luciferase reporter  (InvivoGen)

 
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    Name:
    pNiFty Luc
    Description:
    pNiFty NF κB 5x ELAM AmpR Luc
    Catalog Number:
    pnifty-luc
    Price:
    None
    Size:
    20 µg
    Category:
    pNiFty Luc pNiFty NF κB inducible plasmids TLR Detection Toll Like Receptors TLRs Innate Immunity Sensors Research Fields
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    Structured Review

    InvivoGen luciferase reporter
    pNiFty NF κB 5x ELAM AmpR Luc
    https://www.bioz.com/result/luciferase reporter/product/InvivoGen
    Average 96 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    luciferase reporter - by Bioz Stars, 2020-09
    96/100 stars

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    Related Articles

    Transfection:

    Article Title: Synthesis of a hemin-containing copolymer as a novel immunostimulator that induces IFN-gamma production
    Article Snippet: .. The pNifty-Luc plasmid, which contains the firefly luciferase gene under the nuclear factor (NF)-κB-inducible element; the transfection reagent LyoVec; the alkaline phosphatase substrate QUANTI-Blue; and lipopolysaccharide (LPS) were purchased from InvivoGen (San Diego, CA, USA). .. The pGL4.74 [ hRluc/TK ] plasmid, which encodes the renilla luciferase gene, as well as a dual-luciferase reporter assay system, were purchased from Promega (Madison, WI, USA).

    Article Title: Screen of whole blood responses to flagellin identifies TLR5 variation associated with outcome in melioidosis
    Article Snippet: .. Transfection was performed with 5 × 104 cells/well HEK 293 cells in a 96-well plate with 100 μL transfection reagent containing 0.5 μL Polyfect reagent (Qiagen, Hilden, Germany), 10 ng pNiFty-Luc firefly luciferase reporter (Invivogen, CA), 1 ng pRL-TK Renilla luciferase control reporter (Promega, WI) and 40 ng of plasmid (TLR5 1846T wild type or TLR5 1846C variant or pEF6 control). .. Before transfection, the plasmids were prepared in DMEM without FBS and mixed with Polyfect reagent by vortexing for 10 s, incubated at room temperature for 10 min and the volume adjusted to 100 μL with complete DMEM medium.

    Article Title: Structure Dependent-Immunomodulation by Sugar Beet Arabinans via a SYK Tyrosine Kinase-Dependent Signaling Pathway
    Article Snippet: .. Briefly, HEK293 cells were transfected with one of the mentioned human TLR(s) and pNiFty-luc, a NF-κB luciferase reporter construct (Invivogen, Toulouse, France, catalog numbers 293-htlr2; 293-htlr4a; 293-htlr5; 293-htlr2/6; 293-mtlr1/2; pnifty-luc). .. Additionally, HEK293 cells were transformed with only the pNiFty-luc ( ).

    Article Title: Sensing of bacterial cyclic dinucleotides by the oxidoreductase RECON promotes NF-κB activation and shapes a proinflammatory antibacterial state
    Article Snippet: .. For NF-κB luciferase assays, TIB73 hepatocytes were co-transfected with pNiFty-Luc plasmid (InvivoGen) and eGFP plasmid (internal control) using TransIT-LT1 transfection reagent (Mirus Bio). ..

    Article Title: An Oncolytic Adenovirus Enhanced for Toll-like Receptor 9 Stimulation Increases Antitumor Immune Responses and Tumor Clearance
    Article Snippet: .. Monolayers in triplicates were transfected using SuperFect reagent (Qiagen, Valencia, CA) with 1 µg of reporter plasmid pNiFty-Luc (InvivoGen) which carries the luciferase reporter gene driven by an NFκB-inducible ELAM-1 composite promoter able to respond to TLR9 stimulation. .. Green fluorescent protein-expressing plasmid was used as control of transfection.

    Article Title: Structure and Function of LGP2, a DEX(D/H) Helicase That Regulates the Innate Immunity Response *(D/H) Helicase That Regulates the Innate Immunity Response * S⃞
    Article Snippet: .. Cells at ∼80% confluency were used for transfection with Lipofectamine 2000 (Invitrogen) mixed with plasmids pNiFty-Luc (30 ng, Invivogen) and phRL-TK (5 ng, Promega), which code for the firefly luciferase reporter under a promoter that contains NF-κB elements and the Renilla luciferase driven by the herpesvirus thymidine kinase promoter, respectively. .. Where appropriate, plasmids encoding Rig-I (pUNO-hRIG-I; Invivogen) and/or Lgp2 (pUNO-hLGP2; Invivogen) were added to the transfection mixture.

    Article Title: FGF23 expression is stimulated in transgenic α-Klotho longevity mouse model
    Article Snippet: .. For TNF-α–mediated activation of the NF-κB signaling pathway, HEK293T cells were transiently transfected with either empty expression vector or various Klotho expression constructs along with the NF-κB luciferase reporter plasmid (pNiFty-Luc, InvivoGen) and Renilla luciferase–null as an internal control plasmid. .. For the effects of ADAM17-mediated (TACE-mediated) ectodomain cleavage of full-length membrane α-Klotho on FGF23 signaling, HEK293T cells were transiently transfected with either empty expression vector or TACE expression construct along with membrane α-Klotho, the ERK luciferase reporter, and Renilla luciferase–null as an internal control plasmid.

    Luciferase:

    Article Title: Synthesis of a hemin-containing copolymer as a novel immunostimulator that induces IFN-gamma production
    Article Snippet: .. The pNifty-Luc plasmid, which contains the firefly luciferase gene under the nuclear factor (NF)-κB-inducible element; the transfection reagent LyoVec; the alkaline phosphatase substrate QUANTI-Blue; and lipopolysaccharide (LPS) were purchased from InvivoGen (San Diego, CA, USA). .. The pGL4.74 [ hRluc/TK ] plasmid, which encodes the renilla luciferase gene, as well as a dual-luciferase reporter assay system, were purchased from Promega (Madison, WI, USA).

    Article Title: Screen of whole blood responses to flagellin identifies TLR5 variation associated with outcome in melioidosis
    Article Snippet: .. Transfection was performed with 5 × 104 cells/well HEK 293 cells in a 96-well plate with 100 μL transfection reagent containing 0.5 μL Polyfect reagent (Qiagen, Hilden, Germany), 10 ng pNiFty-Luc firefly luciferase reporter (Invivogen, CA), 1 ng pRL-TK Renilla luciferase control reporter (Promega, WI) and 40 ng of plasmid (TLR5 1846T wild type or TLR5 1846C variant or pEF6 control). .. Before transfection, the plasmids were prepared in DMEM without FBS and mixed with Polyfect reagent by vortexing for 10 s, incubated at room temperature for 10 min and the volume adjusted to 100 μL with complete DMEM medium.

    Article Title: Structure Dependent-Immunomodulation by Sugar Beet Arabinans via a SYK Tyrosine Kinase-Dependent Signaling Pathway
    Article Snippet: .. Briefly, HEK293 cells were transfected with one of the mentioned human TLR(s) and pNiFty-luc, a NF-κB luciferase reporter construct (Invivogen, Toulouse, France, catalog numbers 293-htlr2; 293-htlr4a; 293-htlr5; 293-htlr2/6; 293-mtlr1/2; pnifty-luc). .. Additionally, HEK293 cells were transformed with only the pNiFty-luc ( ).

    Article Title: Sensing of bacterial cyclic dinucleotides by the oxidoreductase RECON promotes NF-κB activation and shapes a proinflammatory antibacterial state
    Article Snippet: .. For NF-κB luciferase assays, TIB73 hepatocytes were co-transfected with pNiFty-Luc plasmid (InvivoGen) and eGFP plasmid (internal control) using TransIT-LT1 transfection reagent (Mirus Bio). ..

    Article Title: An Oncolytic Adenovirus Enhanced for Toll-like Receptor 9 Stimulation Increases Antitumor Immune Responses and Tumor Clearance
    Article Snippet: .. Monolayers in triplicates were transfected using SuperFect reagent (Qiagen, Valencia, CA) with 1 µg of reporter plasmid pNiFty-Luc (InvivoGen) which carries the luciferase reporter gene driven by an NFκB-inducible ELAM-1 composite promoter able to respond to TLR9 stimulation. .. Green fluorescent protein-expressing plasmid was used as control of transfection.

    Article Title: Structure and Function of LGP2, a DEX(D/H) Helicase That Regulates the Innate Immunity Response *(D/H) Helicase That Regulates the Innate Immunity Response * S⃞
    Article Snippet: .. Cells at ∼80% confluency were used for transfection with Lipofectamine 2000 (Invitrogen) mixed with plasmids pNiFty-Luc (30 ng, Invivogen) and phRL-TK (5 ng, Promega), which code for the firefly luciferase reporter under a promoter that contains NF-κB elements and the Renilla luciferase driven by the herpesvirus thymidine kinase promoter, respectively. .. Where appropriate, plasmids encoding Rig-I (pUNO-hRIG-I; Invivogen) and/or Lgp2 (pUNO-hLGP2; Invivogen) were added to the transfection mixture.

    Article Title: FGF23 expression is stimulated in transgenic α-Klotho longevity mouse model
    Article Snippet: .. For TNF-α–mediated activation of the NF-κB signaling pathway, HEK293T cells were transiently transfected with either empty expression vector or various Klotho expression constructs along with the NF-κB luciferase reporter plasmid (pNiFty-Luc, InvivoGen) and Renilla luciferase–null as an internal control plasmid. .. For the effects of ADAM17-mediated (TACE-mediated) ectodomain cleavage of full-length membrane α-Klotho on FGF23 signaling, HEK293T cells were transiently transfected with either empty expression vector or TACE expression construct along with membrane α-Klotho, the ERK luciferase reporter, and Renilla luciferase–null as an internal control plasmid.

    Construct:

    Article Title: Structure Dependent-Immunomodulation by Sugar Beet Arabinans via a SYK Tyrosine Kinase-Dependent Signaling Pathway
    Article Snippet: .. Briefly, HEK293 cells were transfected with one of the mentioned human TLR(s) and pNiFty-luc, a NF-κB luciferase reporter construct (Invivogen, Toulouse, France, catalog numbers 293-htlr2; 293-htlr4a; 293-htlr5; 293-htlr2/6; 293-mtlr1/2; pnifty-luc). .. Additionally, HEK293 cells were transformed with only the pNiFty-luc ( ).

    Article Title: FGF23 expression is stimulated in transgenic α-Klotho longevity mouse model
    Article Snippet: .. For TNF-α–mediated activation of the NF-κB signaling pathway, HEK293T cells were transiently transfected with either empty expression vector or various Klotho expression constructs along with the NF-κB luciferase reporter plasmid (pNiFty-Luc, InvivoGen) and Renilla luciferase–null as an internal control plasmid. .. For the effects of ADAM17-mediated (TACE-mediated) ectodomain cleavage of full-length membrane α-Klotho on FGF23 signaling, HEK293T cells were transiently transfected with either empty expression vector or TACE expression construct along with membrane α-Klotho, the ERK luciferase reporter, and Renilla luciferase–null as an internal control plasmid.

    Activation Assay:

    Article Title: FGF23 expression is stimulated in transgenic α-Klotho longevity mouse model
    Article Snippet: .. For TNF-α–mediated activation of the NF-κB signaling pathway, HEK293T cells were transiently transfected with either empty expression vector or various Klotho expression constructs along with the NF-κB luciferase reporter plasmid (pNiFty-Luc, InvivoGen) and Renilla luciferase–null as an internal control plasmid. .. For the effects of ADAM17-mediated (TACE-mediated) ectodomain cleavage of full-length membrane α-Klotho on FGF23 signaling, HEK293T cells were transiently transfected with either empty expression vector or TACE expression construct along with membrane α-Klotho, the ERK luciferase reporter, and Renilla luciferase–null as an internal control plasmid.

    Expressing:

    Article Title: FGF23 expression is stimulated in transgenic α-Klotho longevity mouse model
    Article Snippet: .. For TNF-α–mediated activation of the NF-κB signaling pathway, HEK293T cells were transiently transfected with either empty expression vector or various Klotho expression constructs along with the NF-κB luciferase reporter plasmid (pNiFty-Luc, InvivoGen) and Renilla luciferase–null as an internal control plasmid. .. For the effects of ADAM17-mediated (TACE-mediated) ectodomain cleavage of full-length membrane α-Klotho on FGF23 signaling, HEK293T cells were transiently transfected with either empty expression vector or TACE expression construct along with membrane α-Klotho, the ERK luciferase reporter, and Renilla luciferase–null as an internal control plasmid.

    Variant Assay:

    Article Title: Screen of whole blood responses to flagellin identifies TLR5 variation associated with outcome in melioidosis
    Article Snippet: .. Transfection was performed with 5 × 104 cells/well HEK 293 cells in a 96-well plate with 100 μL transfection reagent containing 0.5 μL Polyfect reagent (Qiagen, Hilden, Germany), 10 ng pNiFty-Luc firefly luciferase reporter (Invivogen, CA), 1 ng pRL-TK Renilla luciferase control reporter (Promega, WI) and 40 ng of plasmid (TLR5 1846T wild type or TLR5 1846C variant or pEF6 control). .. Before transfection, the plasmids were prepared in DMEM without FBS and mixed with Polyfect reagent by vortexing for 10 s, incubated at room temperature for 10 min and the volume adjusted to 100 μL with complete DMEM medium.

    Plasmid Preparation:

    Article Title: Synthesis of a hemin-containing copolymer as a novel immunostimulator that induces IFN-gamma production
    Article Snippet: .. The pNifty-Luc plasmid, which contains the firefly luciferase gene under the nuclear factor (NF)-κB-inducible element; the transfection reagent LyoVec; the alkaline phosphatase substrate QUANTI-Blue; and lipopolysaccharide (LPS) were purchased from InvivoGen (San Diego, CA, USA). .. The pGL4.74 [ hRluc/TK ] plasmid, which encodes the renilla luciferase gene, as well as a dual-luciferase reporter assay system, were purchased from Promega (Madison, WI, USA).

    Article Title: Screen of whole blood responses to flagellin identifies TLR5 variation associated with outcome in melioidosis
    Article Snippet: .. Transfection was performed with 5 × 104 cells/well HEK 293 cells in a 96-well plate with 100 μL transfection reagent containing 0.5 μL Polyfect reagent (Qiagen, Hilden, Germany), 10 ng pNiFty-Luc firefly luciferase reporter (Invivogen, CA), 1 ng pRL-TK Renilla luciferase control reporter (Promega, WI) and 40 ng of plasmid (TLR5 1846T wild type or TLR5 1846C variant or pEF6 control). .. Before transfection, the plasmids were prepared in DMEM without FBS and mixed with Polyfect reagent by vortexing for 10 s, incubated at room temperature for 10 min and the volume adjusted to 100 μL with complete DMEM medium.

    Article Title: Sensing of bacterial cyclic dinucleotides by the oxidoreductase RECON promotes NF-κB activation and shapes a proinflammatory antibacterial state
    Article Snippet: .. For NF-κB luciferase assays, TIB73 hepatocytes were co-transfected with pNiFty-Luc plasmid (InvivoGen) and eGFP plasmid (internal control) using TransIT-LT1 transfection reagent (Mirus Bio). ..

    Article Title: An Oncolytic Adenovirus Enhanced for Toll-like Receptor 9 Stimulation Increases Antitumor Immune Responses and Tumor Clearance
    Article Snippet: .. Monolayers in triplicates were transfected using SuperFect reagent (Qiagen, Valencia, CA) with 1 µg of reporter plasmid pNiFty-Luc (InvivoGen) which carries the luciferase reporter gene driven by an NFκB-inducible ELAM-1 composite promoter able to respond to TLR9 stimulation. .. Green fluorescent protein-expressing plasmid was used as control of transfection.

    Article Title: FGF23 expression is stimulated in transgenic α-Klotho longevity mouse model
    Article Snippet: .. For TNF-α–mediated activation of the NF-κB signaling pathway, HEK293T cells were transiently transfected with either empty expression vector or various Klotho expression constructs along with the NF-κB luciferase reporter plasmid (pNiFty-Luc, InvivoGen) and Renilla luciferase–null as an internal control plasmid. .. For the effects of ADAM17-mediated (TACE-mediated) ectodomain cleavage of full-length membrane α-Klotho on FGF23 signaling, HEK293T cells were transiently transfected with either empty expression vector or TACE expression construct along with membrane α-Klotho, the ERK luciferase reporter, and Renilla luciferase–null as an internal control plasmid.

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  • 86
    InvivoGen hek htlr luc clones
    (A) (Top) Schematic representation of fractionation of yeast strain S. cerevisiae JC7. Cell lysis followed by differential centrifugation generated SN, a suspension of yeast microsomes. Phenol extraction of SN removed lipids and proteins and resulted in a nucleic acid solution (NA). (Bottom) Next, NA was fractionated by electrophoresis in a native 1% agarose gel. The first and second bands were isolated from the gel and digested in the presence of high-dose RNase A (50 ng/μl). NAB1 was RNase A resistant, while NAB2 and the lower-molecular-mass molecules appearing as smears were digested. Arrow, NAB2; asterisk, lower-molecular-mass molecules. (B) NA, NAB1, and NAB2 were treated with RNase A or not and tested on the <t>HEK-hTLR3-luc</t> cell lines. NAB2, like NA, increased the fold induction of luciferase activity. NAB1 had no effect. Treatment with RNase A abolished TLR3 activation by NA and NAB2. The means ± SEMs of two independent experiments are shown.
    Hek Htlr Luc Clones, supplied by InvivoGen, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hek htlr luc clones/product/InvivoGen
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hek htlr luc clones - by Bioz Stars, 2020-09
    86/100 stars
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    92
    InvivoGen luc
    Cath-D influences TRPS1 transcription repression function A. Transcriptome analysis of TRPS1-silenced and Cath-D-silenced cells. <t>T47D</t> cells were transfected in triplicate with control <t>Luc</t> siRNA (20 μg), TRPS1 or Cath-D siRNA1–3 (20 μg), or with both TRPS1 and Cath-D siRNA2–3 (5 μg of each, total 20 μg). RNAs were extracted 48 h post-transfection and analyzed by microarray hybridization. Panel a: TRPS1-silenced cells versus control. Panel b: Cath-D-silenced cells versus control. Panel c: TRPS1/Cath-D doubly-silenced cells versus control. Panel d: TRPS1/Cath-D doubly-silenced cells versus TRPS1-silenced cells. The significant differences in the mRNA expression of a given gene were determined by dividing the log2 of TRPS1, Cath-D or TRPS1/Cath-D silenced sample signals by the Luc control signal, i.e . log(FC); these data were analyzed using a modified Student’s t -test followed by correction for multiple testing. Over- and under-expressed genes, relative to control, were identified using a threshold for the log(FC) (X-axis) (between 1 or −1), corresponding to fold-changes of 2 and −2, and a threshold for the adjusted p value of 0.05 (Y-axis). B. Cumulative frequency distribution of the difference (fold-change) between TRPS1/Cath-D co-silencing and TRPS1 silencing alone. T47D cells were transfected with Luc siRNA (20 μg), or TRPS1 + Cath-D siRNA2–3 (5 μg of each, total 20 μg) in triplicate transfections. Microarray analysis showed the impact of double silencing on the 53 mRNAs that were significantly up-regulated following silencing of TRPS1 alone ( Table S1 ). Ratio (x-axis), ratio of fold-changes for the 53 mRNAs after TRPS1 and Cath-D co-silencing divided by the fold-changes after TRPS1 silencing.
    Luc, supplied by InvivoGen, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/luc/product/InvivoGen
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    luc - by Bioz Stars, 2020-09
    92/100 stars
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    88
    InvivoGen plasmid p4hre luc odd
    Enhancement of the HIF-1α activity in the tumors of living mice induced by tempol. The stable expression clone, <t>MCF7/HRE-Luc-ODD</t> or MCF7/SV40-Luc, was implanted into the legs of the mice. The tumor-bearing mice were injected intraperitoneally with 100 mM tempol and fasted for 6 h, after which the in vivo bioluminescence emitted from the tumors was measured 3 min after the injection of D-luciferin. The error bars represent the standard deviation (n=3). * P
    Plasmid P4hre Luc Odd, supplied by InvivoGen, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid p4hre luc odd/product/InvivoGen
    Average 88 stars, based on 1 article reviews
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    84
    InvivoGen mda mb 231 d3h2ln luc
    EVs from NFAT3-expressing cells inhibit cell growth and increase apoptosis in cancer cells in cooperation with macrophages. ( A ) <t>MDA-MB-231</t> cells (left panel) and SUM-159-PT cells (right panel) were plated in medium with 3% Matrigel in 96-well ultra low attachment plates for three days to allow the formation of the spheroids formed. Medium was then added to each condition containing the apoptosis indicator (fluorescent caspase 3/7 substrate) supplemented with medium and containing or not the different EVs (3 × 10 8 pp/mL) as indicated. Size of the spheroids and appearance of green fluorescence (apoptosis) was recorded every 2 h on an INCUCYTE device for 4 days. Data are represented as the AUC of the spheroid size (upper panels) and apoptosis (lower panels) over 96 h. Data from one representative experiment of three independent experiments is shown, all data are shown as mean ± SEM (n = 3 technical replicates). (B) Frozen tumor tissues sections of mice xenografted with MDA-MB-231 cells <t>(D3H2LN-LUC)</t> and treated with PBS (−) or EVs T-47D shCtl were co-labelled with Dapi and specific antibodies to mouse macrophages (anti-F4/80 antibody) and to detect the tumor cells with an anti-Pan human cytokeratin (arrows indicate infiltrating mouse macrophages). (C) Hetero-spheroids containing 33% of the murine macrophage cell line RAW 264.7 with either MDA-MB-231 or SUM-159PT were plated in medium with 3% Matrigel in 96 wells ultra low attachment plates for three days to allow the formation of the hetero-spheroids formed. Then medium was added to each condition containing the apoptosis indicator (fluorescent caspase 3/7 substrate) supplemented with medium containing or not the different EVs (3 × 10 8 pp/mL) as indicated in the figure. The size of the spheroids and appearance of green fluorescence (apoptosis) were recorded every 2 h on an INCUCYTE apparatus for 4 days. Data are represented as the AUC of the spheroid size (upper panels) and apoptosis (lower panels) over 96 h. Data from one representative experiment of three independent experiments is shown, all data are shown as mean ± SEM (n = 3 technical replicates; ***p
    Mda Mb 231 D3h2ln Luc, supplied by InvivoGen, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mda mb 231 d3h2ln luc/product/InvivoGen
    Average 84 stars, based on 1 article reviews
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    mda mb 231 d3h2ln luc - by Bioz Stars, 2020-09
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    Image Search Results


    (A) (Top) Schematic representation of fractionation of yeast strain S. cerevisiae JC7. Cell lysis followed by differential centrifugation generated SN, a suspension of yeast microsomes. Phenol extraction of SN removed lipids and proteins and resulted in a nucleic acid solution (NA). (Bottom) Next, NA was fractionated by electrophoresis in a native 1% agarose gel. The first and second bands were isolated from the gel and digested in the presence of high-dose RNase A (50 ng/μl). NAB1 was RNase A resistant, while NAB2 and the lower-molecular-mass molecules appearing as smears were digested. Arrow, NAB2; asterisk, lower-molecular-mass molecules. (B) NA, NAB1, and NAB2 were treated with RNase A or not and tested on the HEK-hTLR3-luc cell lines. NAB2, like NA, increased the fold induction of luciferase activity. NAB1 had no effect. Treatment with RNase A abolished TLR3 activation by NA and NAB2. The means ± SEMs of two independent experiments are shown.

    Journal: Journal of Virology

    Article Title: Yeast Virus-Derived Stimulator of the Innate Immune System Augments the Efficacy of Virus Vector-Based Immunotherapy

    doi: 10.1128/JVI.03819-13

    Figure Lengend Snippet: (A) (Top) Schematic representation of fractionation of yeast strain S. cerevisiae JC7. Cell lysis followed by differential centrifugation generated SN, a suspension of yeast microsomes. Phenol extraction of SN removed lipids and proteins and resulted in a nucleic acid solution (NA). (Bottom) Next, NA was fractionated by electrophoresis in a native 1% agarose gel. The first and second bands were isolated from the gel and digested in the presence of high-dose RNase A (50 ng/μl). NAB1 was RNase A resistant, while NAB2 and the lower-molecular-mass molecules appearing as smears were digested. Arrow, NAB2; asterisk, lower-molecular-mass molecules. (B) NA, NAB1, and NAB2 were treated with RNase A or not and tested on the HEK-hTLR3-luc cell lines. NAB2, like NA, increased the fold induction of luciferase activity. NAB1 had no effect. Treatment with RNase A abolished TLR3 activation by NA and NAB2. The means ± SEMs of two independent experiments are shown.

    Article Snippet: HEK-hTLR-luc clones were seeded in 96-well plates and on the next day were stimulated with the test compounds and their respective specific ligands (all ligands were purchased from InvivoGen): synthetic diacetylated lipoprotein FSL-1 (Pam2CGDPKHPKSF; hTLR2 and hTLR2/6), LPS (hTLR4), poly(I·C) (hTLR3), flagellin (hTLR5), R848 (hTLR7, hTLR8), or ODN2006 (hTLR9) (see ).

    Techniques: Fractionation, Lysis, Centrifugation, Generated, Electrophoresis, Agarose Gel Electrophoresis, Isolation, Luciferase, Activity Assay, Activation Assay

    Cath-D influences TRPS1 transcription repression function A. Transcriptome analysis of TRPS1-silenced and Cath-D-silenced cells. T47D cells were transfected in triplicate with control Luc siRNA (20 μg), TRPS1 or Cath-D siRNA1–3 (20 μg), or with both TRPS1 and Cath-D siRNA2–3 (5 μg of each, total 20 μg). RNAs were extracted 48 h post-transfection and analyzed by microarray hybridization. Panel a: TRPS1-silenced cells versus control. Panel b: Cath-D-silenced cells versus control. Panel c: TRPS1/Cath-D doubly-silenced cells versus control. Panel d: TRPS1/Cath-D doubly-silenced cells versus TRPS1-silenced cells. The significant differences in the mRNA expression of a given gene were determined by dividing the log2 of TRPS1, Cath-D or TRPS1/Cath-D silenced sample signals by the Luc control signal, i.e . log(FC); these data were analyzed using a modified Student’s t -test followed by correction for multiple testing. Over- and under-expressed genes, relative to control, were identified using a threshold for the log(FC) (X-axis) (between 1 or −1), corresponding to fold-changes of 2 and −2, and a threshold for the adjusted p value of 0.05 (Y-axis). B. Cumulative frequency distribution of the difference (fold-change) between TRPS1/Cath-D co-silencing and TRPS1 silencing alone. T47D cells were transfected with Luc siRNA (20 μg), or TRPS1 + Cath-D siRNA2–3 (5 μg of each, total 20 μg) in triplicate transfections. Microarray analysis showed the impact of double silencing on the 53 mRNAs that were significantly up-regulated following silencing of TRPS1 alone ( Table S1 ). Ratio (x-axis), ratio of fold-changes for the 53 mRNAs after TRPS1 and Cath-D co-silencing divided by the fold-changes after TRPS1 silencing.

    Journal: Oncotarget

    Article Title: Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells

    doi:

    Figure Lengend Snippet: Cath-D influences TRPS1 transcription repression function A. Transcriptome analysis of TRPS1-silenced and Cath-D-silenced cells. T47D cells were transfected in triplicate with control Luc siRNA (20 μg), TRPS1 or Cath-D siRNA1–3 (20 μg), or with both TRPS1 and Cath-D siRNA2–3 (5 μg of each, total 20 μg). RNAs were extracted 48 h post-transfection and analyzed by microarray hybridization. Panel a: TRPS1-silenced cells versus control. Panel b: Cath-D-silenced cells versus control. Panel c: TRPS1/Cath-D doubly-silenced cells versus control. Panel d: TRPS1/Cath-D doubly-silenced cells versus TRPS1-silenced cells. The significant differences in the mRNA expression of a given gene were determined by dividing the log2 of TRPS1, Cath-D or TRPS1/Cath-D silenced sample signals by the Luc control signal, i.e . log(FC); these data were analyzed using a modified Student’s t -test followed by correction for multiple testing. Over- and under-expressed genes, relative to control, were identified using a threshold for the log(FC) (X-axis) (between 1 or −1), corresponding to fold-changes of 2 and −2, and a threshold for the adjusted p value of 0.05 (Y-axis). B. Cumulative frequency distribution of the difference (fold-change) between TRPS1/Cath-D co-silencing and TRPS1 silencing alone. T47D cells were transfected with Luc siRNA (20 μg), or TRPS1 + Cath-D siRNA2–3 (5 μg of each, total 20 μg) in triplicate transfections. Microarray analysis showed the impact of double silencing on the 53 mRNAs that were significantly up-regulated following silencing of TRPS1 alone ( Table S1 ). Ratio (x-axis), ratio of fold-changes for the 53 mRNAs after TRPS1 and Cath-D co-silencing divided by the fold-changes after TRPS1 silencing.

    Article Snippet: T47D cells were also transfected with 1 μg Luc, Cath-D, and/or TRPS1 shRNA expression vectors (Invivogen, San Diego, CA, USA) using Nucleofector Technology (Amaxa biosystems, MD, USA).

    Techniques: Transfection, Microarray, Hybridization, Expressing, Modification

    Transcription repression by TRPS1 and Cath-D A. Transcription repression by Cath-D Panel a: Diagram of the L8G5-Luc reporter gene containing the LexA operator sequence and Gal4 binding sites. LexA-VP16 is a transcription transactivator. Gal4 fusion proteins (Gal-Cath-D, Gal- D231N Cath-D) bind to the Gal4 sites to modulate LexA-VP16-induced transcription. Panel b: T47D cells were transfected with pRL-CMV (Renilla) (40 ng), the L8G5-Luc reporter gene (160 ng), the LexA-VP16 expression plasmid (80 ng) and increasing concentrations (100 to 800 ng) of the plasmids encoding the indicated Gal4 fusion proteins. Data are the percentage of the standardized luciferase activity obtained with Gal4 (100 ng) and are the mean ± SD of triplicate transfections. Similar results were obtained in another independent experiment. B. Regulation of the PTHrP promoter activity by TRPS1 and Cath-D. Panel a: The upper left image shows a diagram of the 5′-flanking 4.3 kB region of the human PTHrP gene and the PTHrP promoter-luciferase construct. Exons are indicated by boxes and promoters by arrows. Lower left image: Expression of TRPS1 and Cath-D was analyzed in whole cell extracts from Ctsd− MEFs and T47D cells by western blot analysis. β-actin, loading control. Histogram on the right: Ctsd −/− MEFs were co-transfected with pRL-CMV (Renilla) (40 ng), the pGL2-PTHrP promoter plasmid or pGL2 empty vector (250 ng), and increasing concentrations of Cath-D expression plasmid or pcDNA3 empty vector (200, 300 and 400 ng) in the presence of a non-specific control siRNA (5′AGGUAGUGUAAUCGCCUUGdTdT 3′) or TRPS1 siRNA3 (125 ng). No luciferase activity was detected in cells with the pGL2 empty vector. ** p

    Journal: Oncotarget

    Article Title: Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells

    doi:

    Figure Lengend Snippet: Transcription repression by TRPS1 and Cath-D A. Transcription repression by Cath-D Panel a: Diagram of the L8G5-Luc reporter gene containing the LexA operator sequence and Gal4 binding sites. LexA-VP16 is a transcription transactivator. Gal4 fusion proteins (Gal-Cath-D, Gal- D231N Cath-D) bind to the Gal4 sites to modulate LexA-VP16-induced transcription. Panel b: T47D cells were transfected with pRL-CMV (Renilla) (40 ng), the L8G5-Luc reporter gene (160 ng), the LexA-VP16 expression plasmid (80 ng) and increasing concentrations (100 to 800 ng) of the plasmids encoding the indicated Gal4 fusion proteins. Data are the percentage of the standardized luciferase activity obtained with Gal4 (100 ng) and are the mean ± SD of triplicate transfections. Similar results were obtained in another independent experiment. B. Regulation of the PTHrP promoter activity by TRPS1 and Cath-D. Panel a: The upper left image shows a diagram of the 5′-flanking 4.3 kB region of the human PTHrP gene and the PTHrP promoter-luciferase construct. Exons are indicated by boxes and promoters by arrows. Lower left image: Expression of TRPS1 and Cath-D was analyzed in whole cell extracts from Ctsd− MEFs and T47D cells by western blot analysis. β-actin, loading control. Histogram on the right: Ctsd −/− MEFs were co-transfected with pRL-CMV (Renilla) (40 ng), the pGL2-PTHrP promoter plasmid or pGL2 empty vector (250 ng), and increasing concentrations of Cath-D expression plasmid or pcDNA3 empty vector (200, 300 and 400 ng) in the presence of a non-specific control siRNA (5′AGGUAGUGUAAUCGCCUUGdTdT 3′) or TRPS1 siRNA3 (125 ng). No luciferase activity was detected in cells with the pGL2 empty vector. ** p

    Article Snippet: T47D cells were also transfected with 1 μg Luc, Cath-D, and/or TRPS1 shRNA expression vectors (Invivogen, San Diego, CA, USA) using Nucleofector Technology (Amaxa biosystems, MD, USA).

    Techniques: Sequencing, Binding Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Construct, Western Blot

    Cath-D/TRPS1 dual silencing inhibits cell cycle progression and cell transformation A. Expression of cyclin A and E. T47D cells were transfected with Luc, TRPS1, Cath-D or TRPS1+Cath-D shRNAs. Five days post-transfection, TRPS1, Cath-D, cyclin A and E expression were analyzed in whole cell extracts (10 μg) by western blotting. β-actin: loading control. Similar results were obtained in three independent experiments. B. Cell cycle analysis. Cell cycle progression in T47D cells was analyzed by flow cytometry five days after transfection with the indicated shRNAs. Cell cycle analysis (panels b) and quantification of the subG1, G0G1, S and G2M fractions (panel a). Similar results were obtained in three independent experiments. C. Soft agar colony formation. Three days post-transfection, T47D cells were embedded in soft agar and grown for 8 days. The resulting colonies were stained with p-iodonitrotetrazolium violet and phase-contrast photomicrographs taken. Colonies/well in 6-well plates were counted using Image J (Panel b). Data are the mean ± SD relative to Luc shRNA ( n = 6). ** p

    Journal: Oncotarget

    Article Title: Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells

    doi:

    Figure Lengend Snippet: Cath-D/TRPS1 dual silencing inhibits cell cycle progression and cell transformation A. Expression of cyclin A and E. T47D cells were transfected with Luc, TRPS1, Cath-D or TRPS1+Cath-D shRNAs. Five days post-transfection, TRPS1, Cath-D, cyclin A and E expression were analyzed in whole cell extracts (10 μg) by western blotting. β-actin: loading control. Similar results were obtained in three independent experiments. B. Cell cycle analysis. Cell cycle progression in T47D cells was analyzed by flow cytometry five days after transfection with the indicated shRNAs. Cell cycle analysis (panels b) and quantification of the subG1, G0G1, S and G2M fractions (panel a). Similar results were obtained in three independent experiments. C. Soft agar colony formation. Three days post-transfection, T47D cells were embedded in soft agar and grown for 8 days. The resulting colonies were stained with p-iodonitrotetrazolium violet and phase-contrast photomicrographs taken. Colonies/well in 6-well plates were counted using Image J (Panel b). Data are the mean ± SD relative to Luc shRNA ( n = 6). ** p

    Article Snippet: T47D cells were also transfected with 1 μg Luc, Cath-D, and/or TRPS1 shRNA expression vectors (Invivogen, San Diego, CA, USA) using Nucleofector Technology (Amaxa biosystems, MD, USA).

    Techniques: Transformation Assay, Expressing, Transfection, Western Blot, Cell Cycle Assay, Flow Cytometry, Cytometry, Staining, shRNA

    Nuclear interaction and co-localization of endogenous fully-mature Cath-D and full-length TRPS1 in ER + BCC A. Nuclear Cath-D does not influence TRPS1 proteolysis. Cytosolic, membrane and nuclear fractions of T47D, MCF7 and BT474 cells (10 μg) were resolved by SDS-PAGE. TRPS1 and Cath-D expression in the different fractions were analyzed by immunoblotting. B. Nuclear Cath-D does not affect TRPS1 turnover. T47D cells were transfected with Cath-D siRNA2–3 (10 μg each) or control Luc siRNA (20 μg) for 48 h. Cath-D, TRPS1 and HDAC3 expression in the nuclear fraction was then assessed by WB. HDAC3, control for nuclear fraction. C. Endogenous TRPS1 and Cath-D are co-immunoprecipitated from nuclear fractions. Aliquots of T47D nuclear fraction (100 μg) were immunoprecipitated with the anti-Cath-D antibody M1G8 (IP Cath-D) or control IgG1 (IP IgG). Cath-D (top) and TRPS1 (bottom) were detected by WB. NF, nuclear fraction (10 μg). D. Co-localization of endogenous Cath-D and TRPS1 in the nucleus. Permeabilized T47D cells were double stained with the anti-Cath-D monoclonal antibody M1G8 (red) and an anti-TRPS1 polyclonal antibody (green). DNA was stained with 0.5 μg/ml DAPI (blue). Panel a: DAPI, Cath-D, TRPS1 and merge immunostaining analyzed by confocal microscopy (Z projections (max intensity) of 4 × 0.23 μm slices). Panel b: Cath-D immunostaining and Panel c: double immunostaining patterns. Higher magnifications (slices 1 and 2: 0.23 μm) are shown in the boxed regions. Arrows indicate Cath-D and TRPS1 nuclear co-localization. Scale bar: 10 μm.

    Journal: Oncotarget

    Article Title: Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells

    doi:

    Figure Lengend Snippet: Nuclear interaction and co-localization of endogenous fully-mature Cath-D and full-length TRPS1 in ER + BCC A. Nuclear Cath-D does not influence TRPS1 proteolysis. Cytosolic, membrane and nuclear fractions of T47D, MCF7 and BT474 cells (10 μg) were resolved by SDS-PAGE. TRPS1 and Cath-D expression in the different fractions were analyzed by immunoblotting. B. Nuclear Cath-D does not affect TRPS1 turnover. T47D cells were transfected with Cath-D siRNA2–3 (10 μg each) or control Luc siRNA (20 μg) for 48 h. Cath-D, TRPS1 and HDAC3 expression in the nuclear fraction was then assessed by WB. HDAC3, control for nuclear fraction. C. Endogenous TRPS1 and Cath-D are co-immunoprecipitated from nuclear fractions. Aliquots of T47D nuclear fraction (100 μg) were immunoprecipitated with the anti-Cath-D antibody M1G8 (IP Cath-D) or control IgG1 (IP IgG). Cath-D (top) and TRPS1 (bottom) were detected by WB. NF, nuclear fraction (10 μg). D. Co-localization of endogenous Cath-D and TRPS1 in the nucleus. Permeabilized T47D cells were double stained with the anti-Cath-D monoclonal antibody M1G8 (red) and an anti-TRPS1 polyclonal antibody (green). DNA was stained with 0.5 μg/ml DAPI (blue). Panel a: DAPI, Cath-D, TRPS1 and merge immunostaining analyzed by confocal microscopy (Z projections (max intensity) of 4 × 0.23 μm slices). Panel b: Cath-D immunostaining and Panel c: double immunostaining patterns. Higher magnifications (slices 1 and 2: 0.23 μm) are shown in the boxed regions. Arrows indicate Cath-D and TRPS1 nuclear co-localization. Scale bar: 10 μm.

    Article Snippet: T47D cells were also transfected with 1 μg Luc, Cath-D, and/or TRPS1 shRNA expression vectors (Invivogen, San Diego, CA, USA) using Nucleofector Technology (Amaxa biosystems, MD, USA).

    Techniques: SDS Page, Expressing, Transfection, Western Blot, Immunoprecipitation, Staining, Immunostaining, Confocal Microscopy, Double Immunostaining

    Regulation of nuclear Cath-D accumulation by its molecular partner BAT3 Nuclear (panel A), cytoplasmic (panel B), membrane (panel C) fractions, and whole cell extracts (panel D) (10 μg) from T47D cells transfected with Luc or BAT3 siRNA1 (20 μg), siRNA2 (20 μg), or siRNA(1+2) (10 μg each) were prepared 48 h post-transfection and BAT3, Cath-D, TRPS1, GAPDH, HDAC3 and β-actin expression analyzed by WB. GAPDH and HDAC3, loading markers for cytoplasmic and nuclear fractions. β-actin: whole cell extract loading control.

    Journal: Oncotarget

    Article Title: Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells

    doi:

    Figure Lengend Snippet: Regulation of nuclear Cath-D accumulation by its molecular partner BAT3 Nuclear (panel A), cytoplasmic (panel B), membrane (panel C) fractions, and whole cell extracts (panel D) (10 μg) from T47D cells transfected with Luc or BAT3 siRNA1 (20 μg), siRNA2 (20 μg), or siRNA(1+2) (10 μg each) were prepared 48 h post-transfection and BAT3, Cath-D, TRPS1, GAPDH, HDAC3 and β-actin expression analyzed by WB. GAPDH and HDAC3, loading markers for cytoplasmic and nuclear fractions. β-actin: whole cell extract loading control.

    Article Snippet: T47D cells were also transfected with 1 μg Luc, Cath-D, and/or TRPS1 shRNA expression vectors (Invivogen, San Diego, CA, USA) using Nucleofector Technology (Amaxa biosystems, MD, USA).

    Techniques: Transfection, Expressing, Western Blot

    Enhancement of the HIF-1α activity in the tumors of living mice induced by tempol. The stable expression clone, MCF7/HRE-Luc-ODD or MCF7/SV40-Luc, was implanted into the legs of the mice. The tumor-bearing mice were injected intraperitoneally with 100 mM tempol and fasted for 6 h, after which the in vivo bioluminescence emitted from the tumors was measured 3 min after the injection of D-luciferin. The error bars represent the standard deviation (n=3). * P

    Journal: Oncology Reports

    Article Title: Selective enhancement of hypoxic cell killing by tempol-regulated suicide gene expression

    doi: 10.3892/or.2015.4020

    Figure Lengend Snippet: Enhancement of the HIF-1α activity in the tumors of living mice induced by tempol. The stable expression clone, MCF7/HRE-Luc-ODD or MCF7/SV40-Luc, was implanted into the legs of the mice. The tumor-bearing mice were injected intraperitoneally with 100 mM tempol and fasted for 6 h, after which the in vivo bioluminescence emitted from the tumors was measured 3 min after the injection of D-luciferin. The error bars represent the standard deviation (n=3). * P

    Article Snippet: In addition, in order to express a suicide gene under identical regulation to the luc gene in p4HRE-Luc-ODD, a plasmid p4HRE-fcy::fur-ODD was constructed by replacing the luc gene in the plasmid p4HRE-Luc-ODD with the fcy::fur gene, which encodes cytosine deaminase (CD) and uracil phosphoribosyl-transferase (UPRT), amplified using a plasmid pORF5-fcyfur (InvivoGen, San Diego, CA, USA) as a template with the following pair of primers: 5′-ATACTAGTATCACAGAGGAGACCATGGTCACA-3′ and 5′-ATGGTACCGCGACACAGTAGTATCTGTCCCCAAA-3′.

    Techniques: Activity Assay, Mouse Assay, Expressing, Injection, In Vivo, Standard Deviation

    Tempol enhances the expression of the suicide gene, the fcy::fur fusion gene, in combination with hypoxia or CoCl 2 . (A) Schematic structure of the suicide gene, the fcy::fur fusion gene, regulated by four copies of HREs and ODD under hypoxia. The plasmid p4HRE-fcy::fur-ODD for gene therapy was constructed by replacing the luc of the plasmid p4HRE-Luc-ODD with the fcy::fur fusion gene. The myc-tag was inserted at the C-terminal of the Fcy::Fur-ODD to identify the expression of the fusion proteins in the transfected cells. (B) Western blot analyses of the Fcy::Fur fusion proteins demonstrated enhancement in both the transient and stable expression clones, MCF7/HRE-fcy::fur-ODD, in combination with 2 mM tempol and hypoxia treatment (1.0% O 2 ) for 24 h. (C) Western blot analyses of the Fcy::Fur fusion proteins enhanced by tempol under treatment with 100 µ M of CoCl 2 for 24 h. β-actin served as a loading control for western blotting. ODD, oxygen-dependent degradation domain; HRE, hypoxia-responsive element.

    Journal: Oncology Reports

    Article Title: Selective enhancement of hypoxic cell killing by tempol-regulated suicide gene expression

    doi: 10.3892/or.2015.4020

    Figure Lengend Snippet: Tempol enhances the expression of the suicide gene, the fcy::fur fusion gene, in combination with hypoxia or CoCl 2 . (A) Schematic structure of the suicide gene, the fcy::fur fusion gene, regulated by four copies of HREs and ODD under hypoxia. The plasmid p4HRE-fcy::fur-ODD for gene therapy was constructed by replacing the luc of the plasmid p4HRE-Luc-ODD with the fcy::fur fusion gene. The myc-tag was inserted at the C-terminal of the Fcy::Fur-ODD to identify the expression of the fusion proteins in the transfected cells. (B) Western blot analyses of the Fcy::Fur fusion proteins demonstrated enhancement in both the transient and stable expression clones, MCF7/HRE-fcy::fur-ODD, in combination with 2 mM tempol and hypoxia treatment (1.0% O 2 ) for 24 h. (C) Western blot analyses of the Fcy::Fur fusion proteins enhanced by tempol under treatment with 100 µ M of CoCl 2 for 24 h. β-actin served as a loading control for western blotting. ODD, oxygen-dependent degradation domain; HRE, hypoxia-responsive element.

    Article Snippet: In addition, in order to express a suicide gene under identical regulation to the luc gene in p4HRE-Luc-ODD, a plasmid p4HRE-fcy::fur-ODD was constructed by replacing the luc gene in the plasmid p4HRE-Luc-ODD with the fcy::fur gene, which encodes cytosine deaminase (CD) and uracil phosphoribosyl-transferase (UPRT), amplified using a plasmid pORF5-fcyfur (InvivoGen, San Diego, CA, USA) as a template with the following pair of primers: 5′-ATACTAGTATCACAGAGGAGACCATGGTCACA-3′ and 5′-ATGGTACCGCGACACAGTAGTATCTGTCCCCAAA-3′.

    Techniques: Expressing, Plasmid Preparation, Construct, Transfection, Western Blot, Clone Assay

    HIF1α-inducible properties induced by tempol under various conditions. (A) Schematic structure of the reporter plasmid p4HRE-Luc-ODD. The promoter sequence consisted of four copies of HRE fragments and the TATA-box was fused upstream of the luc gene. The ODD sequence amplified from HIF1α cDNA was inserted downstream of this site. (B) Induction of the Luc activity by tempol under hypoxia or CoCl 2 treatment. MCF7 cells transiently transfected with the plasmid p4HRE-Luc-ODD were allowed to recover for 12 h after transfection under normoxia and exposed to hypoxia (1.0% O 2 ) or treated with 100 µ M of CoCl 2 with or without 2 mM tempol for 24 h and then assayed for Luc activity. The fold induction was calculated by dividing the RLU of each stimulated cell by the RLU of the control cells under normoxia without tempol. Each column presents the average and standard deviation (n=5–7). ** P

    Journal: Oncology Reports

    Article Title: Selective enhancement of hypoxic cell killing by tempol-regulated suicide gene expression

    doi: 10.3892/or.2015.4020

    Figure Lengend Snippet: HIF1α-inducible properties induced by tempol under various conditions. (A) Schematic structure of the reporter plasmid p4HRE-Luc-ODD. The promoter sequence consisted of four copies of HRE fragments and the TATA-box was fused upstream of the luc gene. The ODD sequence amplified from HIF1α cDNA was inserted downstream of this site. (B) Induction of the Luc activity by tempol under hypoxia or CoCl 2 treatment. MCF7 cells transiently transfected with the plasmid p4HRE-Luc-ODD were allowed to recover for 12 h after transfection under normoxia and exposed to hypoxia (1.0% O 2 ) or treated with 100 µ M of CoCl 2 with or without 2 mM tempol for 24 h and then assayed for Luc activity. The fold induction was calculated by dividing the RLU of each stimulated cell by the RLU of the control cells under normoxia without tempol. Each column presents the average and standard deviation (n=5–7). ** P

    Article Snippet: In addition, in order to express a suicide gene under identical regulation to the luc gene in p4HRE-Luc-ODD, a plasmid p4HRE-fcy::fur-ODD was constructed by replacing the luc gene in the plasmid p4HRE-Luc-ODD with the fcy::fur gene, which encodes cytosine deaminase (CD) and uracil phosphoribosyl-transferase (UPRT), amplified using a plasmid pORF5-fcyfur (InvivoGen, San Diego, CA, USA) as a template with the following pair of primers: 5′-ATACTAGTATCACAGAGGAGACCATGGTCACA-3′ and 5′-ATGGTACCGCGACACAGTAGTATCTGTCCCCAAA-3′.

    Techniques: Plasmid Preparation, Sequencing, Amplification, Activity Assay, Transfection, Standard Deviation

    EVs from NFAT3-expressing cells inhibit cell growth and increase apoptosis in cancer cells in cooperation with macrophages. ( A ) MDA-MB-231 cells (left panel) and SUM-159-PT cells (right panel) were plated in medium with 3% Matrigel in 96-well ultra low attachment plates for three days to allow the formation of the spheroids formed. Medium was then added to each condition containing the apoptosis indicator (fluorescent caspase 3/7 substrate) supplemented with medium and containing or not the different EVs (3 × 10 8 pp/mL) as indicated. Size of the spheroids and appearance of green fluorescence (apoptosis) was recorded every 2 h on an INCUCYTE device for 4 days. Data are represented as the AUC of the spheroid size (upper panels) and apoptosis (lower panels) over 96 h. Data from one representative experiment of three independent experiments is shown, all data are shown as mean ± SEM (n = 3 technical replicates). (B) Frozen tumor tissues sections of mice xenografted with MDA-MB-231 cells (D3H2LN-LUC) and treated with PBS (−) or EVs T-47D shCtl were co-labelled with Dapi and specific antibodies to mouse macrophages (anti-F4/80 antibody) and to detect the tumor cells with an anti-Pan human cytokeratin (arrows indicate infiltrating mouse macrophages). (C) Hetero-spheroids containing 33% of the murine macrophage cell line RAW 264.7 with either MDA-MB-231 or SUM-159PT were plated in medium with 3% Matrigel in 96 wells ultra low attachment plates for three days to allow the formation of the hetero-spheroids formed. Then medium was added to each condition containing the apoptosis indicator (fluorescent caspase 3/7 substrate) supplemented with medium containing or not the different EVs (3 × 10 8 pp/mL) as indicated in the figure. The size of the spheroids and appearance of green fluorescence (apoptosis) were recorded every 2 h on an INCUCYTE apparatus for 4 days. Data are represented as the AUC of the spheroid size (upper panels) and apoptosis (lower panels) over 96 h. Data from one representative experiment of three independent experiments is shown, all data are shown as mean ± SEM (n = 3 technical replicates; ***p

    Journal: Scientific Reports

    Article Title: Extracellular vesicles produced by NFAT3-expressing cells hinder tumor growth and metastatic dissemination

    doi: 10.1038/s41598-020-65844-x

    Figure Lengend Snippet: EVs from NFAT3-expressing cells inhibit cell growth and increase apoptosis in cancer cells in cooperation with macrophages. ( A ) MDA-MB-231 cells (left panel) and SUM-159-PT cells (right panel) were plated in medium with 3% Matrigel in 96-well ultra low attachment plates for three days to allow the formation of the spheroids formed. Medium was then added to each condition containing the apoptosis indicator (fluorescent caspase 3/7 substrate) supplemented with medium and containing or not the different EVs (3 × 10 8 pp/mL) as indicated. Size of the spheroids and appearance of green fluorescence (apoptosis) was recorded every 2 h on an INCUCYTE device for 4 days. Data are represented as the AUC of the spheroid size (upper panels) and apoptosis (lower panels) over 96 h. Data from one representative experiment of three independent experiments is shown, all data are shown as mean ± SEM (n = 3 technical replicates). (B) Frozen tumor tissues sections of mice xenografted with MDA-MB-231 cells (D3H2LN-LUC) and treated with PBS (−) or EVs T-47D shCtl were co-labelled with Dapi and specific antibodies to mouse macrophages (anti-F4/80 antibody) and to detect the tumor cells with an anti-Pan human cytokeratin (arrows indicate infiltrating mouse macrophages). (C) Hetero-spheroids containing 33% of the murine macrophage cell line RAW 264.7 with either MDA-MB-231 or SUM-159PT were plated in medium with 3% Matrigel in 96 wells ultra low attachment plates for three days to allow the formation of the hetero-spheroids formed. Then medium was added to each condition containing the apoptosis indicator (fluorescent caspase 3/7 substrate) supplemented with medium containing or not the different EVs (3 × 10 8 pp/mL) as indicated in the figure. The size of the spheroids and appearance of green fluorescence (apoptosis) were recorded every 2 h on an INCUCYTE apparatus for 4 days. Data are represented as the AUC of the spheroid size (upper panels) and apoptosis (lower panels) over 96 h. Data from one representative experiment of three independent experiments is shown, all data are shown as mean ± SEM (n = 3 technical replicates; ***p

    Article Snippet: MDA-MB-231 D3H2LN-LUC were cultured in Eagle’s MEM supplemented with 75ug/mL Zeocin (Invivogen).

    Techniques: Expressing, Multiple Displacement Amplification, Fluorescence, Mouse Assay

    EVs from NFAT3-expressing cells can further increase their anti-tumor effects and are effective in inhibiting tumor growth on a pre-established cancer. ( A ) 5.10 5 MDA-MB-231 cells (D3H2LN-LUC) were injected into the left Fat Pad of each 6-weeks-old female Athymic Nude mouse. Tumor volume : Tumor growth is presented as the mean tumor volume (cm3) ±SEM, from mice injected weekly intra tumor with PBS or with EVs T-47D toCtl (5 × 10 9 pp) or EVs T-47D ΔNFAT3, 7 days after cell xenotransplantation, 10 mice per group. Metastases : Beginning day 35, weekly, bioluminescent images (where the primary tumor was shield with a black tissue) were acquired on the IVIS system to quantify the mean photon flux produced by the metastatic cells. Metastases quantification is presented as the mean photon flux produced by the metastatic cells. Data from one representative experiment of two independent experiments is shown, all data are shown as mean ± SEM (n = 10 technical replicates;, *p

    Journal: Scientific Reports

    Article Title: Extracellular vesicles produced by NFAT3-expressing cells hinder tumor growth and metastatic dissemination

    doi: 10.1038/s41598-020-65844-x

    Figure Lengend Snippet: EVs from NFAT3-expressing cells can further increase their anti-tumor effects and are effective in inhibiting tumor growth on a pre-established cancer. ( A ) 5.10 5 MDA-MB-231 cells (D3H2LN-LUC) were injected into the left Fat Pad of each 6-weeks-old female Athymic Nude mouse. Tumor volume : Tumor growth is presented as the mean tumor volume (cm3) ±SEM, from mice injected weekly intra tumor with PBS or with EVs T-47D toCtl (5 × 10 9 pp) or EVs T-47D ΔNFAT3, 7 days after cell xenotransplantation, 10 mice per group. Metastases : Beginning day 35, weekly, bioluminescent images (where the primary tumor was shield with a black tissue) were acquired on the IVIS system to quantify the mean photon flux produced by the metastatic cells. Metastases quantification is presented as the mean photon flux produced by the metastatic cells. Data from one representative experiment of two independent experiments is shown, all data are shown as mean ± SEM (n = 10 technical replicates;, *p

    Article Snippet: MDA-MB-231 D3H2LN-LUC were cultured in Eagle’s MEM supplemented with 75ug/mL Zeocin (Invivogen).

    Techniques: Expressing, Multiple Displacement Amplification, Injection, Mouse Assay, Produced