recombinant protein Search Results


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  • 99
    Thermo Fisher recombinant proteins
    Recombinant Proteins, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1595 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore recombinant proteins
    Recombinant Proteins, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1870 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore recombinant protein
    Recombinant Protein, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9010 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher tnf α
    Cytokines/chemokines recruitment during MA subsp. abscessus chronic lung infection. ( A ) IFN-γ, ( B ) <t>TNF-α,</t> measured by Mouse Milliplex, were quantified in total the lung of mice. At 7 and 45 days, dots represent cells in individual mice selected from the group of infected mice with MA subsp. abscessus . The data were pooled from two independent experiments. For MA subsp. abscessus , statistical time effect was evaluated with Mann-Whitney Test. Statistical comparison between MA subsp. abscessus and control mice at Day 7 was calculated with Mann-Whitney Test. *p
    Tnf α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 21729 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems netrin 1
    LARP1 regulates <t>Netrin-1</t> microsomal translation. Huh7.5 cells were transfected with control and LARP1-specific siRNAs, and infected by HCV at MOI 0.1 over 4 d. Cells were lysed and analyzed by immunoblotting for LARP1 knockdown as shown in S3 Fig . LARP1 depletion decreases the expression of Netrin-1 protein in microsomes in Huh7.5 cells. LARP1, Netrin-1, and HSP60 proteins were monitored for their partitioning in the cytosolic (free polysomes) and microsome (ER membrane-bound polysomes) compartments, according to the infection status of the cells through sequential extractions followed by immunoblotting. HCV core was used as an infection control.
    Netrin 1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 98/100, based on 927 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher tumor necrosis factor alpha tnf α
    Enzyme linked immunosorbent assay analysis of tumor necrosis <t>factor-alpha,</t> interleukin-1β, interleukin-10 and interferon-gamma expression ( n = 6). A: <t>TNF-α</t> expression in the colon; B: IL-1β expression in the colon; C: IL-10 expression in the colon; D: IFN-γ expression in the colon. a P
    Tumor Necrosis Factor Alpha Tnf α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 925 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fgf 2
    <t>FGF-2</t> induces HA accumulation in primary RPE cells. ( A – D ) FGF-2 induced HA accumulation in primary porcine RPE cells in a dose-dependent manner ( A ) 0 ng/mL, ( B ) 10 ng/mL, ( C ) 25 ng/mL, ( D ) 100 ng/mL. Fluorescence intensity was quantitated by integrated density measurement ( I ) (n ≥ 4, for each cell line). Data are presented as mean ± SEM ( E – H ) Z-plane images show that increased concentrations of FGF-2 induce increased apical accumulation of HA in addition to some peri-cellular and basal deposits of HA at high doses of FGF-2. Green: HA, red: phalloidin; blue: DAPI.
    Fgf 2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 962 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant human tnf α
    The RPE secreted higher levels of GM-CSF after stimulation with HNE and <t>TNF-α.</t> The GM-CSF secreted into the culture supernatant was increased when primary RPE cells were exposed to 4-hydroxynonenal (HNE), an agent that promotes oxidative stress, at 10 µM for 6 h (mean ± SEM, 145.88±5.06 pg/ml versus 123.27±4.05 pg/ml, n=3, Student t test, *p
    Recombinant Human Tnf α, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 1509 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher human recombinant epidermal growth factor
    Persisting antioncogenic efficacy of Emx2 upon neural nestin enhancer-restricted overexpression In vitro kinetic progression of GbmA and GbmB lines C, D. , engineered by lentiviral vectors and TetON technology as in A, B. , and kept as floating cultures, under <t>Fgf2</t> and <t>Egf.</t> Cell numbers were normalized against t =0 values. n is the number of biological replicates. p -value was calculated by t-test (one-tail, unpaired): *** p
    Human Recombinant Epidermal Growth Factor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1562 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems human il 6
    JSI-124 inhibited bleomycin-induced bronchoalveolar inflammatory cell extravasation and lung fibrosis. Wistar rats were administered a single intratracheal dose of bleomycin (BLM; 3.75 U/kg) on day 1. JSI-124 (1 mg/kg/day) or vehicle was administered intraperitoneally from day 14 until the analysis at day 28 ( n = 10 per group). Total bronchoalveolar lavage ( a ) protein, ( b , c ) inflammatory cells, ( d ) lung weight, ( e ) protein, and ( f , g ) <t>IL-6</t> and IL-13 content were measured at day 28. h Masson’s trichrome (upper panels, scale bar: 100 μm) of control, BLM, and BLM + JSI-124 tissue. i Fibrosis Ashcroft scores were assessed as described in the Methods. j Micro-CT images were acquired on day 28 and ( k ) quantified as Hounsfield units. The results are expressed as the mean ± SEM, n = 10. Statistical significance was assessed using a t -test or one-way ANOVA followed by a Bonferroni post-hoc test. * P
    Human Il 6, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 1161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant human interleukin 2
    ( A ) The cytotoxicity of T cells was evaluated by the LDH assay at different ( E ) T ratios. ( C ) Human Th1/Th2 Cytokine Kit determined the expression level of <t>IL-2,</t> IL-4, IL-6, TNF-α, and IFN-γ of DMSO or YN968D1-treated T cells. ( B, D ) The frequency of IFN-γ-producing T cells after co-incubation with gastric cancer cell MKN-45 for 18 hrs was evaluated by IFN-γ ELISPOT kit (Dakewei, China). T cells and MKN-45 were co-incubated at different E: T ratios (10:1, 5:1, 3:1). * means P value
    Recombinant Human Interleukin 2, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant human il 4
    IL-25, but not <t>IL-4,</t> IL-5 or IL-13 induced angiogenesis in vitro. Top panel: representative light photomicrographs (4x original magnification) show formation of primitive vascular tubule structures by human vascular endothelial cells after 11 days of culture (top panel) with medium (A) , VEGF (10 ng/mL) (B) , IL-25 (10 ng/mL) (C) , IL-4 (10 ng/mL) (D) , IL-5 (10 ng/mL) (E) and IL-13 (10 ng/mL) (F) . Bottom panel: computer-assisted quantification of total tubule lengths (G) , numbers of branch points (H) and total numbers of tubules (I) . Bars show the mean ± SEM of three separate experiments performed in duplicate. *p
    Recombinant Human Il 4, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 986 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems il 17f
    Anti-IL-17A antibodies but not <t>anti-IL-17F</t> antibodies render mice susceptible to OPC.
    Il 17f, supplied by R&D Systems, used in various techniques. Bioz Stars score: 91/100, based on 710 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad recombinant proteins
    Anti-IL-17A antibodies but not <t>anti-IL-17F</t> antibodies render mice susceptible to OPC.
    Recombinant Proteins, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 339 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems rmil 4
    HoxC4 deficiency impairs estrogen-mediated enhancement of AID expression and CSR to IgG1 and IgE, as induced by LPS and IL-4 or CD154 and IL-4. A , HoxC4 +/+ and HoxC4 −/− B cells were stimulated with LPS and <t>rmIL-4</t> or with CD154 and rmIL-4
    Rmil 4, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 420 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant human il 6
    MCR expressions during erythroblast differentiation. ( A ) Schematic diagram of an in vitro differentiation protocol for deriving erythroblasts from human HPCs. Human CD34 + HPCs were expanded for 7 days (E0–E7) and stocks were frozen in liquid nitrogen (LN 2 ). The stock cells were differentiated for 3 days (D0–D3) before undergoing maturation (M0–M7). The number of enucleated erythrocytes increased from M5 to M7. ACTH, adrenocorticotropic hormone; EPO, erythropoietin; FL, flt-3 ligand; IL-3, interleukin-3; <t>IL-6,</t> interleukin-6; α-MSH, α-melanocyte stimulating hormone; SCF, stem cell factor; TPO, thrombopoietin. ( B ) May-Grunwald-Giemsa staining of control erythroblasts at day M0–5 day. Cells differentiated to Pro-E stage at day M0 and Baso-E stage at day M3. Arrows, Poly-E; Arrow heads, Orho-E. *, Reticulocyte. Bar 10 μm. ( C ) Conventional RT-PCR for MCRs during erythroblasts differentiation between M0 and M6 day.
    Recombinant Human Il 6, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 996 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems pdgf aa
    Decreased activation of <t>Akt</t> by <t>PDGF-AA</t> in BBS1 M390R/M390R mouse embryonic fibroblast (MEF) cells. A and B : representative Western blots ( A ) and quantification data ( B ) of phosphorylated and total Akt in control and BBS1 M390R/M390R MEF cells stimulated with PDGF-AA or vehicle for 15 min; n = 4/group (performed in duplicate). * P
    Pdgf Aa, supplied by R&D Systems, used in various techniques. Bioz Stars score: 98/100, based on 553 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems mouse il 6
    <t>IL-6</t> is up-regulated in PDGFRα + stromal cells after SO 2 injury. ( A ) RNAs were extracted from whole trachea at 0, 1, 2, and 14 d after injury and subjected to quantitative RT-PCR analysis. The mRNA expression level of cytokines was normalized to Gapdh . ( B ) In situ hybridization combined with immunohistochemistry shows that Il-6 mRNA (red) is expressed in cells in the stroma beneath basal cells (K5 + , green) after SO 2 injury. ( C ) Quantitative PCR analysis of Il-6 expression in sorted stromal cells [ Pdgfr α ( Pdgfra )-GFP + ] and immune cell subpopulations from the trachea at 24 hpi. ( D ) Immunohistochemistry of a trachea section at 24 hpi shows Pdgfra -GFP + cells (GFP + , green) in the stroma beneath the epithelium with basal cells (K5 + , red). ( E ) In situ hybridization and immunohistochemistry show that Pdgfra -GFP + cells (GFP + , green) express Il-6 mRNA (red) at 24 hpi. (Scale bars: B and E , 20 μm; D , 50 μm.) * P
    Mouse Il 6, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 760 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher recombinant protein g sepharose 4b
    SDS–PAGE of 125 I-labelled antigen recognized by mAb C9.1. (a) A-LAK cells derived from C3H/He mice were iodinated with Na 125 I by the lactoperoxidase method. 125 I-labelled membrane proteins were incubated with mAb C9.1 (lanes 1 and 3) or mAb FD441.8 (anti-LFA-1) (lanes 2 and 4), followed by incubation with anti-rat IgG mAb-bound protein <t>G–Sepharose</t> 4B. The immune complexes were analysed under non-reducing (lanes 1 and 2) or reducing (lanes 3 and 4) conditions by SDS–PAGE on 7·5% gels and subjected to autoradiography. (b) A-LAK cells from (C57BL/6×C3H) F 1 mice were radioiodinated and immunoprecipitated with mAb C9.1(lanes 1 and 3) or anti-2B4 mAb (lanes 2 and 4) as in (a). Immune complexes were treated with (lanes 3 and 4) or without (lanes 1 and 2) endoglycosidase F, analysed under reducing conditions by SDS–PAGE on 10% gels and subjected to autoradiography.
    Recombinant Protein G Sepharose 4b, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems human tnf α
    A : KC were isolated from mice at 2 wk after a single subcutaneous injection of iron dextran (25 mg/kg) to assess LPS- or peroxynitrite-stimulated <t>TNF-α</t> expression. Note KC isolated from iron dextran-injected mice release 4–5 times more
    Human Tnf α, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 682 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems igfbp 3
    Depletion of INSR does not inhibit <t>IGFBP-3</t> secretion in hTCEpi cells. Secreted IGFBP-3 in conditioned media was measured using an IGFBP-3 ELISA. (A) hTCEpi cells were treated with siRNA oligonucleotides targeting IGF-1R or INSR. In basal media, knockdown of IGF-1R blunted secretion of IGFBP-3 compared to the non-targeting control (***P
    Igfbp 3, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 297 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Cytokines/chemokines recruitment during MA subsp. abscessus chronic lung infection. ( A ) IFN-γ, ( B ) TNF-α, measured by Mouse Milliplex, were quantified in total the lung of mice. At 7 and 45 days, dots represent cells in individual mice selected from the group of infected mice with MA subsp. abscessus . The data were pooled from two independent experiments. For MA subsp. abscessus , statistical time effect was evaluated with Mann-Whitney Test. Statistical comparison between MA subsp. abscessus and control mice at Day 7 was calculated with Mann-Whitney Test. *p

    Journal: bioRxiv

    Article Title: A new model of chronic Mycobacterium abscessus lung infection in immunocompetent mice

    doi: 10.1101/2020.07.30.228247

    Figure Lengend Snippet: Cytokines/chemokines recruitment during MA subsp. abscessus chronic lung infection. ( A ) IFN-γ, ( B ) TNF-α, measured by Mouse Milliplex, were quantified in total the lung of mice. At 7 and 45 days, dots represent cells in individual mice selected from the group of infected mice with MA subsp. abscessus . The data were pooled from two independent experiments. For MA subsp. abscessus , statistical time effect was evaluated with Mann-Whitney Test. Statistical comparison between MA subsp. abscessus and control mice at Day 7 was calculated with Mann-Whitney Test. *p

    Article Snippet: In particular IFN-γ and TNF-α were evaluated by Mouse Custom ProcartaPlex 9-plex (Invitrogen, Thermo Fisher Scientific) and normalized at 2500 ug/ml of quantified proteins in lung supernatants.

    Techniques: Infection, Mouse Assay, MANN-WHITNEY

    LARP1 regulates Netrin-1 microsomal translation. Huh7.5 cells were transfected with control and LARP1-specific siRNAs, and infected by HCV at MOI 0.1 over 4 d. Cells were lysed and analyzed by immunoblotting for LARP1 knockdown as shown in S3 Fig . LARP1 depletion decreases the expression of Netrin-1 protein in microsomes in Huh7.5 cells. LARP1, Netrin-1, and HSP60 proteins were monitored for their partitioning in the cytosolic (free polysomes) and microsome (ER membrane-bound polysomes) compartments, according to the infection status of the cells through sequential extractions followed by immunoblotting. HCV core was used as an infection control.

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: LARP1 regulates Netrin-1 microsomal translation. Huh7.5 cells were transfected with control and LARP1-specific siRNAs, and infected by HCV at MOI 0.1 over 4 d. Cells were lysed and analyzed by immunoblotting for LARP1 knockdown as shown in S3 Fig . LARP1 depletion decreases the expression of Netrin-1 protein in microsomes in Huh7.5 cells. LARP1, Netrin-1, and HSP60 proteins were monitored for their partitioning in the cytosolic (free polysomes) and microsome (ER membrane-bound polysomes) compartments, according to the infection status of the cells through sequential extractions followed by immunoblotting. HCV core was used as an infection control.

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Transfection, Infection, Expressing

    Netrin-1 mRNA and HCV NS5A are LARP1 interactants. Quantification of ribosomal protein S18 (RPS18) ( A ) and Netrin-1 ( B ) mRNA by qPCR after immunoprecipitation using LARP1 antibody (data are represented as mean ± standard deviation, n = 3, Mann-Whitney test, p

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: Netrin-1 mRNA and HCV NS5A are LARP1 interactants. Quantification of ribosomal protein S18 (RPS18) ( A ) and Netrin-1 ( B ) mRNA by qPCR after immunoprecipitation using LARP1 antibody (data are represented as mean ± standard deviation, n = 3, Mann-Whitney test, p

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Real-time Polymerase Chain Reaction, Immunoprecipitation, Standard Deviation, MANN-WHITNEY

    Netrin-1 increases HCV through the UNC5A receptor. Huh7.5 cells were transfected with siRNA against each UNC5 receptor or with a nontargeting control siRNA and infected at a MOI of 0.1 24 h after seeding. Cells were then trypsinized at day five post-infection before undergoing a second siRNA transfection. Recombinant soluble Netrin-1-Fc was added to the medium 12 h after transfection. Intracellular HCV RNA was quantified by RT-qPCR at each time point (left-hand graphs; data are represented as mean ± standard deviation, n = 3, Wilcoxon test, p

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: Netrin-1 increases HCV through the UNC5A receptor. Huh7.5 cells were transfected with siRNA against each UNC5 receptor or with a nontargeting control siRNA and infected at a MOI of 0.1 24 h after seeding. Cells were then trypsinized at day five post-infection before undergoing a second siRNA transfection. Recombinant soluble Netrin-1-Fc was added to the medium 12 h after transfection. Intracellular HCV RNA was quantified by RT-qPCR at each time point (left-hand graphs; data are represented as mean ± standard deviation, n = 3, Wilcoxon test, p

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Transfection, Infection, Recombinant, Quantitative RT-PCR, Standard Deviation

    Netrin-1 overexpression increases HCV RNA and specific infectivity of HCV virions in vitro whereas Netrin-1 depletion decreases HCV RNA and specific infectivity in vitro. A. Detection of VR1-HA and Netrin-1-HA in transfected Huh7.5 cells by immunoblotting with the anti-HA antibody. B . Netrin-1 overexpression enhances intracellular HCV RNA. Huh7.5 cells were transfected with the VR1-HA or the Netrin-1-HA plasmid and infected at a MOI of 0.1 the day after seeding. Intracellular HCV RNA was quantified by RT-qPCR at each time point (data are shown as mean ± standard deviation, n = 3, Wilcoxon test, p

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: Netrin-1 overexpression increases HCV RNA and specific infectivity of HCV virions in vitro whereas Netrin-1 depletion decreases HCV RNA and specific infectivity in vitro. A. Detection of VR1-HA and Netrin-1-HA in transfected Huh7.5 cells by immunoblotting with the anti-HA antibody. B . Netrin-1 overexpression enhances intracellular HCV RNA. Huh7.5 cells were transfected with the VR1-HA or the Netrin-1-HA plasmid and infected at a MOI of 0.1 the day after seeding. Intracellular HCV RNA was quantified by RT-qPCR at each time point (data are shown as mean ± standard deviation, n = 3, Wilcoxon test, p

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Over Expression, Infection, In Vitro, Transfection, Plasmid Preparation, Quantitative RT-PCR, Standard Deviation

    HCV increases Netrin-1 mRNA and protein. A . Virions depletion experiment. Huh7.5 cells were infected by HCV over 3 d, and the supernatant (SN) was collected, depleted of HCV particles by ultracentrifugation, and added to naïve Huh7.5 cells. Levels of HCV RNA (left) and Netrin-1 mRNA (right) were quantified by RT-qPCR. Data are represented as mean ± standard deviation ( n = 3). B . HCV increases the association of Netrin-1 mRNA with microsomes (endoplasmic reticulum [ER] membrane)-bound polysomes in Huh7.5 cells. Netrin-1 , Glucuronidase ( GUS) , and phosphomannomutase 1 ( PMM1) mRNA were monitored for their partitioning in the cytosolic (free polysomes) and microsome (ER membrane-bound polysomes) compartments according to the infection status of the cells through sequential extractions followed by RT-qPCR. Statistical significance was determined using the Mann-Whitney test, p

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: HCV increases Netrin-1 mRNA and protein. A . Virions depletion experiment. Huh7.5 cells were infected by HCV over 3 d, and the supernatant (SN) was collected, depleted of HCV particles by ultracentrifugation, and added to naïve Huh7.5 cells. Levels of HCV RNA (left) and Netrin-1 mRNA (right) were quantified by RT-qPCR. Data are represented as mean ± standard deviation ( n = 3). B . HCV increases the association of Netrin-1 mRNA with microsomes (endoplasmic reticulum [ER] membrane)-bound polysomes in Huh7.5 cells. Netrin-1 , Glucuronidase ( GUS) , and phosphomannomutase 1 ( PMM1) mRNA were monitored for their partitioning in the cytosolic (free polysomes) and microsome (ER membrane-bound polysomes) compartments according to the infection status of the cells through sequential extractions followed by RT-qPCR. Statistical significance was determined using the Mann-Whitney test, p

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Infection, Quantitative RT-PCR, Standard Deviation, MANN-WHITNEY

    HCV levels correlate with the expression of Netrin-1 in the liver biopsies of HCV-infected patients. A . HCV-positive samples exhibit the highest levels of Netrin-1 mRNA of all the chronic liver disease biopsies. The levels of Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test. B. Positive correlation between intrahepatic levels of HCV and Netrin-1 mRNA. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Spearman test. An outlier test was run to confirm these results. C and D. Netrin-1 mRNA parallels HCV RNA levels upon treatment. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR in paired biopsies, before and after treatment, of partially responding patients ( C,D left panels ) and nonresponding patients (C,D, right panels ). E. Parenchymal Netrin-1 staining is associated with HCV infection status in HCV-infected patients. Uninfected, chronic liver disease samples (non-cirrhotic sample, n = 1; alcohol-related cirrhosis samples, n = 3) and HCV genotype 1-infected cirrhosis samples ( n = 4) were analyzed. Representative images of Netrin-1 staining (upper panels) and HCV E2 staining (lower panels) are shown. F. Netrin-1 protein expression is increased in HCV-positive samples. The level of Netrin-1 was quantified by immunoblotting using recombinant Netrin-1 (rec. Net) as a control. G. The levels of Netrin-1 mRNA are higher in HCV+ versus HCV- biopsies, regardless of the histological stage, from normal liver to HCC. Intrahepatic Netrin-1 mRNA levels were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test, p

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: HCV levels correlate with the expression of Netrin-1 in the liver biopsies of HCV-infected patients. A . HCV-positive samples exhibit the highest levels of Netrin-1 mRNA of all the chronic liver disease biopsies. The levels of Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test. B. Positive correlation between intrahepatic levels of HCV and Netrin-1 mRNA. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Spearman test. An outlier test was run to confirm these results. C and D. Netrin-1 mRNA parallels HCV RNA levels upon treatment. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR in paired biopsies, before and after treatment, of partially responding patients ( C,D left panels ) and nonresponding patients (C,D, right panels ). E. Parenchymal Netrin-1 staining is associated with HCV infection status in HCV-infected patients. Uninfected, chronic liver disease samples (non-cirrhotic sample, n = 1; alcohol-related cirrhosis samples, n = 3) and HCV genotype 1-infected cirrhosis samples ( n = 4) were analyzed. Representative images of Netrin-1 staining (upper panels) and HCV E2 staining (lower panels) are shown. F. Netrin-1 protein expression is increased in HCV-positive samples. The level of Netrin-1 was quantified by immunoblotting using recombinant Netrin-1 (rec. Net) as a control. G. The levels of Netrin-1 mRNA are higher in HCV+ versus HCV- biopsies, regardless of the histological stage, from normal liver to HCC. Intrahepatic Netrin-1 mRNA levels were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test, p

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Expressing, Infection, Quantitative RT-PCR, MANN-WHITNEY, Staining, Recombinant

    HCV induces the expression of Netrin-1 in vitro. A, B, C, D. HCV induces Netrin-1 mRNA in primary human hepatocytes. Cells were infected at a MOI of 1 with HCV genotype 2 strain 4 d after seeding ( n = 4 independent preparations from four different patients, Wilcoxon test, p

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: HCV induces the expression of Netrin-1 in vitro. A, B, C, D. HCV induces Netrin-1 mRNA in primary human hepatocytes. Cells were infected at a MOI of 1 with HCV genotype 2 strain 4 d after seeding ( n = 4 independent preparations from four different patients, Wilcoxon test, p

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Expressing, In Vitro, Infection

    Netrin-1 enhances HCVpp entry. RNAi-mediated knockdown of EGFR. Huh7.5 cells were transfected with anti-EGFR siRNAs #3 and #4 of a previously described study [ 3 ] and infected at a MOI of 0.1. Cells were collected 3 d post-infection and analyzed by RT-qPCR ( A ) and immunoblotting using an anti-EGFR antibody targeting an extracellular epitope of the protein ( B ) (data are represented as mean ± standard deviation, n = 4, Mann-Whitney test, p

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: Netrin-1 enhances HCVpp entry. RNAi-mediated knockdown of EGFR. Huh7.5 cells were transfected with anti-EGFR siRNAs #3 and #4 of a previously described study [ 3 ] and infected at a MOI of 0.1. Cells were collected 3 d post-infection and analyzed by RT-qPCR ( A ) and immunoblotting using an anti-EGFR antibody targeting an extracellular epitope of the protein ( B ) (data are represented as mean ± standard deviation, n = 4, Mann-Whitney test, p

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Transfection, Infection, Quantitative RT-PCR, Standard Deviation, MANN-WHITNEY

    Netrin-1 impedes EGFR recycling. Huh7.5 cells infected with HCV (day four p.i.) were transfected with siRNA (control or Netrin-1-specific) or with plasmids (VR1- or Netrin-1-expressing), serum-starved for 16 h, and incubated with EGF for 5 or 15 min prior to fixation. Cells were subsequently stained for EEA1 and EGFR and visualized by confocal microscopy. A . Li diagrams and Li coefficient calculations ( B ) for Netrin-1 knockdown and forced expression experiments. Pixels present on the left and right sides of the y -axis, i.e., associated with negative and positive staining amplitude values, indicate exclusion and colocalization, respectively. Li coefficients were calculated using the JACop plugin from the imageJ software ( http://rsb.info.nih.gov/ij/plugins/track/jacop.html ). Twelve to 15 random fields were acquired per biological sample, totaling 250–300 cells analyzed for each biological sample in a given experiment ( n = 2). C . Representative immunofluorescence-based localization of EEA1 and EGFR. Nuclei were counterstained with Hoechst 33342. EEA1 (green) and EGFR (red) were detected using Alexa-488 and Alexa-594, respectively. Overlays were generated by the Leica LAS AF software upon image acquisition. Open and solid arrows show partial and total colocalization, respectively. Bar = 5 μm. D . Statistical assessment of the colocalization of EEA1 and EGFR. Red (EGFR) and green (EEA1) fluorescence intensities were measured for each pixel along a 5 μm horizontal line centered around the arrow tips in ( C ) using the Plot Profile function of the ImageJ software. Spearman correlation coefficients for each couple of intensity values are shown. The underlying data for panels in this figure can be found in S1 Data .

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: Netrin-1 impedes EGFR recycling. Huh7.5 cells infected with HCV (day four p.i.) were transfected with siRNA (control or Netrin-1-specific) or with plasmids (VR1- or Netrin-1-expressing), serum-starved for 16 h, and incubated with EGF for 5 or 15 min prior to fixation. Cells were subsequently stained for EEA1 and EGFR and visualized by confocal microscopy. A . Li diagrams and Li coefficient calculations ( B ) for Netrin-1 knockdown and forced expression experiments. Pixels present on the left and right sides of the y -axis, i.e., associated with negative and positive staining amplitude values, indicate exclusion and colocalization, respectively. Li coefficients were calculated using the JACop plugin from the imageJ software ( http://rsb.info.nih.gov/ij/plugins/track/jacop.html ). Twelve to 15 random fields were acquired per biological sample, totaling 250–300 cells analyzed for each biological sample in a given experiment ( n = 2). C . Representative immunofluorescence-based localization of EEA1 and EGFR. Nuclei were counterstained with Hoechst 33342. EEA1 (green) and EGFR (red) were detected using Alexa-488 and Alexa-594, respectively. Overlays were generated by the Leica LAS AF software upon image acquisition. Open and solid arrows show partial and total colocalization, respectively. Bar = 5 μm. D . Statistical assessment of the colocalization of EEA1 and EGFR. Red (EGFR) and green (EEA1) fluorescence intensities were measured for each pixel along a 5 μm horizontal line centered around the arrow tips in ( C ) using the Plot Profile function of the ImageJ software. Spearman correlation coefficients for each couple of intensity values are shown. The underlying data for panels in this figure can be found in S1 Data .

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Infection, Transfection, Expressing, Incubation, Staining, Confocal Microscopy, Software, Immunofluorescence, Generated, Fluorescence

    Netrin-1 is virion-bound and participates in the infectivity of viral particles. HCV particles produced in cells overexpressing Netrin-1 have been preincubated with Netrin-1 antagonists and TCID 50 has been performed. A . Method validation: inhibition of HCV infectivity after incubation with anti-E2 antibody. The anti-E2 antibody was used as a control of neutralization of infectivity. B . Inhibition of HCV infectivity after incubation with three distinct Netrin-1 antagonists (data are represented as mean ± standard deviation, n = 3, Mann-Whitney test, p

    Journal: PLoS Biology

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    doi: 10.1371/journal.pbio.1002421

    Figure Lengend Snippet: Netrin-1 is virion-bound and participates in the infectivity of viral particles. HCV particles produced in cells overexpressing Netrin-1 have been preincubated with Netrin-1 antagonists and TCID 50 has been performed. A . Method validation: inhibition of HCV infectivity after incubation with anti-E2 antibody. The anti-E2 antibody was used as a control of neutralization of infectivity. B . Inhibition of HCV infectivity after incubation with three distinct Netrin-1 antagonists (data are represented as mean ± standard deviation, n = 3, Mann-Whitney test, p

    Article Snippet: Netrin-1 does not affect HCV replication per se.

    Techniques: Infection, Produced, Inhibition, Incubation, Neutralization, Standard Deviation, MANN-WHITNEY

    Enzyme linked immunosorbent assay analysis of tumor necrosis factor-alpha, interleukin-1β, interleukin-10 and interferon-gamma expression ( n = 6). A: TNF-α expression in the colon; B: IL-1β expression in the colon; C: IL-10 expression in the colon; D: IFN-γ expression in the colon. a P

    Journal: World Journal of Gastroenterology

    Article Title: Jianpi Qingchang decoction regulates intestinal motility of dextran sulfate sodium-induced colitis through reducing autophagy of interstitial cells of Cajal

    doi: 10.3748/wjg.v23.i26.4724

    Figure Lengend Snippet: Enzyme linked immunosorbent assay analysis of tumor necrosis factor-alpha, interleukin-1β, interleukin-10 and interferon-gamma expression ( n = 6). A: TNF-α expression in the colon; B: IL-1β expression in the colon; C: IL-10 expression in the colon; D: IFN-γ expression in the colon. a P

    Article Snippet: Materials DSS (MW 36000-50000; MP Biomedicals, Santa Ana, CA, United States); the nine medicinal herbs of JQD raw powder, as shown in Table (Pharmacy Department, Longhua Hospital affiliated to Shanghai University of TCM, Shanghai, China) dissolved in 0.5% carboxymethylcellulose sodium (CMC) solution; mesalazine [5-aminosalicylic acid (5-ASA); Sunflower Pharmaceutical Group, Jiamusi Lu Ling Pharmaceutical Co., Ltd., Liaoning, China; batch number: 111206], external coat removed, broken down, and dissolved in 0.5% CMC solution; fecal occult blood (FOB) (Huashengyuan Medical Science and Technology Co. Ltd., Beijing, China); tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-10 and interferon gamma (IFN-γ) ELISA kits (eBioscience, San Diego, CA, United States); NF-κB antibody (Cell Signaling, Danvers, MA, United States); primer synthesis kit (Yushen Bio-Technique Co. Ltd., Shanghai, China).

    Techniques: Enzyme-linked Immunosorbent Assay, Expressing

    Attenuation of the level of reactive oxygen species (ROS) by genistein. (A) Keratinocytes were stimulated respectively with a proinflammatory “cytokine mix” corresponding to a concentration of 2 ng/mL (ACT 2) or 5 ng/mL (ACT 5) in each compound of the mix and incubated with 10 mM N-acetyl-cysteine (NAC) (ACT 2 + NAC), 100 μM genistein (ACT 2 + GEN), 10 ng/mL TNF-α or 1 μg/mL LPS alone, and with 10 ng/mL TNF-α or 1 μg/mL LPS incubated with 100 μM genistein (TNF-α + GEN or LPS + GEN). DMSO-treated, unstimulated cells were used as the control (NACT). Additional control was used in the form of unstimulated cells treated with 100 μM genistein (GEN). Intracellular ROS levels were examined by a CellROX Deep Red Reagent and confocal fluorescence microscopy. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Results representative of three independent experiments (with scale bars 25 μm) are shown. (B) Analysis of ROS were additionally performed by fluorescent cell analyzer. The data are presented as the means ± standard deviation (SD) from three independent experiments. Significant differences ( p ≤ 0.05) between cell populations not expressing intracellular ROS (ROS [–]) and expressing intracellular ROS (ROS [+]) were observed for all tested conditions, except for LPS where cell numbers of ROS (-) and ROS (+) were comparable. Most important, statistically significant differences of p ≤ 0.05 within ROS groups are indicated with * for samples of TNF-α, TNF-α + GEN, and LPS versus NACT, with † for sample TNF-α + GEN with respect to TNF-α, while with ‡ for sample LPS + GEN referred to LPS. Statistical analysis was performed using ANOVA with Tukey’s HSD test.

    Journal: PLoS ONE

    Article Title: Molecular action of isoflavone genistein in the human epithelial cell line HaCaT

    doi: 10.1371/journal.pone.0192297

    Figure Lengend Snippet: Attenuation of the level of reactive oxygen species (ROS) by genistein. (A) Keratinocytes were stimulated respectively with a proinflammatory “cytokine mix” corresponding to a concentration of 2 ng/mL (ACT 2) or 5 ng/mL (ACT 5) in each compound of the mix and incubated with 10 mM N-acetyl-cysteine (NAC) (ACT 2 + NAC), 100 μM genistein (ACT 2 + GEN), 10 ng/mL TNF-α or 1 μg/mL LPS alone, and with 10 ng/mL TNF-α or 1 μg/mL LPS incubated with 100 μM genistein (TNF-α + GEN or LPS + GEN). DMSO-treated, unstimulated cells were used as the control (NACT). Additional control was used in the form of unstimulated cells treated with 100 μM genistein (GEN). Intracellular ROS levels were examined by a CellROX Deep Red Reagent and confocal fluorescence microscopy. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Results representative of three independent experiments (with scale bars 25 μm) are shown. (B) Analysis of ROS were additionally performed by fluorescent cell analyzer. The data are presented as the means ± standard deviation (SD) from three independent experiments. Significant differences ( p ≤ 0.05) between cell populations not expressing intracellular ROS (ROS [–]) and expressing intracellular ROS (ROS [+]) were observed for all tested conditions, except for LPS where cell numbers of ROS (-) and ROS (+) were comparable. Most important, statistically significant differences of p ≤ 0.05 within ROS groups are indicated with * for samples of TNF-α, TNF-α + GEN, and LPS versus NACT, with † for sample TNF-α + GEN with respect to TNF-α, while with ‡ for sample LPS + GEN referred to LPS. Statistical analysis was performed using ANOVA with Tukey’s HSD test.

    Article Snippet: For further experiments, cells were stimulated with a combination of a proinflammatory “cytokine mix”: IL-1A, IL-17A, IL-22, oncostatin M (OSM), and tumor necrosis factor-α (TNF-α) (Gibco, Thermo Fisher Scientific, CA, USA) 2 ng/mL each or lipopolysaccharide (LPS, from Escherichia coli 055 :B5 , Sigma-Aldrich, St. Louis, USA) 1 μg/mL in the presence or absence of genistein for 24 hours [ ].

    Techniques: Concentration Assay, Activated Clotting Time Assay, Incubation, Fluorescence, Microscopy, Staining, Standard Deviation, Expressing

    FGF-2 induces HA accumulation in primary RPE cells. ( A – D ) FGF-2 induced HA accumulation in primary porcine RPE cells in a dose-dependent manner ( A ) 0 ng/mL, ( B ) 10 ng/mL, ( C ) 25 ng/mL, ( D ) 100 ng/mL. Fluorescence intensity was quantitated by integrated density measurement ( I ) (n ≥ 4, for each cell line). Data are presented as mean ± SEM ( E – H ) Z-plane images show that increased concentrations of FGF-2 induce increased apical accumulation of HA in addition to some peri-cellular and basal deposits of HA at high doses of FGF-2. Green: HA, red: phalloidin; blue: DAPI.

    Journal: Cells

    Article Title: Role of FGF and Hyaluronan in Choroidal Neovascularization in Sorsby Fundus Dystrophy

    doi: 10.3390/cells9030608

    Figure Lengend Snippet: FGF-2 induces HA accumulation in primary RPE cells. ( A – D ) FGF-2 induced HA accumulation in primary porcine RPE cells in a dose-dependent manner ( A ) 0 ng/mL, ( B ) 10 ng/mL, ( C ) 25 ng/mL, ( D ) 100 ng/mL. Fluorescence intensity was quantitated by integrated density measurement ( I ) (n ≥ 4, for each cell line). Data are presented as mean ± SEM ( E – H ) Z-plane images show that increased concentrations of FGF-2 induce increased apical accumulation of HA in addition to some peri-cellular and basal deposits of HA at high doses of FGF-2. Green: HA, red: phalloidin; blue: DAPI.

    Article Snippet: Cells were serum-starved for 24 h before treatment with the FGF Receptor inhibitor BGJ-398 (Selleckchem, Houston, TX, USA, #S2183) for 48 h. Similarly, cells were treated with FGF-2 (Gibco from Thermo Fisher Scientific, #13256-029) with the required cofactor heparin sodium salt (1 μg/mL, Sigma Aldrich, #H3149) for 48 h after serum starving for 24 h.

    Techniques: Fluorescence

    The RPE secreted higher levels of GM-CSF after stimulation with HNE and TNF-α. The GM-CSF secreted into the culture supernatant was increased when primary RPE cells were exposed to 4-hydroxynonenal (HNE), an agent that promotes oxidative stress, at 10 µM for 6 h (mean ± SEM, 145.88±5.06 pg/ml versus 123.27±4.05 pg/ml, n=3, Student t test, *p

    Journal: Molecular Vision

    Article Title: CFH Y402H polymorphism is associated with elevated vitreal GM-CSF and choroidal macrophages in the postmortem human eye

    doi:

    Figure Lengend Snippet: The RPE secreted higher levels of GM-CSF after stimulation with HNE and TNF-α. The GM-CSF secreted into the culture supernatant was increased when primary RPE cells were exposed to 4-hydroxynonenal (HNE), an agent that promotes oxidative stress, at 10 µM for 6 h (mean ± SEM, 145.88±5.06 pg/ml versus 123.27±4.05 pg/ml, n=3, Student t test, *p

    Article Snippet: Then, 200 µl of 1× phenol-free minimum essential media (MEM)/F12 medium (Life Technologies) containing either HNE (Millipore, Etobicoke, Canada) at 10 µM or recombinant human TNF-α (R & D Systems, Minneapolis, MN) at 20 ng/ml was added into each corresponding well and incubated for 6 h. C3a or C5a (R & D Systems) at 5 µg/ml was used to incubate the cell culture for 24 h. After incubation, the resulting supernatants or the cell lysate were collected, centrifuged, and stored at −80 °C for the suspension assay or qPCR.

    Techniques:

    Persisting antioncogenic efficacy of Emx2 upon neural nestin enhancer-restricted overexpression In vitro kinetic progression of GbmA and GbmB lines C, D. , engineered by lentiviral vectors and TetON technology as in A, B. , and kept as floating cultures, under Fgf2 and Egf. Cell numbers were normalized against t =0 values. n is the number of biological replicates. p -value was calculated by t-test (one-tail, unpaired): *** p

    Journal: Oncotarget

    Article Title: Emx2 as a novel tool to suppress glioblastoma

    doi: 10.18632/oncotarget.9322

    Figure Lengend Snippet: Persisting antioncogenic efficacy of Emx2 upon neural nestin enhancer-restricted overexpression In vitro kinetic progression of GbmA and GbmB lines C, D. , engineered by lentiviral vectors and TetON technology as in A, B. , and kept as floating cultures, under Fgf2 and Egf. Cell numbers were normalized against t =0 values. n is the number of biological replicates. p -value was calculated by t-test (one-tail, unpaired): *** p

    Article Snippet: In both cases, mediums were supplemented with 1X Penicillin/Streptomycin, 2 μg/ml human heparin (StemCell Technologies #07980), 20 ng/ml recombinant human EGF (ThermoFisher #PHG0311), 20 ng/ml recombinant human FGF2 (ThermoFisher #PHG0261).

    Techniques: Over Expression, In Vitro

    Population dynamics of Emx2 gain-of-function GBM cultures In vitro kinetic progression of U87MG, T98G, GbmA, GbmB, GbmC, GbmD and GbmE GBM lines C-I ., engineered by lentiviral vectors and TetON technology as in A, B. , and kept as adherent C, D. or floating cultures E-I. , under Fgf2 and Egf. Ki67 + proliferating L, M. and activated-Casp3 + apoptotic N, O. fractions of GbmA, GbmB and GbmC glioblastoma cells, engineered by control ( J. , a-b1) and Emx2 -GOF (J, a-b2) lentiviral sets, and kept as floating cultures according to the timetable in K. Cell numbers were normalized against t =0 values (C-I), or control values L, N. [As for (L, N), absolute average control cell frequencies were: 0.207, 0.155 and 0.131 (Ki67 + , in GbmA, GbmB and GbmC cultures, respectively); 0.001, 0.012 and 0.012 (actCasp3 + , in GbmA, GbmB and GbmC cultures, respectively)]. n is the number of biological replicates. p -value was calculated by t-test (one-tail, unpaired): * p

    Journal: Oncotarget

    Article Title: Emx2 as a novel tool to suppress glioblastoma

    doi: 10.18632/oncotarget.9322

    Figure Lengend Snippet: Population dynamics of Emx2 gain-of-function GBM cultures In vitro kinetic progression of U87MG, T98G, GbmA, GbmB, GbmC, GbmD and GbmE GBM lines C-I ., engineered by lentiviral vectors and TetON technology as in A, B. , and kept as adherent C, D. or floating cultures E-I. , under Fgf2 and Egf. Ki67 + proliferating L, M. and activated-Casp3 + apoptotic N, O. fractions of GbmA, GbmB and GbmC glioblastoma cells, engineered by control ( J. , a-b1) and Emx2 -GOF (J, a-b2) lentiviral sets, and kept as floating cultures according to the timetable in K. Cell numbers were normalized against t =0 values (C-I), or control values L, N. [As for (L, N), absolute average control cell frequencies were: 0.207, 0.155 and 0.131 (Ki67 + , in GbmA, GbmB and GbmC cultures, respectively); 0.001, 0.012 and 0.012 (actCasp3 + , in GbmA, GbmB and GbmC cultures, respectively)]. n is the number of biological replicates. p -value was calculated by t-test (one-tail, unpaired): * p

    Article Snippet: In both cases, mediums were supplemented with 1X Penicillin/Streptomycin, 2 μg/ml human heparin (StemCell Technologies #07980), 20 ng/ml recombinant human EGF (ThermoFisher #PHG0311), 20 ng/ml recombinant human FGF2 (ThermoFisher #PHG0261).

    Techniques: In Vitro

    (a) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 3.2 nM EGF Alexa Fluor 555. After 15 min the cells were acid washed where indicated, fixed and the nuclei stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was assessed in brightfield and fluorescent images taken using confocal microscopy. The right hand panels are enlarged images of the indicated area in the left hand panels. Scale bar = 10 μm. (b) HeLa cells were transfected with either scrambled shRNA or USP17 shRNA1 and 72 hrs post transfection the cells were starved in serum free medium for 3hrs prior to incubation with either 0 nM, 0.32 nM or 3.2 nM recombinant EGF. After 15mins the cells were washed, fixed and stained using an anti-EGFR antibody. EGFR (green) internalisation was assessed in fluorescent images taken using confocal microscopy. The right hand panels are enlarged images of the indicated area in the left hand panels. White arrows point out plasma membrane staining for EGFR. Scale bar = 20 μm.

    Journal: Oncotarget

    Article Title: USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor

    doi:

    Figure Lengend Snippet: (a) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 3.2 nM EGF Alexa Fluor 555. After 15 min the cells were acid washed where indicated, fixed and the nuclei stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was assessed in brightfield and fluorescent images taken using confocal microscopy. The right hand panels are enlarged images of the indicated area in the left hand panels. Scale bar = 10 μm. (b) HeLa cells were transfected with either scrambled shRNA or USP17 shRNA1 and 72 hrs post transfection the cells were starved in serum free medium for 3hrs prior to incubation with either 0 nM, 0.32 nM or 3.2 nM recombinant EGF. After 15mins the cells were washed, fixed and stained using an anti-EGFR antibody. EGFR (green) internalisation was assessed in fluorescent images taken using confocal microscopy. The right hand panels are enlarged images of the indicated area in the left hand panels. White arrows point out plasma membrane staining for EGFR. Scale bar = 20 μm.

    Article Snippet: EGF internalisation Transfected cells were rested for 3hrs in DMEM medium without serum and stimulated with 0.32nM-3.2nM EGF Alexa Fluor 555 (Invitrogen-Molecular Probes, Eugene, USA) or recombinant EGF (Invitrogen-Gibco, Maryland, USA) for the indicated times in the figures.

    Techniques: Transfection, Incubation, Staining, Confocal Microscopy, shRNA, Recombinant

    (a) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF Alexa Fluor 555 where indicated. After 15 min the cells were acid washed, where indicated, fixed and the nuclei stained with DAPI. The cells were then stained using an anti-EGFR antibody and EGF Alexa Fluor 555 (red) and EGFR (green) internalisation was assessed in brightfield and fluorescent images taken using confocal microscopy. The right panels are enlarged images of the indicated area in the left panels. Scale bar = 10 μm. (b) At least 50 cells per condition were blindly scored for three separate experiments based on the presence of EGF Alexa Fluor 555 and EGFR co-staining post acid wash. (c) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF. Whole cell lysates were harvested and levels of phosphorylated ERK1/2, ERK1/2, EGFR and tubulin were assessed by immuno-blotting using anti-pERK1/2, anti-ERK1/2, anti-EGFR and anti-tubulin antibodies. ** p

    Journal: Oncotarget

    Article Title: USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor

    doi:

    Figure Lengend Snippet: (a) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF Alexa Fluor 555 where indicated. After 15 min the cells were acid washed, where indicated, fixed and the nuclei stained with DAPI. The cells were then stained using an anti-EGFR antibody and EGF Alexa Fluor 555 (red) and EGFR (green) internalisation was assessed in brightfield and fluorescent images taken using confocal microscopy. The right panels are enlarged images of the indicated area in the left panels. Scale bar = 10 μm. (b) At least 50 cells per condition were blindly scored for three separate experiments based on the presence of EGF Alexa Fluor 555 and EGFR co-staining post acid wash. (c) HeLa cells were transfected as indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF. Whole cell lysates were harvested and levels of phosphorylated ERK1/2, ERK1/2, EGFR and tubulin were assessed by immuno-blotting using anti-pERK1/2, anti-ERK1/2, anti-EGFR and anti-tubulin antibodies. ** p

    Article Snippet: EGF internalisation Transfected cells were rested for 3hrs in DMEM medium without serum and stimulated with 0.32nM-3.2nM EGF Alexa Fluor 555 (Invitrogen-Molecular Probes, Eugene, USA) or recombinant EGF (Invitrogen-Gibco, Maryland, USA) for the indicated times in the figures.

    Techniques: Transfection, Incubation, Staining, Confocal Microscopy

    (a) HeLa cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF Alexa Fluor 555 and mRNA and protein samples were harvested at the time points indicated. USP17 and GAPDH (loading control) mRNA were then assessed by RT-PCR. USP17 protein levels were assessed by immunoblotting and tubulin was used as a loading control. (b) HeLa cells were transfected as indicated. 72 hrs post transfection mRNA and protein samples were harvested and USP17 mRNA and protein levels assessed as above. (c) HeLa cells were transfected with the constructs indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF Alexa Fluor 555. After 15 min the cells were acid washed, fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The bottom panels are enlarged images of the indicated area in the top panels. Scale bar = 20 μm. (d) At least 50 cells per condition were blindly scored for three separate experiments based on the presence of EGF Alexa Fluor 555 internalisation. (e) A549 cells were transfected as indicated and 72 hrs post transfection the cells were starved in serum free medium for 3hrs prior to incubation with 0.32nM EGF Alexa Fluor 555. After 10 mins the cells were either acid washed or washed with PBS. Subsequently they were fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The middle panels are enlarged images of the indicated area in the upper panels. Scale bar = 25 μm. (f) HeLa cells were transfected as indicated and 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32nM EGF Alexa Fluor 555. After 45 mins the cells were acid washed, fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The lower panels are enlarged images of the indicated area in the upper panels. Scale bar = 10 μm. ** p

    Journal: Oncotarget

    Article Title: USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor

    doi:

    Figure Lengend Snippet: (a) HeLa cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF Alexa Fluor 555 and mRNA and protein samples were harvested at the time points indicated. USP17 and GAPDH (loading control) mRNA were then assessed by RT-PCR. USP17 protein levels were assessed by immunoblotting and tubulin was used as a loading control. (b) HeLa cells were transfected as indicated. 72 hrs post transfection mRNA and protein samples were harvested and USP17 mRNA and protein levels assessed as above. (c) HeLa cells were transfected with the constructs indicated. 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32 nM EGF Alexa Fluor 555. After 15 min the cells were acid washed, fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The bottom panels are enlarged images of the indicated area in the top panels. Scale bar = 20 μm. (d) At least 50 cells per condition were blindly scored for three separate experiments based on the presence of EGF Alexa Fluor 555 internalisation. (e) A549 cells were transfected as indicated and 72 hrs post transfection the cells were starved in serum free medium for 3hrs prior to incubation with 0.32nM EGF Alexa Fluor 555. After 10 mins the cells were either acid washed or washed with PBS. Subsequently they were fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The middle panels are enlarged images of the indicated area in the upper panels. Scale bar = 25 μm. (f) HeLa cells were transfected as indicated and 72 hrs post transfection the cells were starved in serum free medium for 3 hrs prior to incubation with 0.32nM EGF Alexa Fluor 555. After 45 mins the cells were acid washed, fixed and the nuclei were stained with DAPI. EGF Alexa Fluor 555 (red) internalisation was then assessed in brightfield and fluorescent images taken using confocal microscopy. The lower panels are enlarged images of the indicated area in the upper panels. Scale bar = 10 μm. ** p

    Article Snippet: EGF internalisation Transfected cells were rested for 3hrs in DMEM medium without serum and stimulated with 0.32nM-3.2nM EGF Alexa Fluor 555 (Invitrogen-Molecular Probes, Eugene, USA) or recombinant EGF (Invitrogen-Gibco, Maryland, USA) for the indicated times in the figures.

    Techniques: Incubation, Reverse Transcription Polymerase Chain Reaction, Transfection, Construct, Staining, Confocal Microscopy

    JSI-124 inhibited bleomycin-induced bronchoalveolar inflammatory cell extravasation and lung fibrosis. Wistar rats were administered a single intratracheal dose of bleomycin (BLM; 3.75 U/kg) on day 1. JSI-124 (1 mg/kg/day) or vehicle was administered intraperitoneally from day 14 until the analysis at day 28 ( n = 10 per group). Total bronchoalveolar lavage ( a ) protein, ( b , c ) inflammatory cells, ( d ) lung weight, ( e ) protein, and ( f , g ) IL-6 and IL-13 content were measured at day 28. h Masson’s trichrome (upper panels, scale bar: 100 μm) of control, BLM, and BLM + JSI-124 tissue. i Fibrosis Ashcroft scores were assessed as described in the Methods. j Micro-CT images were acquired on day 28 and ( k ) quantified as Hounsfield units. The results are expressed as the mean ± SEM, n = 10. Statistical significance was assessed using a t -test or one-way ANOVA followed by a Bonferroni post-hoc test. * P

    Journal: Respiratory Research

    Article Title: The JAK2 pathway is activated in idiopathic pulmonary fibrosis

    doi: 10.1186/s12931-018-0728-9

    Figure Lengend Snippet: JSI-124 inhibited bleomycin-induced bronchoalveolar inflammatory cell extravasation and lung fibrosis. Wistar rats were administered a single intratracheal dose of bleomycin (BLM; 3.75 U/kg) on day 1. JSI-124 (1 mg/kg/day) or vehicle was administered intraperitoneally from day 14 until the analysis at day 28 ( n = 10 per group). Total bronchoalveolar lavage ( a ) protein, ( b , c ) inflammatory cells, ( d ) lung weight, ( e ) protein, and ( f , g ) IL-6 and IL-13 content were measured at day 28. h Masson’s trichrome (upper panels, scale bar: 100 μm) of control, BLM, and BLM + JSI-124 tissue. i Fibrosis Ashcroft scores were assessed as described in the Methods. j Micro-CT images were acquired on day 28 and ( k ) quantified as Hounsfield units. The results are expressed as the mean ± SEM, n = 10. Statistical significance was assessed using a t -test or one-way ANOVA followed by a Bonferroni post-hoc test. * P

    Article Snippet: IL-6 and IL-13 cytokines were analyzed in the cell culture supernatants of human ATII and fibroblast using commercially available Quantikine® ELISA kits for human IL-6 (Catalog No. D6050; R & D Systems, Madrid, Spain) and IL-13 (Catalog No. D1300B), and in the bronchoalveolar lavage (BAL) fluid of rats using the ELISA rat IL-6 (Catalog No. KRC0061; Invitrogen™, Madrid, Spain) and IL-13 (Catalog No. KRC0132; Invitrogen™) kits according to the manufacturers’ protocols.

    Techniques: Micro-CT

    Effects of JAK2 and STAT3 on ATII to mesenchymal and fibroblast to myofibroblast transitions. Primary ATII and lung fibroblasts were isolated from the lungs of IPF patients. a The cells were incubated with the dual p-JAK2/p-STAT3 inhibitor JSI-124 for 30 min followed by TGF-β1 stimulation for an additional 24 h. IL-6 and IL-13 levels in cell supernatants were measured using ELISA. b Ratios of JAK2/p-JAK2 and STAT3/p-STAT3 were determined by western blotting in ATII cells stimulated for 40 min or 24 h with TGF-β1 in the presence or absence of JSI-124. c , d ATII cells were pre-incubated for 30 min with 1 μM of the p-STAT3 inhibitor 5,15 DPP, the p-JAK2 inhibitor NSC33994, or the dual p-JAK2/p-STAT3 inhibitor JSI-124, and then stimulated for 72 h with TGF-β1 ( c ) or IL-6/IL-13 ( d ). e Levels of IL-6 and IL-13 in primary fibroblasts. f JAK2/p-JAK2 and STAT3/p-STAT3 protein expression in human lung fibroblasts. g , h Primary lung fibroblasts pre-incubated for 30 min with 1 μM of the p-STAT3 inhibitor 5,15 DPP, the p-JAK2 inhibitor NSC33994, or the dual p-JAK2/p-STAT3 inhibitor JSI-124 and stimulated for 72 h with TGF-β1 ( g ) or IL-6/IL-13 ( h ). Representative western blots are shown. The results are expressed as the mean (SEM) of n = 4 (cells from four IPF patients) experiments per condition. Two-way ANOVA followed by post-hoc Bonferroni tests. * P

    Journal: Respiratory Research

    Article Title: The JAK2 pathway is activated in idiopathic pulmonary fibrosis

    doi: 10.1186/s12931-018-0728-9

    Figure Lengend Snippet: Effects of JAK2 and STAT3 on ATII to mesenchymal and fibroblast to myofibroblast transitions. Primary ATII and lung fibroblasts were isolated from the lungs of IPF patients. a The cells were incubated with the dual p-JAK2/p-STAT3 inhibitor JSI-124 for 30 min followed by TGF-β1 stimulation for an additional 24 h. IL-6 and IL-13 levels in cell supernatants were measured using ELISA. b Ratios of JAK2/p-JAK2 and STAT3/p-STAT3 were determined by western blotting in ATII cells stimulated for 40 min or 24 h with TGF-β1 in the presence or absence of JSI-124. c , d ATII cells were pre-incubated for 30 min with 1 μM of the p-STAT3 inhibitor 5,15 DPP, the p-JAK2 inhibitor NSC33994, or the dual p-JAK2/p-STAT3 inhibitor JSI-124, and then stimulated for 72 h with TGF-β1 ( c ) or IL-6/IL-13 ( d ). e Levels of IL-6 and IL-13 in primary fibroblasts. f JAK2/p-JAK2 and STAT3/p-STAT3 protein expression in human lung fibroblasts. g , h Primary lung fibroblasts pre-incubated for 30 min with 1 μM of the p-STAT3 inhibitor 5,15 DPP, the p-JAK2 inhibitor NSC33994, or the dual p-JAK2/p-STAT3 inhibitor JSI-124 and stimulated for 72 h with TGF-β1 ( g ) or IL-6/IL-13 ( h ). Representative western blots are shown. The results are expressed as the mean (SEM) of n = 4 (cells from four IPF patients) experiments per condition. Two-way ANOVA followed by post-hoc Bonferroni tests. * P

    Article Snippet: IL-6 and IL-13 cytokines were analyzed in the cell culture supernatants of human ATII and fibroblast using commercially available Quantikine® ELISA kits for human IL-6 (Catalog No. D6050; R & D Systems, Madrid, Spain) and IL-13 (Catalog No. D1300B), and in the bronchoalveolar lavage (BAL) fluid of rats using the ELISA rat IL-6 (Catalog No. KRC0061; Invitrogen™, Madrid, Spain) and IL-13 (Catalog No. KRC0132; Invitrogen™) kits according to the manufacturers’ protocols.

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

    Fibroblast migration and proliferation in IPF were dependent on JAK2 and STAT3 activation. a , b Primary human fibroblasts from IPF patients were pre-treated for 30 min with 1 μM of the p-STAT3 inhibitor 5,15 DPP, the p-JAK2 inhibitor NSC33994, or the dual p-JAK2/p-STAT3 inhibitor JSI-124 and then cell migration was assessed. A scrape-wound was created by using a sterile p200 pipette tip to make a perpendicular linear scratch in the culture. After the cells had been washed, culture medium with or without pharmacologic modulators and IL-6/IL-13 was added. The size of the wound remaining was analyzed at the indicated times and expressed as a percentage of the initial wound area. c Fibroblast proliferation during 48 h was evaluated by the BrdU assay. Different JAK2 and STAT3 inhibitors were added 30 min before 10% fetal bovine serum (FBS) or ( c ) 50 ng IL-6/IL-13/mL ( d ) was added as the stimulus. Values are expressed as relative absorbance (450 nm) units. The results are expressed as the mean (SEM) of n = 4 (cells from four IPF patients) experiments per condition. Two-way ANOVA followed by post-hoc Bonferroni tests. * P

    Journal: Respiratory Research

    Article Title: The JAK2 pathway is activated in idiopathic pulmonary fibrosis

    doi: 10.1186/s12931-018-0728-9

    Figure Lengend Snippet: Fibroblast migration and proliferation in IPF were dependent on JAK2 and STAT3 activation. a , b Primary human fibroblasts from IPF patients were pre-treated for 30 min with 1 μM of the p-STAT3 inhibitor 5,15 DPP, the p-JAK2 inhibitor NSC33994, or the dual p-JAK2/p-STAT3 inhibitor JSI-124 and then cell migration was assessed. A scrape-wound was created by using a sterile p200 pipette tip to make a perpendicular linear scratch in the culture. After the cells had been washed, culture medium with or without pharmacologic modulators and IL-6/IL-13 was added. The size of the wound remaining was analyzed at the indicated times and expressed as a percentage of the initial wound area. c Fibroblast proliferation during 48 h was evaluated by the BrdU assay. Different JAK2 and STAT3 inhibitors were added 30 min before 10% fetal bovine serum (FBS) or ( c ) 50 ng IL-6/IL-13/mL ( d ) was added as the stimulus. Values are expressed as relative absorbance (450 nm) units. The results are expressed as the mean (SEM) of n = 4 (cells from four IPF patients) experiments per condition. Two-way ANOVA followed by post-hoc Bonferroni tests. * P

    Article Snippet: IL-6 and IL-13 cytokines were analyzed in the cell culture supernatants of human ATII and fibroblast using commercially available Quantikine® ELISA kits for human IL-6 (Catalog No. D6050; R & D Systems, Madrid, Spain) and IL-13 (Catalog No. D1300B), and in the bronchoalveolar lavage (BAL) fluid of rats using the ELISA rat IL-6 (Catalog No. KRC0061; Invitrogen™, Madrid, Spain) and IL-13 (Catalog No. KRC0132; Invitrogen™) kits according to the manufacturers’ protocols.

    Techniques: Migration, Activation Assay, Transferring, BrdU Staining

    Dual JAK2/STAT3 gene silencing suppressed ATII to mesenchymal and fibroblast to myofibroblast transitions. ATII A549 and human lung fibroblast MRC5 cell lines were transfected with control siRNA(−), siRNA-JAK2, siRNA-STAT3, or both siRNA-JAK2/STAT3 and stimulated for 72 h with TG-Fβ1 or IL-6/IL-13 at a concentration of 5 ng/mL. a Total protein and RNA from cell lysates were analyzed by ( a , b ) western blot and ( c ) qPCR. Mesenchymal collagen type I and epithelial E-cadherin markers were measured using ( a ) western blot and ( c ) qPCR. Senescence p21, autophagy LC3I/II, and anti-apoptotic BCL-2 markers were ( a ) measured using western blot and ( b ) quantified by densitometry. Data are expressed as the ratio to β-actin protein and mRNA levels. The results are expressed as the mean (SEM) of n = 4 independent experiments per condition. One-way ANOVA followed by post-hoc Bonferroni tests. * P

    Journal: Respiratory Research

    Article Title: The JAK2 pathway is activated in idiopathic pulmonary fibrosis

    doi: 10.1186/s12931-018-0728-9

    Figure Lengend Snippet: Dual JAK2/STAT3 gene silencing suppressed ATII to mesenchymal and fibroblast to myofibroblast transitions. ATII A549 and human lung fibroblast MRC5 cell lines were transfected with control siRNA(−), siRNA-JAK2, siRNA-STAT3, or both siRNA-JAK2/STAT3 and stimulated for 72 h with TG-Fβ1 or IL-6/IL-13 at a concentration of 5 ng/mL. a Total protein and RNA from cell lysates were analyzed by ( a , b ) western blot and ( c ) qPCR. Mesenchymal collagen type I and epithelial E-cadherin markers were measured using ( a ) western blot and ( c ) qPCR. Senescence p21, autophagy LC3I/II, and anti-apoptotic BCL-2 markers were ( a ) measured using western blot and ( b ) quantified by densitometry. Data are expressed as the ratio to β-actin protein and mRNA levels. The results are expressed as the mean (SEM) of n = 4 independent experiments per condition. One-way ANOVA followed by post-hoc Bonferroni tests. * P

    Article Snippet: IL-6 and IL-13 cytokines were analyzed in the cell culture supernatants of human ATII and fibroblast using commercially available Quantikine® ELISA kits for human IL-6 (Catalog No. D6050; R & D Systems, Madrid, Spain) and IL-13 (Catalog No. D1300B), and in the bronchoalveolar lavage (BAL) fluid of rats using the ELISA rat IL-6 (Catalog No. KRC0061; Invitrogen™, Madrid, Spain) and IL-13 (Catalog No. KRC0132; Invitrogen™) kits according to the manufacturers’ protocols.

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

    ( A ) The cytotoxicity of T cells was evaluated by the LDH assay at different ( E ) T ratios. ( C ) Human Th1/Th2 Cytokine Kit determined the expression level of IL-2, IL-4, IL-6, TNF-α, and IFN-γ of DMSO or YN968D1-treated T cells. ( B, D ) The frequency of IFN-γ-producing T cells after co-incubation with gastric cancer cell MKN-45 for 18 hrs was evaluated by IFN-γ ELISPOT kit (Dakewei, China). T cells and MKN-45 were co-incubated at different E: T ratios (10:1, 5:1, 3:1). * means P value

    Journal: OncoTargets and therapy

    Article Title: Immune-Mediated Antitumor Effect By VEGFR2 Selective Inhibitor For Gastric Cancer

    doi: 10.2147/OTT.S233496

    Figure Lengend Snippet: ( A ) The cytotoxicity of T cells was evaluated by the LDH assay at different ( E ) T ratios. ( C ) Human Th1/Th2 Cytokine Kit determined the expression level of IL-2, IL-4, IL-6, TNF-α, and IFN-γ of DMSO or YN968D1-treated T cells. ( B, D ) The frequency of IFN-γ-producing T cells after co-incubation with gastric cancer cell MKN-45 for 18 hrs was evaluated by IFN-γ ELISPOT kit (Dakewei, China). T cells and MKN-45 were co-incubated at different E: T ratios (10:1, 5:1, 3:1). * means P value

    Article Snippet: We used human anti-CD3 antibody (R & D systems, Minneapolis) at a final concentration of 25ng/mL and recombinant human interleukin-2 (rhIL-2, R & D systems) at a final concentration of 200IU/mL on day 1 for the stimulation and proliferation of T cells.

    Techniques: Lactate Dehydrogenase Assay, Expressing, Incubation, Enzyme-linked Immunospot

    IL-25, but not IL-4, IL-5 or IL-13 induced angiogenesis in vitro. Top panel: representative light photomicrographs (4x original magnification) show formation of primitive vascular tubule structures by human vascular endothelial cells after 11 days of culture (top panel) with medium (A) , VEGF (10 ng/mL) (B) , IL-25 (10 ng/mL) (C) , IL-4 (10 ng/mL) (D) , IL-5 (10 ng/mL) (E) and IL-13 (10 ng/mL) (F) . Bottom panel: computer-assisted quantification of total tubule lengths (G) , numbers of branch points (H) and total numbers of tubules (I) . Bars show the mean ± SEM of three separate experiments performed in duplicate. *p

    Journal: Respiratory Research

    Article Title: IL-25 induces airways angiogenesis and expression of multiple angiogenic factors in a murine asthma model

    doi: 10.1186/s12931-015-0197-3

    Figure Lengend Snippet: IL-25, but not IL-4, IL-5 or IL-13 induced angiogenesis in vitro. Top panel: representative light photomicrographs (4x original magnification) show formation of primitive vascular tubule structures by human vascular endothelial cells after 11 days of culture (top panel) with medium (A) , VEGF (10 ng/mL) (B) , IL-25 (10 ng/mL) (C) , IL-4 (10 ng/mL) (D) , IL-5 (10 ng/mL) (E) and IL-13 (10 ng/mL) (F) . Bottom panel: computer-assisted quantification of total tubule lengths (G) , numbers of branch points (H) and total numbers of tubules (I) . Bars show the mean ± SEM of three separate experiments performed in duplicate. *p

    Article Snippet: Culture medium with recombinant human IL-4, IL-5, IL-13 and IL-25 (10 ng/ml, R & D Systems, Abingdon, UK) [ , ] was replenished on days 4, 7, and 9.

    Techniques: In Vitro

    Anti-IL-17A antibodies but not anti-IL-17F antibodies render mice susceptible to OPC.

    Journal: Journal of Leukocyte Biology

    Article Title: Antibody blockade of IL-17 family cytokines in immunity to acute murine oral mucosal candidiasis

    doi: 10.1189/jlb.4A0915-428R

    Figure Lengend Snippet: Anti-IL-17A antibodies but not anti-IL-17F antibodies render mice susceptible to OPC.

    Article Snippet: As an independent approach to interrogate the role of IL-17 cytokines in OPC, we evaluated IL-17A−/− and IL-17F−/− mice.

    Techniques: Mouse Assay

    HoxC4 deficiency impairs estrogen-mediated enhancement of AID expression and CSR to IgG1 and IgE, as induced by LPS and IL-4 or CD154 and IL-4. A , HoxC4 +/+ and HoxC4 −/− B cells were stimulated with LPS and rmIL-4 or with CD154 and rmIL-4

    Journal: The Journal of Biological Chemistry

    Article Title: Estrogen Receptors Bind to and Activate the HOXC4/HoxC4 Promoter to Potentiate HoxC4-mediated Activation-induced Cytosine Deaminase Induction, Immunoglobulin Class Switch DNA Recombination, and Somatic Hypermutation *

    doi: 10.1074/jbc.M110.169086

    Figure Lengend Snippet: HoxC4 deficiency impairs estrogen-mediated enhancement of AID expression and CSR to IgG1 and IgE, as induced by LPS and IL-4 or CD154 and IL-4. A , HoxC4 +/+ and HoxC4 −/− B cells were stimulated with LPS and rmIL-4 or with CD154 and rmIL-4

    Article Snippet: In addition, consistent with our finding that estrogen alone did not significantly induce HoxC4 expression but powerfully potentiated CD154- or LPS-induced HoxC4 expression and HoxC4-mediated AID induction, an Ab to HoxC4 precipitated a significantly greater amount of Aicda promoter DNA in primary mouse B cells induced by LPS and rmIL-4 in the presence of E2 as compared with B cells cultured under similar conditions but in the absence of E2 or B cells cultured with E2 alone or nil.

    Techniques: Expressing

    MCR expressions during erythroblast differentiation. ( A ) Schematic diagram of an in vitro differentiation protocol for deriving erythroblasts from human HPCs. Human CD34 + HPCs were expanded for 7 days (E0–E7) and stocks were frozen in liquid nitrogen (LN 2 ). The stock cells were differentiated for 3 days (D0–D3) before undergoing maturation (M0–M7). The number of enucleated erythrocytes increased from M5 to M7. ACTH, adrenocorticotropic hormone; EPO, erythropoietin; FL, flt-3 ligand; IL-3, interleukin-3; IL-6, interleukin-6; α-MSH, α-melanocyte stimulating hormone; SCF, stem cell factor; TPO, thrombopoietin. ( B ) May-Grunwald-Giemsa staining of control erythroblasts at day M0–5 day. Cells differentiated to Pro-E stage at day M0 and Baso-E stage at day M3. Arrows, Poly-E; Arrow heads, Orho-E. *, Reticulocyte. Bar 10 μm. ( C ) Conventional RT-PCR for MCRs during erythroblasts differentiation between M0 and M6 day.

    Journal: PLoS ONE

    Article Title: Melanocortins Contribute to Sequential Differentiation and Enucleation of Human Erythroblasts via Melanocortin Receptors 1, 2 and 5

    doi: 10.1371/journal.pone.0123232

    Figure Lengend Snippet: MCR expressions during erythroblast differentiation. ( A ) Schematic diagram of an in vitro differentiation protocol for deriving erythroblasts from human HPCs. Human CD34 + HPCs were expanded for 7 days (E0–E7) and stocks were frozen in liquid nitrogen (LN 2 ). The stock cells were differentiated for 3 days (D0–D3) before undergoing maturation (M0–M7). The number of enucleated erythrocytes increased from M5 to M7. ACTH, adrenocorticotropic hormone; EPO, erythropoietin; FL, flt-3 ligand; IL-3, interleukin-3; IL-6, interleukin-6; α-MSH, α-melanocyte stimulating hormone; SCF, stem cell factor; TPO, thrombopoietin. ( B ) May-Grunwald-Giemsa staining of control erythroblasts at day M0–5 day. Cells differentiated to Pro-E stage at day M0 and Baso-E stage at day M3. Arrows, Poly-E; Arrow heads, Orho-E. *, Reticulocyte. Bar 10 μm. ( C ) Conventional RT-PCR for MCRs during erythroblasts differentiation between M0 and M6 day.

    Article Snippet: In the second passage (for differentiation; D0–D3 in ), cell stocks were thawed and cultured at 2 × 105 cells/ml in HPGM supplemented with 3 U/ml human EPO (Kyowa Hakko Kirin), 25 ng/ml SCF, 10 ng/ml recombinant human IL-3 (PeproTech), and 10 ng/ml recombinant human IL-6 (R & D Systems) for 3 days ( ).

    Techniques: In Vitro, Staining, Reverse Transcription Polymerase Chain Reaction

    Decreased activation of Akt by PDGF-AA in BBS1 M390R/M390R mouse embryonic fibroblast (MEF) cells. A and B : representative Western blots ( A ) and quantification data ( B ) of phosphorylated and total Akt in control and BBS1 M390R/M390R MEF cells stimulated with PDGF-AA or vehicle for 15 min; n = 4/group (performed in duplicate). * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: The Bardet-Biedl syndrome protein complex regulates cell migration and tissue repair through a Cullin-3/RhoA pathway

    doi: 10.1152/ajpcell.00498.2018

    Figure Lengend Snippet: Decreased activation of Akt by PDGF-AA in BBS1 M390R/M390R mouse embryonic fibroblast (MEF) cells. A and B : representative Western blots ( A ) and quantification data ( B ) of phosphorylated and total Akt in control and BBS1 M390R/M390R MEF cells stimulated with PDGF-AA or vehicle for 15 min; n = 4/group (performed in duplicate). * P

    Article Snippet: To understand the molecular mechanisms involved in cell migration defects in BBS1M390R/M390R MEF, we investigated PDGF receptor-α signaling by assessing the activation of Akt by PDGF-AA.

    Techniques: Activation Assay, Western Blot

    IL-6 is up-regulated in PDGFRα + stromal cells after SO 2 injury. ( A ) RNAs were extracted from whole trachea at 0, 1, 2, and 14 d after injury and subjected to quantitative RT-PCR analysis. The mRNA expression level of cytokines was normalized to Gapdh . ( B ) In situ hybridization combined with immunohistochemistry shows that Il-6 mRNA (red) is expressed in cells in the stroma beneath basal cells (K5 + , green) after SO 2 injury. ( C ) Quantitative PCR analysis of Il-6 expression in sorted stromal cells [ Pdgfr α ( Pdgfra )-GFP + ] and immune cell subpopulations from the trachea at 24 hpi. ( D ) Immunohistochemistry of a trachea section at 24 hpi shows Pdgfra -GFP + cells (GFP + , green) in the stroma beneath the epithelium with basal cells (K5 + , red). ( E ) In situ hybridization and immunohistochemistry show that Pdgfra -GFP + cells (GFP + , green) express Il-6 mRNA (red) at 24 hpi. (Scale bars: B and E , 20 μm; D , 50 μm.) * P

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

    Article Title: IL-6/STAT3 promotes regeneration of airway ciliated cells from basal stem cells

    doi: 10.1073/pnas.1409781111

    Figure Lengend Snippet: IL-6 is up-regulated in PDGFRα + stromal cells after SO 2 injury. ( A ) RNAs were extracted from whole trachea at 0, 1, 2, and 14 d after injury and subjected to quantitative RT-PCR analysis. The mRNA expression level of cytokines was normalized to Gapdh . ( B ) In situ hybridization combined with immunohistochemistry shows that Il-6 mRNA (red) is expressed in cells in the stroma beneath basal cells (K5 + , green) after SO 2 injury. ( C ) Quantitative PCR analysis of Il-6 expression in sorted stromal cells [ Pdgfr α ( Pdgfra )-GFP + ] and immune cell subpopulations from the trachea at 24 hpi. ( D ) Immunohistochemistry of a trachea section at 24 hpi shows Pdgfra -GFP + cells (GFP + , green) in the stroma beneath the epithelium with basal cells (K5 + , red). ( E ) In situ hybridization and immunohistochemistry show that Pdgfra -GFP + cells (GFP + , green) express Il-6 mRNA (red) at 24 hpi. (Scale bars: B and E , 20 μm; D , 50 μm.) * P

    Article Snippet: Mouse ALI cultures at day 7 were exposed to mouse IL-6 (20 ng/mL; R & D Systems) at 37 °C for 4 h. Approximately 4 × 106 cells were fixed at room temperature for 10 min and scraped off the inserts.

    Techniques: Quantitative RT-PCR, Expressing, In Situ Hybridization, Immunohistochemistry, Real-time Polymerase Chain Reaction

    IL-6/STAT3 signaling is activated in tracheal epithelium during repair. ( A ) Schematic of the SO 2 injury model. After exposure to SO 2 , luminal cells die. Basal cells spread, proliferate, and generate early progenitors. These progenitors differentiate into ciliated and secretory cells, and repair is complete in 2 wk. ( B ) Longitudinal midline sections stained with antibodies to p-STAT3 (red) and p63 (green), a marker for basal cells. ( C ) Expression of p-STAT3 (red) and FOXJ1 (green) during epithelial repair. Note the coexpression of p-STAT3 and FOXJ1 at 3 dpi. (Scale bars: B and C .)

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

    Article Title: IL-6/STAT3 promotes regeneration of airway ciliated cells from basal stem cells

    doi: 10.1073/pnas.1409781111

    Figure Lengend Snippet: IL-6/STAT3 signaling is activated in tracheal epithelium during repair. ( A ) Schematic of the SO 2 injury model. After exposure to SO 2 , luminal cells die. Basal cells spread, proliferate, and generate early progenitors. These progenitors differentiate into ciliated and secretory cells, and repair is complete in 2 wk. ( B ) Longitudinal midline sections stained with antibodies to p-STAT3 (red) and p63 (green), a marker for basal cells. ( C ) Expression of p-STAT3 (red) and FOXJ1 (green) during epithelial repair. Note the coexpression of p-STAT3 and FOXJ1 at 3 dpi. (Scale bars: B and C .)

    Article Snippet: Mouse ALI cultures at day 7 were exposed to mouse IL-6 (20 ng/mL; R & D Systems) at 37 °C for 4 h. Approximately 4 × 106 cells were fixed at room temperature for 10 min and scraped off the inserts.

    Techniques: Staining, Marker, Expressing

    Effect of IL-6/STAT3 on tracheal epithelial repair in vivo. ( A ) Schematic of gain-of-function ( K5-CreER T2 ; Socs3 flox/flox ; Rosa-YFP ) model. Floxed alleles are deleted, and the YFP reporter is activated in basal cells with three doses of Tmx. One week later, mice are exposed to SO 2 and tracheas are harvested at 6 dpi. ( B ) Representative midline sections of tracheas (ventral) stained with YFP (lineage label, green) and a-tub (ciliated cells, red) in control ( K5-CreER T2 ; Rosa-YFP ) and gain-of-function ( K5-CreER T2 ; Socs3 flox/flox ; Rosa-YFP ) mice. A similar analysis was carried out using antibodies to K5 for basal cells and SCGB1A1 and SCGB3A2 for secretory cells, respectively. ( C ) Percentage of total lineage-labeled cells (YFP + ) throughout the trachea that are ciliated, secretory, or basal cells. Blue and red bars show K5-CreER T2 ; Rosa-YFP and K5-CreER T2 ; Socs3 flox/flox ; Rosa-YFP , respectively. ( D ) FOXJ1 staining (green) of airway epithelium at 4 dpi in WT and Il-6 null mice. ( E ) SCGB3A2 staining (green) of airway epithelium at 4 dpi in WT and Il-6 null mice. ( F ) In Il-6 null mice, there is a reduction of ciliated cells (FOXJ1 + ) and an increase of secretory cells (SCGB3A2 + ) after SO 2 injury (4 dpi). * P

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

    Article Title: IL-6/STAT3 promotes regeneration of airway ciliated cells from basal stem cells

    doi: 10.1073/pnas.1409781111

    Figure Lengend Snippet: Effect of IL-6/STAT3 on tracheal epithelial repair in vivo. ( A ) Schematic of gain-of-function ( K5-CreER T2 ; Socs3 flox/flox ; Rosa-YFP ) model. Floxed alleles are deleted, and the YFP reporter is activated in basal cells with three doses of Tmx. One week later, mice are exposed to SO 2 and tracheas are harvested at 6 dpi. ( B ) Representative midline sections of tracheas (ventral) stained with YFP (lineage label, green) and a-tub (ciliated cells, red) in control ( K5-CreER T2 ; Rosa-YFP ) and gain-of-function ( K5-CreER T2 ; Socs3 flox/flox ; Rosa-YFP ) mice. A similar analysis was carried out using antibodies to K5 for basal cells and SCGB1A1 and SCGB3A2 for secretory cells, respectively. ( C ) Percentage of total lineage-labeled cells (YFP + ) throughout the trachea that are ciliated, secretory, or basal cells. Blue and red bars show K5-CreER T2 ; Rosa-YFP and K5-CreER T2 ; Socs3 flox/flox ; Rosa-YFP , respectively. ( D ) FOXJ1 staining (green) of airway epithelium at 4 dpi in WT and Il-6 null mice. ( E ) SCGB3A2 staining (green) of airway epithelium at 4 dpi in WT and Il-6 null mice. ( F ) In Il-6 null mice, there is a reduction of ciliated cells (FOXJ1 + ) and an increase of secretory cells (SCGB3A2 + ) after SO 2 injury (4 dpi). * P

    Article Snippet: Mouse ALI cultures at day 7 were exposed to mouse IL-6 (20 ng/mL; R & D Systems) at 37 °C for 4 h. Approximately 4 × 106 cells were fixed at room temperature for 10 min and scraped off the inserts.

    Techniques: In Vivo, Mouse Assay, Staining, Labeling

    Effect of IL-6 on regeneration of human epithelium in ALI culture. ( A ) Schematic of ALI culture of primary HBE cells. ( B ) Whole-mount staining of day 21 cultures for ciliated (α-tubulin, green) and secretory (SCGB3A1, red) cells. Nuclei are blue (DAPI). (Scale bar: 100 μm.) ( C ) Quantification of whole-mount staining, shown as a fold change over untreated culture. The α-tubulin + or SCGB3A1 + cells were counted in four randomly chosen areas (0.18 mm 2 ) per filter. Values are mean ± SD for cultures from three different donors. * P

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

    Article Title: IL-6/STAT3 promotes regeneration of airway ciliated cells from basal stem cells

    doi: 10.1073/pnas.1409781111

    Figure Lengend Snippet: Effect of IL-6 on regeneration of human epithelium in ALI culture. ( A ) Schematic of ALI culture of primary HBE cells. ( B ) Whole-mount staining of day 21 cultures for ciliated (α-tubulin, green) and secretory (SCGB3A1, red) cells. Nuclei are blue (DAPI). (Scale bar: 100 μm.) ( C ) Quantification of whole-mount staining, shown as a fold change over untreated culture. The α-tubulin + or SCGB3A1 + cells were counted in four randomly chosen areas (0.18 mm 2 ) per filter. Values are mean ± SD for cultures from three different donors. * P

    Article Snippet: Mouse ALI cultures at day 7 were exposed to mouse IL-6 (20 ng/mL; R & D Systems) at 37 °C for 4 h. Approximately 4 × 106 cells were fixed at room temperature for 10 min and scraped off the inserts.

    Techniques: Staining

    IL-6 enhances Foxj1-GFP expression in the mouse tracheosphere culture assay. ( A ) Schematic of the assay. NGFR + basal cells from Foxj1-GFP tracheas were cultured in 50% Matrigel in 96-well inserts. ( Right ) Section of a typical sphere with acetylated tubulin + (a-tub) ciliated (magenta) and Splunc + secretory cells (green). IHC, immunohistochemistry. The effect of IL-6 ( B ) and STAT3 inhibitor ( C ) on Foxj1 -GFP expression is shown. Differential interference contrast images ( Upper ) and fluorescent images ( Lower ) of the same spheres are shown. ( D ) Quantification by FACS at day 11 of the percentage of GFP + cells in dissociated spheres treated with IL-6 (0, 1, and 10 ng/mL). ( E ) Quantification at different times of GFP + cells in spheres cultured with or without IL-6 (1 ng/mL). ( F ) Representative sections of spheres at day 14 treated with IL-6 ( Left , 10 ng/mL) or S3I-201 ( Right , 200 μM, days 4–7). Both sections were stained with antibodies to a-tub + (magenta) and Splunc + (green). * P

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

    Article Title: IL-6/STAT3 promotes regeneration of airway ciliated cells from basal stem cells

    doi: 10.1073/pnas.1409781111

    Figure Lengend Snippet: IL-6 enhances Foxj1-GFP expression in the mouse tracheosphere culture assay. ( A ) Schematic of the assay. NGFR + basal cells from Foxj1-GFP tracheas were cultured in 50% Matrigel in 96-well inserts. ( Right ) Section of a typical sphere with acetylated tubulin + (a-tub) ciliated (magenta) and Splunc + secretory cells (green). IHC, immunohistochemistry. The effect of IL-6 ( B ) and STAT3 inhibitor ( C ) on Foxj1 -GFP expression is shown. Differential interference contrast images ( Upper ) and fluorescent images ( Lower ) of the same spheres are shown. ( D ) Quantification by FACS at day 11 of the percentage of GFP + cells in dissociated spheres treated with IL-6 (0, 1, and 10 ng/mL). ( E ) Quantification at different times of GFP + cells in spheres cultured with or without IL-6 (1 ng/mL). ( F ) Representative sections of spheres at day 14 treated with IL-6 ( Left , 10 ng/mL) or S3I-201 ( Right , 200 μM, days 4–7). Both sections were stained with antibodies to a-tub + (magenta) and Splunc + (green). * P

    Article Snippet: Mouse ALI cultures at day 7 were exposed to mouse IL-6 (20 ng/mL; R & D Systems) at 37 °C for 4 h. Approximately 4 × 106 cells were fixed at room temperature for 10 min and scraped off the inserts.

    Techniques: Expressing, Cell Culture, Immunohistochemistry, FACS, Staining

    IL-6 enhances expression of cilia-related genes and inhibits Notch1 expression in mouse ALI culture. ( A ) Schematic of ALI culture of mouse tracheal epithelial cells. At day 7, IL-6 (10 ng/mL) was added to culture medium in the lower chamber. Cells were harvested after 6, 12, and 24 h, and total RNA was extracted. ( B ) Quantitative RT-PCR shows that IL-6 treatment promotes the expression of the known target gene Socs3 and ciliogenesis-related genes, such as Multicilin ( Mcidas) and Foxj1 . IL-6 treatment also inhibits Notch1 and promotes expression of Cdc20b , the host gene for miR-449a/b. No significant changes were observed in the expression of Notch2 , Dll1 , or Jagged1 . ( C ) ChIP assay shows that p-STAT3 binding to promoter regions of Socs3, Foxj1 , Mcidas , and Notch1 is increased after IL-6 stimulation. * P

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

    Article Title: IL-6/STAT3 promotes regeneration of airway ciliated cells from basal stem cells

    doi: 10.1073/pnas.1409781111

    Figure Lengend Snippet: IL-6 enhances expression of cilia-related genes and inhibits Notch1 expression in mouse ALI culture. ( A ) Schematic of ALI culture of mouse tracheal epithelial cells. At day 7, IL-6 (10 ng/mL) was added to culture medium in the lower chamber. Cells were harvested after 6, 12, and 24 h, and total RNA was extracted. ( B ) Quantitative RT-PCR shows that IL-6 treatment promotes the expression of the known target gene Socs3 and ciliogenesis-related genes, such as Multicilin ( Mcidas) and Foxj1 . IL-6 treatment also inhibits Notch1 and promotes expression of Cdc20b , the host gene for miR-449a/b. No significant changes were observed in the expression of Notch2 , Dll1 , or Jagged1 . ( C ) ChIP assay shows that p-STAT3 binding to promoter regions of Socs3, Foxj1 , Mcidas , and Notch1 is increased after IL-6 stimulation. * P

    Article Snippet: Mouse ALI cultures at day 7 were exposed to mouse IL-6 (20 ng/mL; R & D Systems) at 37 °C for 4 h. Approximately 4 × 106 cells were fixed at room temperature for 10 min and scraped off the inserts.

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

    Model for regulation of ciliogenesis in airway epithelium by STAT3. ( Upper ) After injury, STAT3 in both basal cells and progenitors is activated by IL-6 secreted from PDGFRα + stromal cells. Ciliogenesis is likely promoted both at the level of cell fate determination and at the level of differentiation/maturation of the progenitors of multiciliated cells. ( Lower ) Schematic model for how STAT3 may directly regulate ciliogenesis-related genes during repair of the tracheal epithelium.

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

    Article Title: IL-6/STAT3 promotes regeneration of airway ciliated cells from basal stem cells

    doi: 10.1073/pnas.1409781111

    Figure Lengend Snippet: Model for regulation of ciliogenesis in airway epithelium by STAT3. ( Upper ) After injury, STAT3 in both basal cells and progenitors is activated by IL-6 secreted from PDGFRα + stromal cells. Ciliogenesis is likely promoted both at the level of cell fate determination and at the level of differentiation/maturation of the progenitors of multiciliated cells. ( Lower ) Schematic model for how STAT3 may directly regulate ciliogenesis-related genes during repair of the tracheal epithelium.

    Article Snippet: Mouse ALI cultures at day 7 were exposed to mouse IL-6 (20 ng/mL; R & D Systems) at 37 °C for 4 h. Approximately 4 × 106 cells were fixed at room temperature for 10 min and scraped off the inserts.

    Techniques:

    SDS–PAGE of 125 I-labelled antigen recognized by mAb C9.1. (a) A-LAK cells derived from C3H/He mice were iodinated with Na 125 I by the lactoperoxidase method. 125 I-labelled membrane proteins were incubated with mAb C9.1 (lanes 1 and 3) or mAb FD441.8 (anti-LFA-1) (lanes 2 and 4), followed by incubation with anti-rat IgG mAb-bound protein G–Sepharose 4B. The immune complexes were analysed under non-reducing (lanes 1 and 2) or reducing (lanes 3 and 4) conditions by SDS–PAGE on 7·5% gels and subjected to autoradiography. (b) A-LAK cells from (C57BL/6×C3H) F 1 mice were radioiodinated and immunoprecipitated with mAb C9.1(lanes 1 and 3) or anti-2B4 mAb (lanes 2 and 4) as in (a). Immune complexes were treated with (lanes 3 and 4) or without (lanes 1 and 2) endoglycosidase F, analysed under reducing conditions by SDS–PAGE on 10% gels and subjected to autoradiography.

    Journal: Immunology

    Article Title: Characterization of a surface membrane molecule expressed by natural killer cells in most inbred mouse strains: monoclonal antibody C9.1 identifies an allelic form of the 2B4 antigen

    doi: 10.1046/j.1365-2567.1999.00709.x

    Figure Lengend Snippet: SDS–PAGE of 125 I-labelled antigen recognized by mAb C9.1. (a) A-LAK cells derived from C3H/He mice were iodinated with Na 125 I by the lactoperoxidase method. 125 I-labelled membrane proteins were incubated with mAb C9.1 (lanes 1 and 3) or mAb FD441.8 (anti-LFA-1) (lanes 2 and 4), followed by incubation with anti-rat IgG mAb-bound protein G–Sepharose 4B. The immune complexes were analysed under non-reducing (lanes 1 and 2) or reducing (lanes 3 and 4) conditions by SDS–PAGE on 7·5% gels and subjected to autoradiography. (b) A-LAK cells from (C57BL/6×C3H) F 1 mice were radioiodinated and immunoprecipitated with mAb C9.1(lanes 1 and 3) or anti-2B4 mAb (lanes 2 and 4) as in (a). Immune complexes were treated with (lanes 3 and 4) or without (lanes 1 and 2) endoglycosidase F, analysed under reducing conditions by SDS–PAGE on 10% gels and subjected to autoradiography.

    Article Snippet: Biotinylated rabbit anti-mouse immunoglobulins and recombinant protein G–Sepharose 4B were purchased from Zymed Laboratories, Inc. (San Francisco, CA).

    Techniques: SDS Page, Derivative Assay, Mouse Assay, Incubation, Autoradiography, Immunoprecipitation

    A : KC were isolated from mice at 2 wk after a single subcutaneous injection of iron dextran (25 mg/kg) to assess LPS- or peroxynitrite-stimulated TNF-α expression. Note KC isolated from iron dextran-injected mice release 4–5 times more

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Hepatic macrophage iron aggravates experimental alcoholic steatohepatitis

    doi: 10.1152/ajpgi.90327.2008

    Figure Lengend Snippet: A : KC were isolated from mice at 2 wk after a single subcutaneous injection of iron dextran (25 mg/kg) to assess LPS- or peroxynitrite-stimulated TNF-α expression. Note KC isolated from iron dextran-injected mice release 4–5 times more

    Article Snippet: Release of TNF-α into the media by cultured PBMs or KC was determined by commercially available immunoassay kits for rodent and human TNF-α following the manufacturer's instruction (R & D Systems).

    Techniques: Isolation, Mouse Assay, Injection, Expressing

    A : normal rat cultured KC were pretreated with the different concentrations of iron dextran and stimulated with LPS to determine the effects of iron loading on TNF-α release. Note iron dextran treatment alone does not affect the basal TNF-α

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Hepatic macrophage iron aggravates experimental alcoholic steatohepatitis

    doi: 10.1152/ajpgi.90327.2008

    Figure Lengend Snippet: A : normal rat cultured KC were pretreated with the different concentrations of iron dextran and stimulated with LPS to determine the effects of iron loading on TNF-α release. Note iron dextran treatment alone does not affect the basal TNF-α

    Article Snippet: Release of TNF-α into the media by cultured PBMs or KC was determined by commercially available immunoassay kits for rodent and human TNF-α following the manufacturer's instruction (R & D Systems).

    Techniques: Cell Culture

    Depletion of INSR does not inhibit IGFBP-3 secretion in hTCEpi cells. Secreted IGFBP-3 in conditioned media was measured using an IGFBP-3 ELISA. (A) hTCEpi cells were treated with siRNA oligonucleotides targeting IGF-1R or INSR. In basal media, knockdown of IGF-1R blunted secretion of IGFBP-3 compared to the non-targeting control (***P

    Journal: Journal of cellular physiology

    Article Title: Mutual regulation between IGF-1R and IGFBP-3 in human corneal epithelial cells

    doi: 10.1002/jcp.26948

    Figure Lengend Snippet: Depletion of INSR does not inhibit IGFBP-3 secretion in hTCEpi cells. Secreted IGFBP-3 in conditioned media was measured using an IGFBP-3 ELISA. (A) hTCEpi cells were treated with siRNA oligonucleotides targeting IGF-1R or INSR. In basal media, knockdown of IGF-1R blunted secretion of IGFBP-3 compared to the non-targeting control (***P

    Article Snippet: Similar to expression in conditioned media, IGFBP-3 was increased in whole cell lysates from basal media compared to growth media.

    Techniques: Enzyme-linked Immunosorbent Assay

    IGFBP-3 regulates IGF-1R nuclear translocation. hTCEpi cells were cultured in basal media and transfected with siRNAs for IGFBP-3 with or without 100 ng/ml rhIGFBP-3. (A) Cells were lysed and fractionated into cytosolic, soluble and insoluble nuclear fractions. Immunoblotting for IGF-1R showed an increase in IGF-1R expression in basal media compared to growth media in all fractions. The increase in IGF-1R was higher in the nuclear fraction after IGFBP-3 knockdown and further increased in the insoluble fraction after addition of exogenous rhIGFBP-3. Immunoblotting for IGFBP-3 confirmed knockdown and uptake of rhIGFBP-3. Immunoblotting for GAPDH, SP1 and Histone H3 were used for cytosolic, soluble nucleus, and insoluble nucleus fractionation controls, respectively. Data representative of 3 independent experiments. Low and high exposures shown for IGF-1R immunoblot. (B) Immunofluorescence for IGF-1R (green) and IGFBP-3 (red). Nuclei were counterstained with DAPI (blue). There was an increase in IGF-1R expression and nuclear accumulation in cells cultured in basal media compared to growth media. IGF-1R remained in the nucleus following IGFBP-3 knockdown. There was a robust increase in nuclear IGF-1R in the nucleus after rescue with exogenous rhIGFBP-3. Scale bar: 10 μm. KGM: keratinocyte growth media; KBM: keratinocyte basal media; BP3: IGFBP-3; CTRL: control. Data representative of 3 independent experiments.

    Journal: Journal of cellular physiology

    Article Title: Mutual regulation between IGF-1R and IGFBP-3 in human corneal epithelial cells

    doi: 10.1002/jcp.26948

    Figure Lengend Snippet: IGFBP-3 regulates IGF-1R nuclear translocation. hTCEpi cells were cultured in basal media and transfected with siRNAs for IGFBP-3 with or without 100 ng/ml rhIGFBP-3. (A) Cells were lysed and fractionated into cytosolic, soluble and insoluble nuclear fractions. Immunoblotting for IGF-1R showed an increase in IGF-1R expression in basal media compared to growth media in all fractions. The increase in IGF-1R was higher in the nuclear fraction after IGFBP-3 knockdown and further increased in the insoluble fraction after addition of exogenous rhIGFBP-3. Immunoblotting for IGFBP-3 confirmed knockdown and uptake of rhIGFBP-3. Immunoblotting for GAPDH, SP1 and Histone H3 were used for cytosolic, soluble nucleus, and insoluble nucleus fractionation controls, respectively. Data representative of 3 independent experiments. Low and high exposures shown for IGF-1R immunoblot. (B) Immunofluorescence for IGF-1R (green) and IGFBP-3 (red). Nuclei were counterstained with DAPI (blue). There was an increase in IGF-1R expression and nuclear accumulation in cells cultured in basal media compared to growth media. IGF-1R remained in the nucleus following IGFBP-3 knockdown. There was a robust increase in nuclear IGF-1R in the nucleus after rescue with exogenous rhIGFBP-3. Scale bar: 10 μm. KGM: keratinocyte growth media; KBM: keratinocyte basal media; BP3: IGFBP-3; CTRL: control. Data representative of 3 independent experiments.

    Article Snippet: Similar to expression in conditioned media, IGFBP-3 was increased in whole cell lysates from basal media compared to growth media.

    Techniques: Translocation Assay, Cell Culture, Transfection, Expressing, Fractionation, Immunofluorescence

    Schematic summary of IGF-1R and IGFBP-3 in corneal epithelial cells in response to stress induced by growth factor withdrawal. INSR, IGF-1R, and Hybrid-R are increased when cultured in basal media devoid of growth factors. This effect is mediated by the action of insulin at the plasma membrane. The induction of cellular stress triggers an increase in secreted IGFBP-3 into the extracellular space. During the growth phase, IGFBP-3 mediates cell cycle arrest. During stress however, IGFBP-3 mediates translocation of IGF-1R to the insoluble nucleus and potentially increases DNA binding and transcriptional modulation. In contrast to other studies, IGF-1R is SUMOylated by SUMO2/3, which may function to drive the translocation of IGF-1R from the soluble to insoluble nuclear fraction. BP3: IGFBP-3.

    Journal: Journal of cellular physiology

    Article Title: Mutual regulation between IGF-1R and IGFBP-3 in human corneal epithelial cells

    doi: 10.1002/jcp.26948

    Figure Lengend Snippet: Schematic summary of IGF-1R and IGFBP-3 in corneal epithelial cells in response to stress induced by growth factor withdrawal. INSR, IGF-1R, and Hybrid-R are increased when cultured in basal media devoid of growth factors. This effect is mediated by the action of insulin at the plasma membrane. The induction of cellular stress triggers an increase in secreted IGFBP-3 into the extracellular space. During the growth phase, IGFBP-3 mediates cell cycle arrest. During stress however, IGFBP-3 mediates translocation of IGF-1R to the insoluble nucleus and potentially increases DNA binding and transcriptional modulation. In contrast to other studies, IGF-1R is SUMOylated by SUMO2/3, which may function to drive the translocation of IGF-1R from the soluble to insoluble nuclear fraction. BP3: IGFBP-3.

    Article Snippet: Similar to expression in conditioned media, IGFBP-3 was increased in whole cell lysates from basal media compared to growth media.

    Techniques: Cell Culture, Translocation Assay, Binding Assay

    IGF-1R, CAV-1, and CLTC regulate IGFBP-3 secretion in conditioned media. An IGFBP-3 ELISA was used to analyze the concentration of secreted IGFBP-3. (A) hTCEpi cells treated with siRNA oligonucleotides targeting IGF-1R significantly decreased IGFBP-3 secretion into culture media compared to the non-targeting control (**P

    Journal: Journal of cellular physiology

    Article Title: Mutual regulation between IGF-1R and IGFBP-3 in human corneal epithelial cells

    doi: 10.1002/jcp.26948

    Figure Lengend Snippet: IGF-1R, CAV-1, and CLTC regulate IGFBP-3 secretion in conditioned media. An IGFBP-3 ELISA was used to analyze the concentration of secreted IGFBP-3. (A) hTCEpi cells treated with siRNA oligonucleotides targeting IGF-1R significantly decreased IGFBP-3 secretion into culture media compared to the non-targeting control (**P

    Article Snippet: Similar to expression in conditioned media, IGFBP-3 was increased in whole cell lysates from basal media compared to growth media.

    Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

    SUMO-2/3 but not SUMO-1 triggers IGF-1R nuclear translocation and DNA binding in corneal epithelial cells. (A) Immunoblotting for SUMO-1 and SUMO-2/3 showed a decrease in SUMO-1 in basal media compared with growth. Instead in basal media, there was an observable increase in SUMO-2/3. Data representative of 3 independent experiments. (B) Proximal Ligation Assay (PLA) shows an increase in IGF-1R SUMOylation by SUMO-2/3 (green) in the cytoplasm and nucleus in basal media compared with growth media. Scale bar: 23 μm. Data representative of 3 independent experiments. (C) The global SUMOylation assay confirmed a significant decrease in SUMOylated IGF-1R in nuclear extracts after knock down of IGFBP-3 in basal media (siRNA control versus siRNA IGFBP3 *P

    Journal: Journal of cellular physiology

    Article Title: Mutual regulation between IGF-1R and IGFBP-3 in human corneal epithelial cells

    doi: 10.1002/jcp.26948

    Figure Lengend Snippet: SUMO-2/3 but not SUMO-1 triggers IGF-1R nuclear translocation and DNA binding in corneal epithelial cells. (A) Immunoblotting for SUMO-1 and SUMO-2/3 showed a decrease in SUMO-1 in basal media compared with growth. Instead in basal media, there was an observable increase in SUMO-2/3. Data representative of 3 independent experiments. (B) Proximal Ligation Assay (PLA) shows an increase in IGF-1R SUMOylation by SUMO-2/3 (green) in the cytoplasm and nucleus in basal media compared with growth media. Scale bar: 23 μm. Data representative of 3 independent experiments. (C) The global SUMOylation assay confirmed a significant decrease in SUMOylated IGF-1R in nuclear extracts after knock down of IGFBP-3 in basal media (siRNA control versus siRNA IGFBP3 *P

    Article Snippet: Similar to expression in conditioned media, IGFBP-3 was increased in whole cell lysates from basal media compared to growth media.

    Techniques: Translocation Assay, Binding Assay, Ligation, Proximity Ligation Assay

    Knockdown of IGF-1R attenuates IGFBP-3 secretion in HCECs. Secreted IGFBP-3 in conditioned media was measured using an IGFBP-3 ELISA. (A) HCECs were treated with siRNA oligonucleotides targeting IGF-1R. In basal media, IGF-1R knockdown resulted in a large decrease in secreted IGFBP-3 compared to the non-targeting control (***P

    Journal: Journal of cellular physiology

    Article Title: Mutual regulation between IGF-1R and IGFBP-3 in human corneal epithelial cells

    doi: 10.1002/jcp.26948

    Figure Lengend Snippet: Knockdown of IGF-1R attenuates IGFBP-3 secretion in HCECs. Secreted IGFBP-3 in conditioned media was measured using an IGFBP-3 ELISA. (A) HCECs were treated with siRNA oligonucleotides targeting IGF-1R. In basal media, IGF-1R knockdown resulted in a large decrease in secreted IGFBP-3 compared to the non-targeting control (***P

    Article Snippet: Similar to expression in conditioned media, IGFBP-3 was increased in whole cell lysates from basal media compared to growth media.

    Techniques: Enzyme-linked Immunosorbent Assay

    IGFBP-3 induces cell cycle arrest but does not alter cellular respiration. (A, C and E) Representative graphs of cell count as a function of PI intensity in (A) basal media; (C) growth media; (E) growth media supplemented with 100 ng/ml rhIGFBP-3. (B) hTCEpi cells cultured in basal media showed an increase in the proportion of cells in G 0 /G 1 compared to growth conditions (***P

    Journal: Journal of cellular physiology

    Article Title: Mutual regulation between IGF-1R and IGFBP-3 in human corneal epithelial cells

    doi: 10.1002/jcp.26948

    Figure Lengend Snippet: IGFBP-3 induces cell cycle arrest but does not alter cellular respiration. (A, C and E) Representative graphs of cell count as a function of PI intensity in (A) basal media; (C) growth media; (E) growth media supplemented with 100 ng/ml rhIGFBP-3. (B) hTCEpi cells cultured in basal media showed an increase in the proportion of cells in G 0 /G 1 compared to growth conditions (***P

    Article Snippet: Similar to expression in conditioned media, IGFBP-3 was increased in whole cell lysates from basal media compared to growth media.

    Techniques: Cell Counting, Cell Culture

    IGFBP-3 regulates nuclear accumulation of IGF-1R. HCECs were treated with siRNA oligonucleotides targeting IGFBP-3 in basal media. (A, B) An IGFBP-3 ELISA was used to confirm knockdown of IGFBP-3 in whole cell lysates (A, ***P

    Journal: Journal of cellular physiology

    Article Title: Mutual regulation between IGF-1R and IGFBP-3 in human corneal epithelial cells

    doi: 10.1002/jcp.26948

    Figure Lengend Snippet: IGFBP-3 regulates nuclear accumulation of IGF-1R. HCECs were treated with siRNA oligonucleotides targeting IGFBP-3 in basal media. (A, B) An IGFBP-3 ELISA was used to confirm knockdown of IGFBP-3 in whole cell lysates (A, ***P

    Article Snippet: Similar to expression in conditioned media, IGFBP-3 was increased in whole cell lysates from basal media compared to growth media.

    Techniques: Enzyme-linked Immunosorbent Assay

    Knockdown of IGFBP-3 increases expression of IGF-1R in whole cell lysates. hTCEpi cells were treated with siRNA oligonucleotides targeting IGFBP-3 in basal media. (A, B) An IGFBP-3 ELISA was used to confirm knockdown of IGFBP-3 in whole cell lysates (A, ***P

    Journal: Journal of cellular physiology

    Article Title: Mutual regulation between IGF-1R and IGFBP-3 in human corneal epithelial cells

    doi: 10.1002/jcp.26948

    Figure Lengend Snippet: Knockdown of IGFBP-3 increases expression of IGF-1R in whole cell lysates. hTCEpi cells were treated with siRNA oligonucleotides targeting IGFBP-3 in basal media. (A, B) An IGFBP-3 ELISA was used to confirm knockdown of IGFBP-3 in whole cell lysates (A, ***P

    Article Snippet: Similar to expression in conditioned media, IGFBP-3 was increased in whole cell lysates from basal media compared to growth media.

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay