astrocytes , , .

    Techniques: Expressing, Produced

    Effect of PFB on soluble adhesion molecules expressed by NHA with 24 h or 96 h stimulation with IL-1β. ( A , B ) PFB exposure decreased siCAM and sVCAM expression in both unstimulated and IL-1β-stimulated NHA. ( C ) NCAM expression did not differ upon exposure to IL-1β. Values given as cytokine concentration, mean +/− SD. Experiment was performed in triplicate. ## p

    Journal: Scientific Reports

    Article Title: Palm Fruit Bioactives modulate human astrocyte activity in vitro altering the cytokine secretome reducing levels of TNFα, RANTES and IP-10

    doi: 10.1038/s41598-018-34763-3

    Figure Lengend Snippet: Effect of PFB on soluble adhesion molecules expressed by NHA with 24 h or 96 h stimulation with IL-1β. ( A , B ) PFB exposure decreased siCAM and sVCAM expression in both unstimulated and IL-1β-stimulated NHA. ( C ) NCAM expression did not differ upon exposure to IL-1β. Values given as cytokine concentration, mean +/− SD. Experiment was performed in triplicate. ## p

    Article Snippet: At this time, to induce an inflammatory condition, cells were treated with recombinant human IL-1β (Sigma-Aldrich, St Louis, Missouri, USA) at a concentration of 20 ng/mL as commonly used for the activation of astrocytes , , .

    Techniques: Expressing, Concentration Assay

    Elevated Reactive Oxygen Species production in NHA stimulated by IL-1β is reduced by treatment with Palm Fruit Bioactives, as measured by DCFDA Assay after 24 h. No change is observed between ROS production between untreated NHA and the highest concentration of PFB treatment used, while increased ROS production from stimulation is decreased by PFB. Fluorescence intensity correlates with level of oxidative stress per condition in the DCDFA Assay. Values given as mean fluorescence intensity +/− standard deviation of three replicates. Student’s t-test, ### p

    Journal: Scientific Reports

    Article Title: Palm Fruit Bioactives modulate human astrocyte activity in vitro altering the cytokine secretome reducing levels of TNFα, RANTES and IP-10

    doi: 10.1038/s41598-018-34763-3

    Figure Lengend Snippet: Elevated Reactive Oxygen Species production in NHA stimulated by IL-1β is reduced by treatment with Palm Fruit Bioactives, as measured by DCFDA Assay after 24 h. No change is observed between ROS production between untreated NHA and the highest concentration of PFB treatment used, while increased ROS production from stimulation is decreased by PFB. Fluorescence intensity correlates with level of oxidative stress per condition in the DCDFA Assay. Values given as mean fluorescence intensity +/− standard deviation of three replicates. Student’s t-test, ### p

    Article Snippet: At this time, to induce an inflammatory condition, cells were treated with recombinant human IL-1β (Sigma-Aldrich, St Louis, Missouri, USA) at a concentration of 20 ng/mL as commonly used for the activation of astrocytes , , .

    Techniques: Concentration Assay, Fluorescence, Standard Deviation

    Effects of phenolic vs. non-phenolic fractions of PFB on ( A ) IP10, ( B ) RANTES, and ( C ) TNFα levels in IL-1β-stimulated NHA. Concentrations of IP10, RANTES, TNFα for untreated and IL-1β stimulated controls, and IL-1β for different treatment conditions: PFB, non-phenolic PFB fraction, and phenolic PFB fraction at 20 and 40 μL/mL concentrations for each condition. Mean and standard deviation given for three replicates. Student’s t-test, ### p

    Journal: Scientific Reports

    Article Title: Palm Fruit Bioactives modulate human astrocyte activity in vitro altering the cytokine secretome reducing levels of TNFα, RANTES and IP-10

    doi: 10.1038/s41598-018-34763-3

    Figure Lengend Snippet: Effects of phenolic vs. non-phenolic fractions of PFB on ( A ) IP10, ( B ) RANTES, and ( C ) TNFα levels in IL-1β-stimulated NHA. Concentrations of IP10, RANTES, TNFα for untreated and IL-1β stimulated controls, and IL-1β for different treatment conditions: PFB, non-phenolic PFB fraction, and phenolic PFB fraction at 20 and 40 μL/mL concentrations for each condition. Mean and standard deviation given for three replicates. Student’s t-test, ### p

    Article Snippet: At this time, to induce an inflammatory condition, cells were treated with recombinant human IL-1β (Sigma-Aldrich, St Louis, Missouri, USA) at a concentration of 20 ng/mL as commonly used for the activation of astrocytes , , .

    Techniques: Standard Deviation

    cGAS-dependent inflammasome activation is necessary for the control of MCMV. WT, Aim2 −/− , cGAS −/− , or Ifnar −/− BMDMs were infected with MCMV at multiplicity of infection 1. (a) IL-1β ELISA of supernatants from BMDMs 1 d postinfection. (b and c) WT, Ifnar −/− , or Ifnar −/− cGAS −/− BMDMs were infected with MCMV at multiplicity of infection 1. (b) IL-1β ELISA of supernatants from BMDMs 1–3 d postinfection. (c) Relative MCMV genome copy number from BMDMs 2 d postinfection. (d and e) WT and Ifnar −/− BMDMs were infected in the presence or absence of caspase-1 inhibitor, Z-YVAD, and assayed for IL-1β (d) or relative MCVM genome copy (e). In e, left and right panels are from the same experiment. n = 2 independent experiments. Shown is a representative experiment with technical replicates of six; mean ± SEM. (f and g) WT, Ifnar −/− , or Ifnar −/− cGAS −/− mice were infected with MCMV for 36 h. IL-18 serum levels (f), IFNγ spleen levels (g), and relative MCMV genome copy number from spleens after infection (h). n = 8. Error bars, SEM; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: A noncanonical function of cGAMP in inflammasome priming and activation

    doi: 10.1084/jem.20171749

    Figure Lengend Snippet: cGAS-dependent inflammasome activation is necessary for the control of MCMV. WT, Aim2 −/− , cGAS −/− , or Ifnar −/− BMDMs were infected with MCMV at multiplicity of infection 1. (a) IL-1β ELISA of supernatants from BMDMs 1 d postinfection. (b and c) WT, Ifnar −/− , or Ifnar −/− cGAS −/− BMDMs were infected with MCMV at multiplicity of infection 1. (b) IL-1β ELISA of supernatants from BMDMs 1–3 d postinfection. (c) Relative MCMV genome copy number from BMDMs 2 d postinfection. (d and e) WT and Ifnar −/− BMDMs were infected in the presence or absence of caspase-1 inhibitor, Z-YVAD, and assayed for IL-1β (d) or relative MCVM genome copy (e). In e, left and right panels are from the same experiment. n = 2 independent experiments. Shown is a representative experiment with technical replicates of six; mean ± SEM. (f and g) WT, Ifnar −/− , or Ifnar −/− cGAS −/− mice were infected with MCMV for 36 h. IL-18 serum levels (f), IFNγ spleen levels (g), and relative MCMV genome copy number from spleens after infection (h). n = 8. Error bars, SEM; *, P

    Article Snippet: Human IL-1β was detected with the OptEIA kit set II (557953; BD Biosciences).

    Techniques: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Mouse Assay

    cGAMP induces AIM2-NLRP3-ASC inflammasome. IL-1β ELISAs from supernatants of LPS-primed BMDMs transfected with the indicated PAMP or DAMP. BMDMs were obtained from WT or Aim2 −/− (a), Nlrp3 −/− (b), Asc −/− (c), Casp1 −/− (d), Nlrc4 −/− (e), and Casp11 −/− and Ice −/− (f). NT, not transfected with PAMP; TFXN, transfected with transfection reagent only. n = 3 independent experiments ± SEM. **, P

    Journal: The Journal of Experimental Medicine

    Article Title: A noncanonical function of cGAMP in inflammasome priming and activation

    doi: 10.1084/jem.20171749

    Figure Lengend Snippet: cGAMP induces AIM2-NLRP3-ASC inflammasome. IL-1β ELISAs from supernatants of LPS-primed BMDMs transfected with the indicated PAMP or DAMP. BMDMs were obtained from WT or Aim2 −/− (a), Nlrp3 −/− (b), Asc −/− (c), Casp1 −/− (d), Nlrc4 −/− (e), and Casp11 −/− and Ice −/− (f). NT, not transfected with PAMP; TFXN, transfected with transfection reagent only. n = 3 independent experiments ± SEM. **, P

    Article Snippet: Human IL-1β was detected with the OptEIA kit set II (557953; BD Biosciences).

    Techniques: Transfection

    cGAMP alone is sufficient to induce the inflammasome in vivo . (a) IL-1β from BALF obtained from WT mice treated intranasally with cGAMP followed by saline or with cGAMP followed by cGAMP. (b) IL-1β from BALF obtained from WT, Aim2 −/− , Nlrp3 −/− , or Aim2 −/− Nlrp3 −/− DKO mice intranasally treated with cGAMP. (c and d) IFN-β ELISA from BALF or serum from WT and gene-deficient mice as in b. For cGAMP/cGAMP-treated: WT, n = 17; Aim2 −/− , n = 10; Nlrp3 −/− , n = 8; Aim2 −/− Nlrp3 −/− DKO, n = 8; for saline treated: WT, n = 15; Aim2 −/− , n = 9; Nlrp3 −/− , n = 8; Aim2 −/− Nlrp3 −/− DKO, n = 7; for cGAMP followed by saline: n = 6 for all strains. Error bars, SEM; ns, not significant; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: A noncanonical function of cGAMP in inflammasome priming and activation

    doi: 10.1084/jem.20171749

    Figure Lengend Snippet: cGAMP alone is sufficient to induce the inflammasome in vivo . (a) IL-1β from BALF obtained from WT mice treated intranasally with cGAMP followed by saline or with cGAMP followed by cGAMP. (b) IL-1β from BALF obtained from WT, Aim2 −/− , Nlrp3 −/− , or Aim2 −/− Nlrp3 −/− DKO mice intranasally treated with cGAMP. (c and d) IFN-β ELISA from BALF or serum from WT and gene-deficient mice as in b. For cGAMP/cGAMP-treated: WT, n = 17; Aim2 −/− , n = 10; Nlrp3 −/− , n = 8; Aim2 −/− Nlrp3 −/− DKO, n = 8; for saline treated: WT, n = 15; Aim2 −/− , n = 9; Nlrp3 −/− , n = 8; Aim2 −/− Nlrp3 −/− DKO, n = 7; for cGAMP followed by saline: n = 6 for all strains. Error bars, SEM; ns, not significant; *, P

    Article Snippet: Human IL-1β was detected with the OptEIA kit set II (557953; BD Biosciences).

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

    cGAMP-induced inflammasome activation after LPS priming is partially dependent on Sting. (a and b)WT and Ifnar −/− (a) or Sting gt/gt (b) BMDMs were LPS-primed followed by transfection with cGAMP as indicated for 6 h, and supernatants were measured for IL-1β. (c–g) mRNA levels as determined by qPCR of Aim2 (c), Nlrp3 (d), Casp1 (e), Il1b (f), and Ifnb (g). (h and i) Immunoblots of lysates (h) or supernatants (i) from the same experiment. n = 5 independent experiments ± SEM. ***, P

    Journal: The Journal of Experimental Medicine

    Article Title: A noncanonical function of cGAMP in inflammasome priming and activation

    doi: 10.1084/jem.20171749

    Figure Lengend Snippet: cGAMP-induced inflammasome activation after LPS priming is partially dependent on Sting. (a and b)WT and Ifnar −/− (a) or Sting gt/gt (b) BMDMs were LPS-primed followed by transfection with cGAMP as indicated for 6 h, and supernatants were measured for IL-1β. (c–g) mRNA levels as determined by qPCR of Aim2 (c), Nlrp3 (d), Casp1 (e), Il1b (f), and Ifnb (g). (h and i) Immunoblots of lysates (h) or supernatants (i) from the same experiment. n = 5 independent experiments ± SEM. ***, P

    Article Snippet: Human IL-1β was detected with the OptEIA kit set II (557953; BD Biosciences).

    Techniques: Activation Assay, Transfection, Real-time Polymerase Chain Reaction, Western Blot

    cGAMP induces complexes containing both NLRP3 and AIM2. WT, Aim2 −/− , Nlrp3 −/− , or Aim2 −/− Nlrp3 −/− DKO BMDMs were LPS-primed followed by transfection with cGAMP or indicated PAMPs or DAMPs. (a–c) IL-1β ELISA (a) and cell death (b and c) as measured by adenylate cyclase release in supernatants. n = 3 independent experiments, mean ± SEM. ***, P

    Journal: The Journal of Experimental Medicine

    Article Title: A noncanonical function of cGAMP in inflammasome priming and activation

    doi: 10.1084/jem.20171749

    Figure Lengend Snippet: cGAMP induces complexes containing both NLRP3 and AIM2. WT, Aim2 −/− , Nlrp3 −/− , or Aim2 −/− Nlrp3 −/− DKO BMDMs were LPS-primed followed by transfection with cGAMP or indicated PAMPs or DAMPs. (a–c) IL-1β ELISA (a) and cell death (b and c) as measured by adenylate cyclase release in supernatants. n = 3 independent experiments, mean ± SEM. ***, P

    Article Snippet: Human IL-1β was detected with the OptEIA kit set II (557953; BD Biosciences).

    Techniques: Transfection, Enzyme-linked Immunosorbent Assay

    cGAS is important for DNA-induced inflammasome. (a) Pathways that can be activated by dsDNA to activate the inflammasome. To the left is the published AIM2-dependent pathway. To the right is the proposed pathway mediated by cGAMP. (b) Relative cGAS as determined by qPCR of JAWSII cells containing empty vector (sh-ev) or shRNA targeting cGAS (sh-cGAS). (c) IFN-β or (d) IL-1β levels of supernatants from JAWSII DCs primed with LPS then transfected with dAdT, transfection reagent alone (TFXN), or not treated (NT). (e and f) IL-1β (e) or IFN-β (f) levels of supernatants from BMDM primed with LPS then transfected with the indicated PAMP or DAMP, or not transfected (NT). n = 3 independent experiments, mean ± SEM. ***, P

    Journal: The Journal of Experimental Medicine

    Article Title: A noncanonical function of cGAMP in inflammasome priming and activation

    doi: 10.1084/jem.20171749

    Figure Lengend Snippet: cGAS is important for DNA-induced inflammasome. (a) Pathways that can be activated by dsDNA to activate the inflammasome. To the left is the published AIM2-dependent pathway. To the right is the proposed pathway mediated by cGAMP. (b) Relative cGAS as determined by qPCR of JAWSII cells containing empty vector (sh-ev) or shRNA targeting cGAS (sh-cGAS). (c) IFN-β or (d) IL-1β levels of supernatants from JAWSII DCs primed with LPS then transfected with dAdT, transfection reagent alone (TFXN), or not treated (NT). (e and f) IL-1β (e) or IFN-β (f) levels of supernatants from BMDM primed with LPS then transfected with the indicated PAMP or DAMP, or not transfected (NT). n = 3 independent experiments, mean ± SEM. ***, P

    Article Snippet: Human IL-1β was detected with the OptEIA kit set II (557953; BD Biosciences).

    Techniques: Real-time Polymerase Chain Reaction, Plasmid Preparation, shRNA, Transfection

    cGAMP induces inflammasome activation. (a and b) IFN-β (a) and IL-1β (b) ELISAs of BMDM supernatants primed with LPS then transfected with various dosages of cGAMP ( n = 3 independent experiments, mean ± SEM). (c) Western blots of supernatants and cell lysates from b. (d) IL-18 ELISA of BMDMs treated as described in a. (e–g) ELISAs of primary human macrophages (e and f) and human DCs (g) treated as depicted for BMDMs (individual donors ± SD). casp-1, caspase-1; Mφ, macrophage, NT, not transfected with PAMP; Tfxn, transfected with transfection reagent only. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: A noncanonical function of cGAMP in inflammasome priming and activation

    doi: 10.1084/jem.20171749

    Figure Lengend Snippet: cGAMP induces inflammasome activation. (a and b) IFN-β (a) and IL-1β (b) ELISAs of BMDM supernatants primed with LPS then transfected with various dosages of cGAMP ( n = 3 independent experiments, mean ± SEM). (c) Western blots of supernatants and cell lysates from b. (d) IL-18 ELISA of BMDMs treated as described in a. (e–g) ELISAs of primary human macrophages (e and f) and human DCs (g) treated as depicted for BMDMs (individual donors ± SD). casp-1, caspase-1; Mφ, macrophage, NT, not transfected with PAMP; Tfxn, transfected with transfection reagent only. *, P

    Article Snippet: Human IL-1β was detected with the OptEIA kit set II (557953; BD Biosciences).

    Techniques: Activation Assay, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

    cGAMP alone can serve as the priming signal by increasing mRNA levels for inflammasome components in a Sting- and Ifnar-dependent fashion. BMDMs were transfected with increasing amounts of 2′3′-cGAMP, dAdT, or addition of 200 ng/ml LPS and analyzed after 6 h. (a–f) qPCR transcript levels of inflammasome components Aim2 , Nlrp3 , Casp1 , Il1b , and Asc in addition to Ifnb produced by BMDM after treatment. n = 3 independent experiments. Shown is a representative with technical replicates of three, mean ± SD. (g–j) IFN-β (g and h) and IL-1β (i and j) in supernatants from BMDMs treated with increasing amounts of 2′3′-cGAMP for 6 h (g and i) and 19 h (h and j). NT, not treated. WT, Ifnar −/− , or Sting gt/gt BMDMs were transfected with cGAMP, transfection reagent alone (TFXN), or not treated (NT) for 19 h. (k–m) IL-1β (k), IFN-β (l), or Tnf (m) levels of supernatants after treatment. (n–r) Transcript levels as measured by qPCR for inflammasome components Aim2 , Nlrp3 , Casp1 , Il1b , and Asc produced by BMDMs after treatment. n = 3 independent experiments, mean ± SEM; ns, not significant; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: A noncanonical function of cGAMP in inflammasome priming and activation

    doi: 10.1084/jem.20171749

    Figure Lengend Snippet: cGAMP alone can serve as the priming signal by increasing mRNA levels for inflammasome components in a Sting- and Ifnar-dependent fashion. BMDMs were transfected with increasing amounts of 2′3′-cGAMP, dAdT, or addition of 200 ng/ml LPS and analyzed after 6 h. (a–f) qPCR transcript levels of inflammasome components Aim2 , Nlrp3 , Casp1 , Il1b , and Asc in addition to Ifnb produced by BMDM after treatment. n = 3 independent experiments. Shown is a representative with technical replicates of three, mean ± SD. (g–j) IFN-β (g and h) and IL-1β (i and j) in supernatants from BMDMs treated with increasing amounts of 2′3′-cGAMP for 6 h (g and i) and 19 h (h and j). NT, not treated. WT, Ifnar −/− , or Sting gt/gt BMDMs were transfected with cGAMP, transfection reagent alone (TFXN), or not treated (NT) for 19 h. (k–m) IL-1β (k), IFN-β (l), or Tnf (m) levels of supernatants after treatment. (n–r) Transcript levels as measured by qPCR for inflammasome components Aim2 , Nlrp3 , Casp1 , Il1b , and Asc produced by BMDMs after treatment. n = 3 independent experiments, mean ± SEM; ns, not significant; *, P

    Article Snippet: Human IL-1β was detected with the OptEIA kit set II (557953; BD Biosciences).

    Techniques: Transfection, Real-time Polymerase Chain Reaction, Produced

    HF ameliorates LPS-induced production of IL-1β in macrophages by affecting mRNA stability and processing of mature IL-1β. (A–B) IL-1β production from LPS (500 ng/ml)-primed or -unprimed BMDMs treated with different concentrations of HF or MAZ1310 (control) for 6 h. ATP (5 mM) was added to the LPS-stimulated macrophage cultures for 30 min at the end of time point ( S1 Data ). Statistical significance was determined by student t test. * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005. (C) IL-1β production from LPS-primed macrophages stimulated with MSU (150 ug/ml), or ALU (200 ug/ml) for 6 h, or infected with S . typhimurium (MOI 10) in presence or absence of HF ( S1 Data ). (D) IL-1β and caspase-1 (pro and active) expression by immunoblot analysis from LPS-primed BMDMs treated with HF as indicated; β-actin was used as loading control. (E) ROS levels detected by CM-H2DCFDA staining in macrophages treated with HF or LPS plus HF. (F, G) qRT-PCR analysis of mature IL-1β and pre–IL-1β mRNA levels in J774A.1 cells stimulated with LPS or LPS plus HF ( S1 Data ). (H) Analysis of IL-1β mRNA levels by qRT-PCR in LPS-primed macrophages treated with Act-D for 2 h followed by HF treatment for an additional 2 h ( S1 Data ). * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005 were considered statistically significant. Data are representative of 1 of 3–4 independent experiments. Act-D, actinomycin-D; ALU, aluminum hydroxide; BMDM, bone marrow–derived macrophage; HF, Halofuginone; IL-1β, interleukin 1β; LPS, lipopolysaccharide; Lys., cell lysates; MOI, multiplicity of infection; MSU, monosodium urate; qRT-PCR, quantitative reverse transcription PCR; ROS, reactive oxygen species; Sup., culture supernatant.

    Journal: PLoS Biology

    Article Title: Amino acid starvation sensing dampens IL-1β production by activating riboclustering and autophagy

    doi: 10.1371/journal.pbio.2005317

    Figure Lengend Snippet: HF ameliorates LPS-induced production of IL-1β in macrophages by affecting mRNA stability and processing of mature IL-1β. (A–B) IL-1β production from LPS (500 ng/ml)-primed or -unprimed BMDMs treated with different concentrations of HF or MAZ1310 (control) for 6 h. ATP (5 mM) was added to the LPS-stimulated macrophage cultures for 30 min at the end of time point ( S1 Data ). Statistical significance was determined by student t test. * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005. (C) IL-1β production from LPS-primed macrophages stimulated with MSU (150 ug/ml), or ALU (200 ug/ml) for 6 h, or infected with S . typhimurium (MOI 10) in presence or absence of HF ( S1 Data ). (D) IL-1β and caspase-1 (pro and active) expression by immunoblot analysis from LPS-primed BMDMs treated with HF as indicated; β-actin was used as loading control. (E) ROS levels detected by CM-H2DCFDA staining in macrophages treated with HF or LPS plus HF. (F, G) qRT-PCR analysis of mature IL-1β and pre–IL-1β mRNA levels in J774A.1 cells stimulated with LPS or LPS plus HF ( S1 Data ). (H) Analysis of IL-1β mRNA levels by qRT-PCR in LPS-primed macrophages treated with Act-D for 2 h followed by HF treatment for an additional 2 h ( S1 Data ). * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005 were considered statistically significant. Data are representative of 1 of 3–4 independent experiments. Act-D, actinomycin-D; ALU, aluminum hydroxide; BMDM, bone marrow–derived macrophage; HF, Halofuginone; IL-1β, interleukin 1β; LPS, lipopolysaccharide; Lys., cell lysates; MOI, multiplicity of infection; MSU, monosodium urate; qRT-PCR, quantitative reverse transcription PCR; ROS, reactive oxygen species; Sup., culture supernatant.

    Article Snippet: The antibodies used for immunoblot analysis were rabbit anti–eIF2α-P, anti–GCN2-P (Cell Signaling Technology), rabbit anti–IL-1β (Santa Cruz), rabbit anti pro–caspase-1 (Santa Cruz), goat anti–cleaved caspase-1 (Santa Cruz), rabbit anti–β-actin (Sigma-Aldrich), goat anti–TIA-1/TIAR (Santa Cruz), rabbit anti-LC3 (Cell Signaling Technology), and mouse anti-Flag (Sigma-Aldrich).

    Techniques: Infection, Expressing, Staining, Quantitative RT-PCR, Activated Clotting Time Assay, Derivative Assay, Polymerase Chain Reaction

    HF attenuates LPS-induced IL-1β production through GCN2-dependent activation of PTR events such as riboclustering and SG formation. (A) Immunoblot analysis of GCN2 and eIF2-α phosphorylation in the lysates of macrophages treated or untreated with varying concentrations of HF for 3 h. (B) Confocal microscopy imaging of SGs, indicated by white arrows in macrophages stimulated with HF (20 nM) for 3 h; nuclei were stained with DAPI (blue). Scale bars, 10 μm. (C) Quantification of average number of SGs per cell, from 5 different fields taken from the results in panel B ( S1 Data ). (D) Confocal microscopy imaging of SGs in WT (top) or GCN2 −/− MEFs (bottom). (E) Quantification of an average number of SGs per cell in WT or GCN2 −/− cells treated with HF ( S1 Data ). (F, G) IL-1β (panel F) or TNF-α (panel G) levels by ELISA in the culture supernatants of WT or GCN2 −/− BMDMs primed with LPS for 3 h followed by HF treatment. ATP (5 mM) was added to the cultures for 30 min at the end of the experiment ( S1 Data ). * P

    Journal: PLoS Biology

    Article Title: Amino acid starvation sensing dampens IL-1β production by activating riboclustering and autophagy

    doi: 10.1371/journal.pbio.2005317

    Figure Lengend Snippet: HF attenuates LPS-induced IL-1β production through GCN2-dependent activation of PTR events such as riboclustering and SG formation. (A) Immunoblot analysis of GCN2 and eIF2-α phosphorylation in the lysates of macrophages treated or untreated with varying concentrations of HF for 3 h. (B) Confocal microscopy imaging of SGs, indicated by white arrows in macrophages stimulated with HF (20 nM) for 3 h; nuclei were stained with DAPI (blue). Scale bars, 10 μm. (C) Quantification of average number of SGs per cell, from 5 different fields taken from the results in panel B ( S1 Data ). (D) Confocal microscopy imaging of SGs in WT (top) or GCN2 −/− MEFs (bottom). (E) Quantification of an average number of SGs per cell in WT or GCN2 −/− cells treated with HF ( S1 Data ). (F, G) IL-1β (panel F) or TNF-α (panel G) levels by ELISA in the culture supernatants of WT or GCN2 −/− BMDMs primed with LPS for 3 h followed by HF treatment. ATP (5 mM) was added to the cultures for 30 min at the end of the experiment ( S1 Data ). * P

    Article Snippet: The antibodies used for immunoblot analysis were rabbit anti–eIF2α-P, anti–GCN2-P (Cell Signaling Technology), rabbit anti–IL-1β (Santa Cruz), rabbit anti pro–caspase-1 (Santa Cruz), goat anti–cleaved caspase-1 (Santa Cruz), rabbit anti–β-actin (Sigma-Aldrich), goat anti–TIA-1/TIAR (Santa Cruz), rabbit anti-LC3 (Cell Signaling Technology), and mouse anti-Flag (Sigma-Aldrich).

    Techniques: Activation Assay, Confocal Microscopy, Imaging, Staining, Enzyme-linked Immunosorbent Assay

    HF mitigates the severity of DSS-induced colitis in mice. (A) Body weight (percentage of initial body weight) of mice ( n = 5) ( S1 Data ). (B) Quantification of IL-1β levels by ELISA in serum samples of the indicated mice ( S1 Data ). ** P ≤ 0.0015. (C) Visualization of rectal bleeding. (D) Visualization of typical colon length in control, HF-, DSS-, and DSS plus HF–treated mice. (E) Measurement of colon length (cm) ( S1 Data ). (F) Visualization of mucosal epithelium erosion and crypt loss in colon sections (HE-stained) as indicated. (G) Immunoblot analysis of IL-1β levels in the large intestine tissue samples. (H) Densitometric analysis of pro–IL-1β levels from colon tissues of mice subjected to treatments as indicated ( n = 4) ( S1 Data ). β-actin was used as loading control. Data are representative of 1 of 3 separate experiments. DSS, dextran sulfate sodium; HE, hematoxylin–eosin; HF, Halofuginone; IL-1β, interleukin 1β.

    Journal: PLoS Biology

    Article Title: Amino acid starvation sensing dampens IL-1β production by activating riboclustering and autophagy

    doi: 10.1371/journal.pbio.2005317

    Figure Lengend Snippet: HF mitigates the severity of DSS-induced colitis in mice. (A) Body weight (percentage of initial body weight) of mice ( n = 5) ( S1 Data ). (B) Quantification of IL-1β levels by ELISA in serum samples of the indicated mice ( S1 Data ). ** P ≤ 0.0015. (C) Visualization of rectal bleeding. (D) Visualization of typical colon length in control, HF-, DSS-, and DSS plus HF–treated mice. (E) Measurement of colon length (cm) ( S1 Data ). (F) Visualization of mucosal epithelium erosion and crypt loss in colon sections (HE-stained) as indicated. (G) Immunoblot analysis of IL-1β levels in the large intestine tissue samples. (H) Densitometric analysis of pro–IL-1β levels from colon tissues of mice subjected to treatments as indicated ( n = 4) ( S1 Data ). β-actin was used as loading control. Data are representative of 1 of 3 separate experiments. DSS, dextran sulfate sodium; HE, hematoxylin–eosin; HF, Halofuginone; IL-1β, interleukin 1β.

    Article Snippet: The antibodies used for immunoblot analysis were rabbit anti–eIF2α-P, anti–GCN2-P (Cell Signaling Technology), rabbit anti–IL-1β (Santa Cruz), rabbit anti pro–caspase-1 (Santa Cruz), goat anti–cleaved caspase-1 (Santa Cruz), rabbit anti–β-actin (Sigma-Aldrich), goat anti–TIA-1/TIAR (Santa Cruz), rabbit anti-LC3 (Cell Signaling Technology), and mouse anti-Flag (Sigma-Aldrich).

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

    IL-1β transcripts targeted to SGs are degraded by the activation of autophagy during HF treatment. (A) Confocal microscopy imaging of LC3 punctates (indicated by white arrows) in BMDMs left untreated (media control) or treated with HF (20 nM) at time points indicated. Scale bars, 5 μm. (B) LC3 punctate counts per cell ( S1 Data ). (C) Immunoblot analysis of autophagy marker, LC3 in J774A.1 macrophages left untreated or treated with HF in the presence or absence of autophagy inhibitor Baf. (D) Immunoblotting of LC3 in the lysates of J774A.1 macrophages treated with LPS plus HF or LPS alone. ATP was added to the cultures for 30’ at the end of the experiment. β-actin was used as a loading control. (E) Immunoblot analysis of p62/SQSTM1 expression in J774A.1 macrophages stimulated with LPS plus ATP or LPS plus HF plus ATP in the presence or absence of autophagy inhibitor Baf ( S1 data ). (F) Immunoblot analysis of TIA-1/TIAR expression in J774A.1 macrophages treated with HF or LPS in the presence or absence of Baf (10 nM). (G–I) Quantification of IL-1β levels using ELISA in culture supernatants of macrophages stimulated with LPS (500 ng/ml) alone or LPS (500 ng/ml) plus HF (20 nM) in the presence or absence of pharmacological inhibitors of autophagy, 3-MA, Wortmannin (500 nM), or Baf (10 nM). ATP (5 mM) was added for 30 min at the end of the experiment ( S1 Data ). Statistical significance was determined by student t test. * P ≤ 0.05, ** P ≤ 0.005. (J, K) qRT-PCR analysis of IL-1β in HEK293T cells transfected with pCMV6-IL-1β or cotransfected with pCMV6-IL-1β plus pEYFP-TIA-1/pEYFP-TIAR followed by rapamycin (100 nM) or Baf treatment ( S1 Data ). *** P ≤ 0.0005. (L) qRT-PCR analysis of IL-1β mRNA in the RIP material of LPS-primed macrophages treated with HF in presence of Wortmannin (500 nM) or CQ (25 μM) ( S1 Data ). Data are representative of 1 of 3 independent experiments. 3-MA, 3-methyl adenine; Baf, Bafilomycin A1; BMDM, bone marrow–derived macrophage; CQ, chloroquine; HEK293T, human embryonic kidney cells 293T; HF, Halofuginone; IL-1β, interleukin 1β; LPS, lipopolysaccharide; p62/SQSTM1, sequestosome 1; qRT-PCR, quantitative reverse transcription PCR; RIP, RNA immunoprecipitation; SG, stress granule; TIA-1, T cell–restricted intracellular antigen-1; TIAR, TIA-1–related.

    Journal: PLoS Biology

    Article Title: Amino acid starvation sensing dampens IL-1β production by activating riboclustering and autophagy

    doi: 10.1371/journal.pbio.2005317

    Figure Lengend Snippet: IL-1β transcripts targeted to SGs are degraded by the activation of autophagy during HF treatment. (A) Confocal microscopy imaging of LC3 punctates (indicated by white arrows) in BMDMs left untreated (media control) or treated with HF (20 nM) at time points indicated. Scale bars, 5 μm. (B) LC3 punctate counts per cell ( S1 Data ). (C) Immunoblot analysis of autophagy marker, LC3 in J774A.1 macrophages left untreated or treated with HF in the presence or absence of autophagy inhibitor Baf. (D) Immunoblotting of LC3 in the lysates of J774A.1 macrophages treated with LPS plus HF or LPS alone. ATP was added to the cultures for 30’ at the end of the experiment. β-actin was used as a loading control. (E) Immunoblot analysis of p62/SQSTM1 expression in J774A.1 macrophages stimulated with LPS plus ATP or LPS plus HF plus ATP in the presence or absence of autophagy inhibitor Baf ( S1 data ). (F) Immunoblot analysis of TIA-1/TIAR expression in J774A.1 macrophages treated with HF or LPS in the presence or absence of Baf (10 nM). (G–I) Quantification of IL-1β levels using ELISA in culture supernatants of macrophages stimulated with LPS (500 ng/ml) alone or LPS (500 ng/ml) plus HF (20 nM) in the presence or absence of pharmacological inhibitors of autophagy, 3-MA, Wortmannin (500 nM), or Baf (10 nM). ATP (5 mM) was added for 30 min at the end of the experiment ( S1 Data ). Statistical significance was determined by student t test. * P ≤ 0.05, ** P ≤ 0.005. (J, K) qRT-PCR analysis of IL-1β in HEK293T cells transfected with pCMV6-IL-1β or cotransfected with pCMV6-IL-1β plus pEYFP-TIA-1/pEYFP-TIAR followed by rapamycin (100 nM) or Baf treatment ( S1 Data ). *** P ≤ 0.0005. (L) qRT-PCR analysis of IL-1β mRNA in the RIP material of LPS-primed macrophages treated with HF in presence of Wortmannin (500 nM) or CQ (25 μM) ( S1 Data ). Data are representative of 1 of 3 independent experiments. 3-MA, 3-methyl adenine; Baf, Bafilomycin A1; BMDM, bone marrow–derived macrophage; CQ, chloroquine; HEK293T, human embryonic kidney cells 293T; HF, Halofuginone; IL-1β, interleukin 1β; LPS, lipopolysaccharide; p62/SQSTM1, sequestosome 1; qRT-PCR, quantitative reverse transcription PCR; RIP, RNA immunoprecipitation; SG, stress granule; TIA-1, T cell–restricted intracellular antigen-1; TIAR, TIA-1–related.

    Article Snippet: The antibodies used for immunoblot analysis were rabbit anti–eIF2α-P, anti–GCN2-P (Cell Signaling Technology), rabbit anti–IL-1β (Santa Cruz), rabbit anti pro–caspase-1 (Santa Cruz), goat anti–cleaved caspase-1 (Santa Cruz), rabbit anti–β-actin (Sigma-Aldrich), goat anti–TIA-1/TIAR (Santa Cruz), rabbit anti-LC3 (Cell Signaling Technology), and mouse anti-Flag (Sigma-Aldrich).

    Techniques: Activation Assay, Confocal Microscopy, Imaging, Marker, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Transfection, Derivative Assay, Polymerase Chain Reaction, Immunoprecipitation

    HF treatment promotes IL-1β mRNA degradation by targeting it to SGs through recruitment of TIA-1/TIAR. (A) Immunoblot analysis of RBPs, TIA-1/TIAR, in the lysates of J774A.1 macrophages, left untreated or treated with varying concentrations of HF for 3-h, 6-h, and 12-h time points. β-actin was used as loading control. (B) J774A.1 macrophages were transfected with either control siRNA or TIA-1/TIAR siRNA. After 24 h, cells were either left untreated or stimulated with LPS (500 ng/ml) or LPS (500 ng/ml) plus HF (20 nM) for 6 h, ATP (5 mM) was added to the macrophage cultures for 30 min at the end of time point and were assayed for IL-1β levels in culture supernatants by ELISA ( S1 Data ). (C) IL-1β expression in the whole-cell lysates of HEK293T cells transiently expressing various combinations of plasmids (pcDNA3, pcDNA3-IL-1β, and pEYFP-TIA-1/TIAR) as indicated (top). β-actin was used as loading control. (D) RT-PCR analysis of IL-1β expression in RIP material (pull-down using TIA-1/TIAR or IgG) of lysates from J774A.1 macrophages left untreated or treated with LPS or LPS plus HF. (E) Immunoblot analysis of pro–IL-1β or pro–caspase-1 expression in macrophages treated with LPS or LPS plus HF; β–actin was used as a loading control. (F) qRT-PCR analysis of IL-1β mRNA in the RIP material from LPS-primed J774A.1 macrophages treated with the indicated concentrations of HF ( S1 Data ). (G) qRT-PCR analysis of IL-1β mRNA in HEK293T cells transfected with constructs pCMV6-IL1β or pCMV6 IL-1β Δ3'UTR ARE, or cotransfected along with pEYFP-TIA-1/TIAR ( S1 Data ). Statistical significance was determined by student t test. * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005. Data are representative of 1 of 3 independent experiments. HEK293T, human embryonic kidney cells 293T; HF, Halofuginone; IgG, immunoglobulin G; IL-1β, interleukin 1β; LPS, lipopolysaccharide; qRT-PCR, quantitative reverse transcription PCR; RBP, RNA-binding protein; RIP, RNA immunoprecipitation; siRNA, small interfering RNA; TIA-1, T cell–restricted intracellular antigen-1; TIAR, TIA-1–related.

    Journal: PLoS Biology

    Article Title: Amino acid starvation sensing dampens IL-1β production by activating riboclustering and autophagy

    doi: 10.1371/journal.pbio.2005317

    Figure Lengend Snippet: HF treatment promotes IL-1β mRNA degradation by targeting it to SGs through recruitment of TIA-1/TIAR. (A) Immunoblot analysis of RBPs, TIA-1/TIAR, in the lysates of J774A.1 macrophages, left untreated or treated with varying concentrations of HF for 3-h, 6-h, and 12-h time points. β-actin was used as loading control. (B) J774A.1 macrophages were transfected with either control siRNA or TIA-1/TIAR siRNA. After 24 h, cells were either left untreated or stimulated with LPS (500 ng/ml) or LPS (500 ng/ml) plus HF (20 nM) for 6 h, ATP (5 mM) was added to the macrophage cultures for 30 min at the end of time point and were assayed for IL-1β levels in culture supernatants by ELISA ( S1 Data ). (C) IL-1β expression in the whole-cell lysates of HEK293T cells transiently expressing various combinations of plasmids (pcDNA3, pcDNA3-IL-1β, and pEYFP-TIA-1/TIAR) as indicated (top). β-actin was used as loading control. (D) RT-PCR analysis of IL-1β expression in RIP material (pull-down using TIA-1/TIAR or IgG) of lysates from J774A.1 macrophages left untreated or treated with LPS or LPS plus HF. (E) Immunoblot analysis of pro–IL-1β or pro–caspase-1 expression in macrophages treated with LPS or LPS plus HF; β–actin was used as a loading control. (F) qRT-PCR analysis of IL-1β mRNA in the RIP material from LPS-primed J774A.1 macrophages treated with the indicated concentrations of HF ( S1 Data ). (G) qRT-PCR analysis of IL-1β mRNA in HEK293T cells transfected with constructs pCMV6-IL1β or pCMV6 IL-1β Δ3'UTR ARE, or cotransfected along with pEYFP-TIA-1/TIAR ( S1 Data ). Statistical significance was determined by student t test. * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005. Data are representative of 1 of 3 independent experiments. HEK293T, human embryonic kidney cells 293T; HF, Halofuginone; IgG, immunoglobulin G; IL-1β, interleukin 1β; LPS, lipopolysaccharide; qRT-PCR, quantitative reverse transcription PCR; RBP, RNA-binding protein; RIP, RNA immunoprecipitation; siRNA, small interfering RNA; TIA-1, T cell–restricted intracellular antigen-1; TIAR, TIA-1–related.

    Article Snippet: The antibodies used for immunoblot analysis were rabbit anti–eIF2α-P, anti–GCN2-P (Cell Signaling Technology), rabbit anti–IL-1β (Santa Cruz), rabbit anti pro–caspase-1 (Santa Cruz), goat anti–cleaved caspase-1 (Santa Cruz), rabbit anti–β-actin (Sigma-Aldrich), goat anti–TIA-1/TIAR (Santa Cruz), rabbit anti-LC3 (Cell Signaling Technology), and mouse anti-Flag (Sigma-Aldrich).

    Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Construct, Polymerase Chain Reaction, RNA Binding Assay, Immunoprecipitation, Small Interfering RNA

    Proposed model of the mechanism by which HF suppresses IL-1β expression. HF induces activation of the amino acid starvation sensor, GCN2, which triggers SG formation and autophagy. SG formation regulates IL-1β expression at mRNA level via translational silencers, TIA-1/TIAR and RBPs. Later, these SGs are cleared by autophagy. On the other hand, the activated autophagy process decreases ROS levels, thereby inhibiting inflammasome activation, leading to decrease in active caspase-1 and secretion of mature IL-1β. eIF2, eukaryotic initiation factor 2; GCN2, general control nonderepressible 2 kinase; GDP, guanosine diphosphate; GTP, guanosine triphosphate; HF, Halofuginone; IL-1β, interleukin 1β; RBP, RNA-binding protein; ROS, reactive oxygen species; SG, stress granule; TIA-1, T cell–restricted intracellular antigen-1; TIAR, TIA-1–related.

    Journal: PLoS Biology

    Article Title: Amino acid starvation sensing dampens IL-1β production by activating riboclustering and autophagy

    doi: 10.1371/journal.pbio.2005317

    Figure Lengend Snippet: Proposed model of the mechanism by which HF suppresses IL-1β expression. HF induces activation of the amino acid starvation sensor, GCN2, which triggers SG formation and autophagy. SG formation regulates IL-1β expression at mRNA level via translational silencers, TIA-1/TIAR and RBPs. Later, these SGs are cleared by autophagy. On the other hand, the activated autophagy process decreases ROS levels, thereby inhibiting inflammasome activation, leading to decrease in active caspase-1 and secretion of mature IL-1β. eIF2, eukaryotic initiation factor 2; GCN2, general control nonderepressible 2 kinase; GDP, guanosine diphosphate; GTP, guanosine triphosphate; HF, Halofuginone; IL-1β, interleukin 1β; RBP, RNA-binding protein; ROS, reactive oxygen species; SG, stress granule; TIA-1, T cell–restricted intracellular antigen-1; TIAR, TIA-1–related.

    Article Snippet: The antibodies used for immunoblot analysis were rabbit anti–eIF2α-P, anti–GCN2-P (Cell Signaling Technology), rabbit anti–IL-1β (Santa Cruz), rabbit anti pro–caspase-1 (Santa Cruz), goat anti–cleaved caspase-1 (Santa Cruz), rabbit anti–β-actin (Sigma-Aldrich), goat anti–TIA-1/TIAR (Santa Cruz), rabbit anti-LC3 (Cell Signaling Technology), and mouse anti-Flag (Sigma-Aldrich).

    Techniques: Expressing, Activation Assay, RNA Binding Assay

    Regulation of type II collagen and MMP-13 by IL-1β in rat chondrocytes. (A) Reverse transcription-quantitative polymerase chain reaction and (B) western blot analyses demonstrating that IL-1β stimulation decreases type II collagen and increases MMP-13 mRNA and protein expression levels. GAPDH served as an internal control. Data are presented as the mean ± standard deviation. **P

    Journal: Molecular Medicine Reports

    Article Title: Regulation of type II collagen, matrix metalloproteinase-13 and cell proliferation by interleukin-1β is mediated by curcumin via inhibition of NF-κB signaling in rat chondrocytes

    doi: 10.3892/mmr.2017.6771

    Figure Lengend Snippet: Regulation of type II collagen and MMP-13 by IL-1β in rat chondrocytes. (A) Reverse transcription-quantitative polymerase chain reaction and (B) western blot analyses demonstrating that IL-1β stimulation decreases type II collagen and increases MMP-13 mRNA and protein expression levels. GAPDH served as an internal control. Data are presented as the mean ± standard deviation. **P

    Article Snippet: To acquire the optimum timepoint and concentration of curcumin for the treatment of chondrocytes, chondrocytes were co-treated with either of the following: i) 10 ng/ml IL-1β and 50 µM curcumin for various time periods (0, 12, 24, 36 or 48 h); or ii) 10 ng/ml IL-1β and various concentrations of curcumin (0, 25, 50, 75, or 100 µM) for 36 h. To investigate p65/RelA translocation and IκBα phosphorylation, chondrocytes were pretreated with 10 ng/ml IL-1β in the presence or absence of 50 µM curcumin for 0, 10, 20, 30, 40 or 60 min, after which nuclear and cytoplasmic extracts were prepared using a nuclear and cytoplasmic protein extraction kit (P0028; Beyotime Institute of Biotechnology) following the manufacturer's protocol.

    Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Standard Deviation

    Curcumin inhibits IL-1β-induced IκBα phosphorylation in rat chondrocytes. Western blot analysis and quantification of IκBα and p-IκBα protein expression levels following treatment with either (A) IL-1β alone or (B) IL-1β and curcumin for various durations. GAPDH served as an internal control. Data are presented as the mean ± standard deviation; **P

    Journal: Molecular Medicine Reports

    Article Title: Regulation of type II collagen, matrix metalloproteinase-13 and cell proliferation by interleukin-1β is mediated by curcumin via inhibition of NF-κB signaling in rat chondrocytes

    doi: 10.3892/mmr.2017.6771

    Figure Lengend Snippet: Curcumin inhibits IL-1β-induced IκBα phosphorylation in rat chondrocytes. Western blot analysis and quantification of IκBα and p-IκBα protein expression levels following treatment with either (A) IL-1β alone or (B) IL-1β and curcumin for various durations. GAPDH served as an internal control. Data are presented as the mean ± standard deviation; **P

    Article Snippet: To acquire the optimum timepoint and concentration of curcumin for the treatment of chondrocytes, chondrocytes were co-treated with either of the following: i) 10 ng/ml IL-1β and 50 µM curcumin for various time periods (0, 12, 24, 36 or 48 h); or ii) 10 ng/ml IL-1β and various concentrations of curcumin (0, 25, 50, 75, or 100 µM) for 36 h. To investigate p65/RelA translocation and IκBα phosphorylation, chondrocytes were pretreated with 10 ng/ml IL-1β in the presence or absence of 50 µM curcumin for 0, 10, 20, 30, 40 or 60 min, after which nuclear and cytoplasmic extracts were prepared using a nuclear and cytoplasmic protein extraction kit (P0028; Beyotime Institute of Biotechnology) following the manufacturer's protocol.

    Techniques: Western Blot, Expressing, Standard Deviation

    Curcumin inhibits IL-1β-induced nuclear translocation of nuclear factor-κB subunit p65/RelA. Western blot analysis of p65/RelA protein expression levels in chondrocytes treated with either (A) IL-1β alone or (B) IL-1β and curcumin for various durations. Quantification of (C) cytoplasmic and (D) nuclear p65/RelA protein expression levels. GAPDH and lamin B1 served as internal controls. Data are presented as the mean ± standard deviation; **P

    Journal: Molecular Medicine Reports

    Article Title: Regulation of type II collagen, matrix metalloproteinase-13 and cell proliferation by interleukin-1β is mediated by curcumin via inhibition of NF-κB signaling in rat chondrocytes

    doi: 10.3892/mmr.2017.6771

    Figure Lengend Snippet: Curcumin inhibits IL-1β-induced nuclear translocation of nuclear factor-κB subunit p65/RelA. Western blot analysis of p65/RelA protein expression levels in chondrocytes treated with either (A) IL-1β alone or (B) IL-1β and curcumin for various durations. Quantification of (C) cytoplasmic and (D) nuclear p65/RelA protein expression levels. GAPDH and lamin B1 served as internal controls. Data are presented as the mean ± standard deviation; **P

    Article Snippet: To acquire the optimum timepoint and concentration of curcumin for the treatment of chondrocytes, chondrocytes were co-treated with either of the following: i) 10 ng/ml IL-1β and 50 µM curcumin for various time periods (0, 12, 24, 36 or 48 h); or ii) 10 ng/ml IL-1β and various concentrations of curcumin (0, 25, 50, 75, or 100 µM) for 36 h. To investigate p65/RelA translocation and IκBα phosphorylation, chondrocytes were pretreated with 10 ng/ml IL-1β in the presence or absence of 50 µM curcumin for 0, 10, 20, 30, 40 or 60 min, after which nuclear and cytoplasmic extracts were prepared using a nuclear and cytoplasmic protein extraction kit (P0028; Beyotime Institute of Biotechnology) following the manufacturer's protocol.

    Techniques: Translocation Assay, Western Blot, Expressing, Standard Deviation

    Effect of curcumin on chondrocyte proliferation activity. Isolated rat articular chondrocytes were treated with 10 ng/ml interleukin-1β for 24 h, and subsequently co-treated with 50 µM curcumin, 0.1 mmol/l PDTC, or curcumin plus PDTC. Proliferation of chondrocytes at 12, 24, 36 and 48 h was assessed by Cell Counting kit-8 assays. Data are presented as the mean ± standard deviation. **P

    Journal: Molecular Medicine Reports

    Article Title: Regulation of type II collagen, matrix metalloproteinase-13 and cell proliferation by interleukin-1β is mediated by curcumin via inhibition of NF-κB signaling in rat chondrocytes

    doi: 10.3892/mmr.2017.6771

    Figure Lengend Snippet: Effect of curcumin on chondrocyte proliferation activity. Isolated rat articular chondrocytes were treated with 10 ng/ml interleukin-1β for 24 h, and subsequently co-treated with 50 µM curcumin, 0.1 mmol/l PDTC, or curcumin plus PDTC. Proliferation of chondrocytes at 12, 24, 36 and 48 h was assessed by Cell Counting kit-8 assays. Data are presented as the mean ± standard deviation. **P

    Article Snippet: To acquire the optimum timepoint and concentration of curcumin for the treatment of chondrocytes, chondrocytes were co-treated with either of the following: i) 10 ng/ml IL-1β and 50 µM curcumin for various time periods (0, 12, 24, 36 or 48 h); or ii) 10 ng/ml IL-1β and various concentrations of curcumin (0, 25, 50, 75, or 100 µM) for 36 h. To investigate p65/RelA translocation and IκBα phosphorylation, chondrocytes were pretreated with 10 ng/ml IL-1β in the presence or absence of 50 µM curcumin for 0, 10, 20, 30, 40 or 60 min, after which nuclear and cytoplasmic extracts were prepared using a nuclear and cytoplasmic protein extraction kit (P0028; Beyotime Institute of Biotechnology) following the manufacturer's protocol.

    Techniques: Activity Assay, Isolation, Cell Counting, Standard Deviation

    Effect of curcumin and PDTC on protein and mRNA expression levels in interleukin-1β-treated chondrocytes. (A) Western blot analysis of MMP-13, type II collagen, IκBα and p-IκBα protein expression levels. GAPDH served as an internal control. (B) Quantification of MMP-13, type II collagen, IκBα and NF-κB subunit p65/RelA mRNA expression levels, as assessed by reverse transcription-quantitative polymerase chain reaction analysis. Data are presented as the mean ± standard deviation; **P

    Journal: Molecular Medicine Reports

    Article Title: Regulation of type II collagen, matrix metalloproteinase-13 and cell proliferation by interleukin-1β is mediated by curcumin via inhibition of NF-κB signaling in rat chondrocytes

    doi: 10.3892/mmr.2017.6771

    Figure Lengend Snippet: Effect of curcumin and PDTC on protein and mRNA expression levels in interleukin-1β-treated chondrocytes. (A) Western blot analysis of MMP-13, type II collagen, IκBα and p-IκBα protein expression levels. GAPDH served as an internal control. (B) Quantification of MMP-13, type II collagen, IκBα and NF-κB subunit p65/RelA mRNA expression levels, as assessed by reverse transcription-quantitative polymerase chain reaction analysis. Data are presented as the mean ± standard deviation; **P

    Article Snippet: To acquire the optimum timepoint and concentration of curcumin for the treatment of chondrocytes, chondrocytes were co-treated with either of the following: i) 10 ng/ml IL-1β and 50 µM curcumin for various time periods (0, 12, 24, 36 or 48 h); or ii) 10 ng/ml IL-1β and various concentrations of curcumin (0, 25, 50, 75, or 100 µM) for 36 h. To investigate p65/RelA translocation and IκBα phosphorylation, chondrocytes were pretreated with 10 ng/ml IL-1β in the presence or absence of 50 µM curcumin for 0, 10, 20, 30, 40 or 60 min, after which nuclear and cytoplasmic extracts were prepared using a nuclear and cytoplasmic protein extraction kit (P0028; Beyotime Institute of Biotechnology) following the manufacturer's protocol.

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation

    Immunofluorescence staining of nuclear factor-κB subunit p65/RelA. Curcumin and PDTC block IL-1β-induced nuclear translocation of p65/RelA. PDTC, pyrrolidine dithiocarbamate; IL-1β, interleukin-1β.

    Journal: Molecular Medicine Reports

    Article Title: Regulation of type II collagen, matrix metalloproteinase-13 and cell proliferation by interleukin-1β is mediated by curcumin via inhibition of NF-κB signaling in rat chondrocytes

    doi: 10.3892/mmr.2017.6771

    Figure Lengend Snippet: Immunofluorescence staining of nuclear factor-κB subunit p65/RelA. Curcumin and PDTC block IL-1β-induced nuclear translocation of p65/RelA. PDTC, pyrrolidine dithiocarbamate; IL-1β, interleukin-1β.

    Article Snippet: To acquire the optimum timepoint and concentration of curcumin for the treatment of chondrocytes, chondrocytes were co-treated with either of the following: i) 10 ng/ml IL-1β and 50 µM curcumin for various time periods (0, 12, 24, 36 or 48 h); or ii) 10 ng/ml IL-1β and various concentrations of curcumin (0, 25, 50, 75, or 100 µM) for 36 h. To investigate p65/RelA translocation and IκBα phosphorylation, chondrocytes were pretreated with 10 ng/ml IL-1β in the presence or absence of 50 µM curcumin for 0, 10, 20, 30, 40 or 60 min, after which nuclear and cytoplasmic extracts were prepared using a nuclear and cytoplasmic protein extraction kit (P0028; Beyotime Institute of Biotechnology) following the manufacturer's protocol.

    Techniques: Immunofluorescence, Staining, Blocking Assay, Translocation Assay