pgl3 basic vector Search Results


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  • 91
    Promega vector pgl3 basic
    Impact of the −50T > C variant in <t>pGL3</t> basic-derived constructs. A) Partial structures of constructs used in reporter assays. B) Gene reporter assays in PC12 and EOMA cells. Cells were transiently co-transfected with the reporter constructs containing the rs62620038 (position −50) variant or empty vector, as indicated. Each bar represents the average fold-induction relative to the empty vector.
    Vector Pgl3 Basic, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 3043 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 3043 article reviews
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    92
    Addgene inc pgl3 basic vector
    MALAT1 epigenetically regulates EF1A1. A. p EEF1A1 -luciferase assay. The EEF1A1 promoter (p EEF1A1 ) sequence was cloned into the upstream of luciferase gene. Luciferase reporter assay was performed in CTL group, shNC group and shMALAT1 group by co-transfecting respectively with <t>pGL3-Basic</t> vector or luciferase reporter vector. Data were adjusted over the negative control (CTL) and were represented as means ± SD. ** P
    Pgl3 Basic Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgl3 basic vector/product/Addgene inc
    Average 92 stars, based on 108 article reviews
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    92
    Thermo Fisher pgl3 basic vector
    PLK1 is a target of miR-100. (A) Western blot analysis of PLK1 protein expression in SKOV-3 cells transfected with pre-miR-100 (pre-miR-NC) or anti-miR-100 (anti-miR-NC). (B) PLK1 3′-UTR and corresponding fragments were inserted into the region immediately downstream of the luciferase gene in <t>pGL3-Basic</t> vector and validated by DNA sequencing. The sequences of the predicted miR-100 binding sites within the PLK1 3′-UTR, including wild-type UTR or UTR segments containing mutant binding site are shown. (C) Detection of luciferase activity. For luciferase reporter assays in 6-well plates, 1-mg luciferase reporter plasmid containing either wild-type (wt) or mutant (mut) PLK1 3′-UTR, and 200 pmol of pre-miR-100, anti-miR-100, pre-miR-NC or anti-miR-100 were transfected. The parental luciferase plasmid was also transfected as a control. At 24 h after transfection, cells were assayed using the Luciferase Gene Reporter Assay kit. The data are presented as the mean ± SD of three experiments. * P
    Pgl3 Basic Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 957 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgl3 basic vector/product/Thermo Fisher
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    93
    TaKaRa pgl3 basic vector
    Foxo4 negatively regulate MC5R transcription in αMSH inhibited inflammation in mice adipocytes (A) Fragments of MC5R promoter fused to a luciferase reporter plasmid or <t>PGL3-basic</t> (control) were co-transfected into cells together with Renlilla plasmid and pAd-Foxo4 (n=3). Luciferase activity was corrected for Renilla luciferase activity and normalized to control activity (n=3). (B) Chromatin immunoprecipitation (ChIP) analysis of Foxo4 and MC5R interaction. (C, D) After pAd-Foxo4 together with αMSH or pc-MC5R in LPS/saline treatment, MC5R mRNA level was determined in adipocytes (n=3). (E) When adipocytes were treated with pAd-Foxo4 and αMSH in LPS/saline treatment, mRNA levels of Foxo4 , IL-6 , TNFα , MCP-1 and Leptin were analyzed (n=3). Values are means ± SD. vs. control group, * P
    Pgl3 Basic Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 374 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgl3 basic vector/product/TaKaRa
    Average 93 stars, based on 374 article reviews
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    92
    Stratagene pgl3 basic vector
    Analysis of transcriptional activity of putative intron locus promoter elements. (A) Diagram of genomic location of the viral sequences cloned into <t>pGL3-reporter</t> plasmids. (B) pGL3-reporter constructs were co-transfected into mouse fibroblasts and luciferase induction was assayed 24 h p.i. following either mock co-infection, (C) UV inactivated co-infection, or MCMV co-infection (MOI = 0.05).
    Pgl3 Basic Vector, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Ribobio pgl3 basic vector
    Analysis of transcriptional activity of putative intron locus promoter elements. (A) Diagram of genomic location of the viral sequences cloned into <t>pGL3-reporter</t> plasmids. (B) pGL3-reporter constructs were co-transfected into mouse fibroblasts and luciferase induction was assayed 24 h p.i. following either mock co-infection, (C) UV inactivated co-infection, or MCMV co-infection (MOI = 0.05).
    Pgl3 Basic Vector, supplied by Ribobio, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Promega pgl3 basic vectors
    USF1 transcriptionally elevates TGF-β1 expression in breast cancer. a The relative luciferase activities were analyzed after co-transfection with various TGF-β1 promoter reporters or the <t>pGL3-basic</t> vector and USF1 or control vector. b Schematic of the location of E-box motif bound by USF1 in TGF-β1 promoter region. c Wild-type (WT) or mutant (Mut) TGF-β1 luciferase reporter vector was co-transfected with USF1 or control vector into MCF-7 and MDA-MB-231 cells, after 48 h of co-transfection, the luciferase activities were tested. d ChIP-qPCR analysis of USF1 binding to the TGF-β1 promoter region in MCF-7 and MDA-MB-231 cells. RNA polymerase II (RNAPII) was used as a positive control. e qRT-PCR (left) and immunoblot (right) analysis of TGF-β1 mRNA expression in USF1-overexpressing MCF-7 cells and USF1 knockdown MDA-MB-231 cells. β-actin was used as a loading control. f The correlation between USF1 and TGF-β1 expression in breast cancer tissues from TCGA database was analyzed by Spearman correlation coefficients ( r = 0.223, n = 1109, p
    Pgl3 Basic Vectors, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 436 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Genecopoeia pgl3 basic vector
    USF1 transcriptionally elevates TGF-β1 expression in breast cancer. a The relative luciferase activities were analyzed after co-transfection with various TGF-β1 promoter reporters or the <t>pGL3-basic</t> vector and USF1 or control vector. b Schematic of the location of E-box motif bound by USF1 in TGF-β1 promoter region. c Wild-type (WT) or mutant (Mut) TGF-β1 luciferase reporter vector was co-transfected with USF1 or control vector into MCF-7 and MDA-MB-231 cells, after 48 h of co-transfection, the luciferase activities were tested. d ChIP-qPCR analysis of USF1 binding to the TGF-β1 promoter region in MCF-7 and MDA-MB-231 cells. RNA polymerase II (RNAPII) was used as a positive control. e qRT-PCR (left) and immunoblot (right) analysis of TGF-β1 mRNA expression in USF1-overexpressing MCF-7 cells and USF1 knockdown MDA-MB-231 cells. β-actin was used as a loading control. f The correlation between USF1 and TGF-β1 expression in breast cancer tissues from TCGA database was analyzed by Spearman correlation coefficients ( r = 0.223, n = 1109, p
    Pgl3 Basic Vector, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Genechem pgl3 basic vector
    USF1 transcriptionally elevates TGF-β1 expression in breast cancer. a The relative luciferase activities were analyzed after co-transfection with various TGF-β1 promoter reporters or the <t>pGL3-basic</t> vector and USF1 or control vector. b Schematic of the location of E-box motif bound by USF1 in TGF-β1 promoter region. c Wild-type (WT) or mutant (Mut) TGF-β1 luciferase reporter vector was co-transfected with USF1 or control vector into MCF-7 and MDA-MB-231 cells, after 48 h of co-transfection, the luciferase activities were tested. d ChIP-qPCR analysis of USF1 binding to the TGF-β1 promoter region in MCF-7 and MDA-MB-231 cells. RNA polymerase II (RNAPII) was used as a positive control. e qRT-PCR (left) and immunoblot (right) analysis of TGF-β1 mRNA expression in USF1-overexpressing MCF-7 cells and USF1 knockdown MDA-MB-231 cells. β-actin was used as a loading control. f The correlation between USF1 and TGF-β1 expression in breast cancer tissues from TCGA database was analyzed by Spearman correlation coefficients ( r = 0.223, n = 1109, p
    Pgl3 Basic Vector, supplied by Genechem, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgl3 basic vector/product/Genechem
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    92
    OMEGA Engineering pgl3 basic vector
    USF1 transcriptionally elevates TGF-β1 expression in breast cancer. a The relative luciferase activities were analyzed after co-transfection with various TGF-β1 promoter reporters or the <t>pGL3-basic</t> vector and USF1 or control vector. b Schematic of the location of E-box motif bound by USF1 in TGF-β1 promoter region. c Wild-type (WT) or mutant (Mut) TGF-β1 luciferase reporter vector was co-transfected with USF1 or control vector into MCF-7 and MDA-MB-231 cells, after 48 h of co-transfection, the luciferase activities were tested. d ChIP-qPCR analysis of USF1 binding to the TGF-β1 promoter region in MCF-7 and MDA-MB-231 cells. RNA polymerase II (RNAPII) was used as a positive control. e qRT-PCR (left) and immunoblot (right) analysis of TGF-β1 mRNA expression in USF1-overexpressing MCF-7 cells and USF1 knockdown MDA-MB-231 cells. β-actin was used as a loading control. f The correlation between USF1 and TGF-β1 expression in breast cancer tissues from TCGA database was analyzed by Spearman correlation coefficients ( r = 0.223, n = 1109, p
    Pgl3 Basic Vector, supplied by OMEGA Engineering, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgl3 basic vector/product/OMEGA Engineering
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    92
    GenePharma Company pgl3 basic vector
    USF1 transcriptionally elevates TGF-β1 expression in breast cancer. a The relative luciferase activities were analyzed after co-transfection with various TGF-β1 promoter reporters or the <t>pGL3-basic</t> vector and USF1 or control vector. b Schematic of the location of E-box motif bound by USF1 in TGF-β1 promoter region. c Wild-type (WT) or mutant (Mut) TGF-β1 luciferase reporter vector was co-transfected with USF1 or control vector into MCF-7 and MDA-MB-231 cells, after 48 h of co-transfection, the luciferase activities were tested. d ChIP-qPCR analysis of USF1 binding to the TGF-β1 promoter region in MCF-7 and MDA-MB-231 cells. RNA polymerase II (RNAPII) was used as a positive control. e qRT-PCR (left) and immunoblot (right) analysis of TGF-β1 mRNA expression in USF1-overexpressing MCF-7 cells and USF1 knockdown MDA-MB-231 cells. β-actin was used as a loading control. f The correlation between USF1 and TGF-β1 expression in breast cancer tissues from TCGA database was analyzed by Spearman correlation coefficients ( r = 0.223, n = 1109, p
    Pgl3 Basic Vector, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 92/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgl3 basic vector/product/GenePharma Company
    Average 92 stars, based on 39 article reviews
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    90
    Promega promoterless pgl3 basic vector
    Functional analysis of the MTG16 promoter by sequential 5'-deletion . The following 5'deleted reporter constructs were generated from the <t>pGL3</t> -820-57 (-820) construct and examined after expression in erythroid HEL/TF-1, erythroid/megakaryocytic UT-7 and megakaryocytic MEG-01cells: pGL3 -668-57 (-668), pGL3 -512-57 (-512), pGL3 -359-57 (-359), pGL3 -339-57 (-339), pGL3 -299-57 (-299) and pGL3 -219-57 (-219). <t>Promoterless</t> pGL3/basic and the pGL3/SV40-promoter are used as negative and positive control, respectively. The luciferase activity was normalized against pGL3/SV40- promoter activity. Results are shown for 3 to 5 separate transfections; bars represent the mean and the error bars show SEM.
    Promoterless Pgl3 Basic Vector, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 758 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/promoterless pgl3 basic vector/product/Promega
    Average 90 stars, based on 758 article reviews
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    88
    Promega compatible pgl3 basic vector
    Functional analysis of the MTG16 promoter by sequential 5'-deletion . The following 5'deleted reporter constructs were generated from the <t>pGL3</t> -820-57 (-820) construct and examined after expression in erythroid HEL/TF-1, erythroid/megakaryocytic UT-7 and megakaryocytic MEG-01cells: pGL3 -668-57 (-668), pGL3 -512-57 (-512), pGL3 -359-57 (-359), pGL3 -339-57 (-339), pGL3 -299-57 (-299) and pGL3 -219-57 (-219). <t>Promoterless</t> pGL3/basic and the pGL3/SV40-promoter are used as negative and positive control, respectively. The luciferase activity was normalized against pGL3/SV40- promoter activity. Results are shown for 3 to 5 separate transfections; bars represent the mean and the error bars show SEM.
    Compatible Pgl3 Basic Vector, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 88 stars, based on 7 article reviews
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    Image Search Results


    Impact of the −50T > C variant in pGL3 basic-derived constructs. A) Partial structures of constructs used in reporter assays. B) Gene reporter assays in PC12 and EOMA cells. Cells were transiently co-transfected with the reporter constructs containing the rs62620038 (position −50) variant or empty vector, as indicated. Each bar represents the average fold-induction relative to the empty vector.

    Journal: PLoS ONE

    Article Title: A PTG Variant Contributes to a Milder Phenotype in Lafora Disease

    doi: 10.1371/journal.pone.0021294

    Figure Lengend Snippet: Impact of the −50T > C variant in pGL3 basic-derived constructs. A) Partial structures of constructs used in reporter assays. B) Gene reporter assays in PC12 and EOMA cells. Cells were transiently co-transfected with the reporter constructs containing the rs62620038 (position −50) variant or empty vector, as indicated. Each bar represents the average fold-induction relative to the empty vector.

    Article Snippet: Luciferase constructs Analysis of PPP1R3C 5′-UTR was performed by insertion of a 890 pb fragment of the promoter region into vector pGL3 basic (Promega) using NcoI and HindII sites.

    Techniques: Variant Assay, Derivative Assay, Construct, Transfection, Plasmid Preparation

    HBx Increased the Expression of AKR1C1 by Enhancing the Activity of Its Promoter A) 0.5 μg of pCH9/3091 was cotransfected with 0.5 μg of pGL3-Basic-AKR1C1-P and 0.2 μg of pRL-TK (Renilla expression vector) into HepG2 cells and luciferase assay was performed. Luciferase activity was normalized with the renilla luciferase activity in cell lysate. B) Four HBV expression plasmids (pCMV-Sport6-HBs, pCMV-Sport6-HBx, pCMV-Sport6-HBc, and pCMV-Sport6-HBp), pGL3-Basic-AKR1C1-P and pRL-TK were cotransfected into HepG2 cells, pCMV-Sport6 was used as the control and luciferase activity was measured. C) HBx recombinant adenovirus and GFP control recombinant adenovirus were used to infect the HepG2 cells and AKR1C1 expression were measured with RT-PCR. β-actin was used as a control. D) 0.2 μg of pAKR1C1-641/+59, 0.5 μg of pCMV-Sport6-HBx and 0.1 μg of pRL-TK were co-transfected into HepG2 cells. The pCMV-Sport6 group was used as a negative control. The values are means ± SD of three independent experiments.

    Journal: Hepatitis Monthly

    Article Title: Hepatitis B Virus X Protein Up-Regulates AKR1C1 Expression Through Nuclear Factor-Y in Human Hepatocarcinoma Cells

    doi: 10.5812/hepatmon.8792

    Figure Lengend Snippet: HBx Increased the Expression of AKR1C1 by Enhancing the Activity of Its Promoter A) 0.5 μg of pCH9/3091 was cotransfected with 0.5 μg of pGL3-Basic-AKR1C1-P and 0.2 μg of pRL-TK (Renilla expression vector) into HepG2 cells and luciferase assay was performed. Luciferase activity was normalized with the renilla luciferase activity in cell lysate. B) Four HBV expression plasmids (pCMV-Sport6-HBs, pCMV-Sport6-HBx, pCMV-Sport6-HBc, and pCMV-Sport6-HBp), pGL3-Basic-AKR1C1-P and pRL-TK were cotransfected into HepG2 cells, pCMV-Sport6 was used as the control and luciferase activity was measured. C) HBx recombinant adenovirus and GFP control recombinant adenovirus were used to infect the HepG2 cells and AKR1C1 expression were measured with RT-PCR. β-actin was used as a control. D) 0.2 μg of pAKR1C1-641/+59, 0.5 μg of pCMV-Sport6-HBx and 0.1 μg of pRL-TK were co-transfected into HepG2 cells. The pCMV-Sport6 group was used as a negative control. The values are means ± SD of three independent experiments.

    Article Snippet: PCR amplified promoter fragments -641/+59, -349/+59, -180/+59, -152/+59, -128/+59, -88/+59 and -49/+59 were cloned in the Kpn I and Xho I sites of the pGL3-basic vector ( , ).

    Techniques: Expressing, Activity Assay, Plasmid Preparation, Luciferase, Recombinant, Reverse Transcription Polymerase Chain Reaction, Transfection, Negative Control

    In vitro footprinting of the VEGF promoter region with DNase I. ( A ) Autoradiograms showing DNase I cleavage sites on the top strand of a supercoiled pGL3-VEGF plasmid. The plasmid DNA was incubated in the absence of salt (lane 1), or in the presence of 100 mM KCl without (lane 2) and with (lane 3) 1 µM telomestatin at 37°C for 1 h before digesting with DNase I. DNase I cleavage sites were mapped using linear amplification by PCR with 32 P-labeled gene-specific plasmid DNA pretreated with DNase I. Arrows A and B indicate the hypersensitive cleavage sites to nucleases. ( B ) Densitometric scanning of the autoradiogram in (A). The bars indicate the guanine repeats involved in the formation of the G-quadruplex structures. Arrows A and B indicate the hypersensitive cleavage sites to nucleases. ( C ) Autoradiograms showing DNase I cleavage sites on the bottom strand of a supercoiled pGL3-VEGF plasmid. The designation of lanes 1–3 was as in ( A ) above. DNase I cleavage sites were mapped using linear amplification by PCR with 32 P-labeled gene-specific plasmid DNA pretreated with DNase I. The vertical bar next to the gel indicates the polypyrimidine tract.

    Journal: Nucleic Acids Research

    Article Title: Facilitation of a structural transition in the polypurine/polypyrimidine tract within the proximal promoter region of the human VEGF gene by the presence of potassium and G-quadruplex-interactive agents

    doi: 10.1093/nar/gki917

    Figure Lengend Snippet: In vitro footprinting of the VEGF promoter region with DNase I. ( A ) Autoradiograms showing DNase I cleavage sites on the top strand of a supercoiled pGL3-VEGF plasmid. The plasmid DNA was incubated in the absence of salt (lane 1), or in the presence of 100 mM KCl without (lane 2) and with (lane 3) 1 µM telomestatin at 37°C for 1 h before digesting with DNase I. DNase I cleavage sites were mapped using linear amplification by PCR with 32 P-labeled gene-specific plasmid DNA pretreated with DNase I. Arrows A and B indicate the hypersensitive cleavage sites to nucleases. ( B ) Densitometric scanning of the autoradiogram in (A). The bars indicate the guanine repeats involved in the formation of the G-quadruplex structures. Arrows A and B indicate the hypersensitive cleavage sites to nucleases. ( C ) Autoradiograms showing DNase I cleavage sites on the bottom strand of a supercoiled pGL3-VEGF plasmid. The designation of lanes 1–3 was as in ( A ) above. DNase I cleavage sites were mapped using linear amplification by PCR with 32 P-labeled gene-specific plasmid DNA pretreated with DNase I. The vertical bar next to the gel indicates the polypyrimidine tract.

    Article Snippet: Plasmid DNA For in vitro footprinting of the VEGF promoter region, we used the supercoiled form of the luciferase reporter plasmid pGL3-VEGFP, which was constructed by ligating an 837 bp VEGF promoter region (−787 to +50 relative to the transcription initiation site) into the KpnI and NheI sites of pGL3-basic basic vector (Promega, Madison, WI), as described previously ( ).

    Techniques: In Vitro, Footprinting, Plasmid Preparation, Incubation, Amplification, Polymerase Chain Reaction, Labeling

    In vitro footprinting of the VEGF promoter region with S1 nuclease. ( A ) Autoradiograms showing S1 nuclease cleavage sites on the top strand of a supercoiled pGL3-VEGF plasmid. Arrow A indicates the hypersensitive cleavage sites to S1 nuclease. ( B ) Densitometric scanning of the autoradiogram in (A). The plasmid DNA was incubated in the absence of salt (lane 1) or in the presence of 100 mM KCl without (lane 2) and with (lane 3) 1 µM telomestatin at 37°C for 1 h before digesting with S1 nuclease. S1 nuclease cleavage sites were mapped using linear amplification by PCR with 32 P-labeled gene-specific primers on plasmid DNA pretreated with S1 nuclease. Arrow A indicates the hypersensitive cleavage sites to S1 nuclease. ( C ) Autoradiograms showing S1 cleavage sites on the bottom strand of a supercoiled pGL3-VEGF plasmid. The designation of lanes 1–3 was as in (A) above. The vertical bar next to the gel indicates the polypyrimidine tract and the arrows indicate the S1 nuclease hypersensitivity sites.

    Journal: Nucleic Acids Research

    Article Title: Facilitation of a structural transition in the polypurine/polypyrimidine tract within the proximal promoter region of the human VEGF gene by the presence of potassium and G-quadruplex-interactive agents

    doi: 10.1093/nar/gki917

    Figure Lengend Snippet: In vitro footprinting of the VEGF promoter region with S1 nuclease. ( A ) Autoradiograms showing S1 nuclease cleavage sites on the top strand of a supercoiled pGL3-VEGF plasmid. Arrow A indicates the hypersensitive cleavage sites to S1 nuclease. ( B ) Densitometric scanning of the autoradiogram in (A). The plasmid DNA was incubated in the absence of salt (lane 1) or in the presence of 100 mM KCl without (lane 2) and with (lane 3) 1 µM telomestatin at 37°C for 1 h before digesting with S1 nuclease. S1 nuclease cleavage sites were mapped using linear amplification by PCR with 32 P-labeled gene-specific primers on plasmid DNA pretreated with S1 nuclease. Arrow A indicates the hypersensitive cleavage sites to S1 nuclease. ( C ) Autoradiograms showing S1 cleavage sites on the bottom strand of a supercoiled pGL3-VEGF plasmid. The designation of lanes 1–3 was as in (A) above. The vertical bar next to the gel indicates the polypyrimidine tract and the arrows indicate the S1 nuclease hypersensitivity sites.

    Article Snippet: Plasmid DNA For in vitro footprinting of the VEGF promoter region, we used the supercoiled form of the luciferase reporter plasmid pGL3-VEGFP, which was constructed by ligating an 837 bp VEGF promoter region (−787 to +50 relative to the transcription initiation site) into the KpnI and NheI sites of pGL3-basic basic vector (Promega, Madison, WI), as described previously ( ).

    Techniques: In Vitro, Footprinting, Plasmid Preparation, Incubation, Amplification, Polymerase Chain Reaction, Labeling

    In vitro footprinting of the mutant VEGF promoter region with DNase I and S1 nuclease. Autoradiograms showing DNase I (lanes 1–3) and S1 (lanes 4–6) cleavage sites on the top strand of a supercoiled pGL3-VEGFM17 plasmid. This plasmid was incubated in the absence of salt (lanes 1 and 4) or in the presence of 100 mM KCl without (lanes 2 and 5) and with (lanes 3 and 6) 1 µM telomestatin at 37°C for 1 h before digesting with nucleases. Nuclease cleavage sites were mapped using linear amplification by PCR with 32 P-labeled gene-specific primers on mutant plasmid DNA pretreated with S1 nuclease or DNase I.

    Journal: Nucleic Acids Research

    Article Title: Facilitation of a structural transition in the polypurine/polypyrimidine tract within the proximal promoter region of the human VEGF gene by the presence of potassium and G-quadruplex-interactive agents

    doi: 10.1093/nar/gki917

    Figure Lengend Snippet: In vitro footprinting of the mutant VEGF promoter region with DNase I and S1 nuclease. Autoradiograms showing DNase I (lanes 1–3) and S1 (lanes 4–6) cleavage sites on the top strand of a supercoiled pGL3-VEGFM17 plasmid. This plasmid was incubated in the absence of salt (lanes 1 and 4) or in the presence of 100 mM KCl without (lanes 2 and 5) and with (lanes 3 and 6) 1 µM telomestatin at 37°C for 1 h before digesting with nucleases. Nuclease cleavage sites were mapped using linear amplification by PCR with 32 P-labeled gene-specific primers on mutant plasmid DNA pretreated with S1 nuclease or DNase I.

    Article Snippet: Plasmid DNA For in vitro footprinting of the VEGF promoter region, we used the supercoiled form of the luciferase reporter plasmid pGL3-VEGFP, which was constructed by ligating an 837 bp VEGF promoter region (−787 to +50 relative to the transcription initiation site) into the KpnI and NheI sites of pGL3-basic basic vector (Promega, Madison, WI), as described previously ( ).

    Techniques: In Vitro, Footprinting, Mutagenesis, Plasmid Preparation, Incubation, Amplification, Polymerase Chain Reaction, Labeling

    p300 overexpression increased MMP-1 promoter activity. A, Schematic structure of MMP-1 promoter constructs. B, HDFs were transiently transfected with a series of 5′ deletion hMMP1/luciferase reporter plasmids (MMP1-1959luc, MMP1-939luc, and MMP1-207luc) together with 200 ng of pCMV-wt-p300 plasmid or pCMV and pCMV-E1A as controls. The cells were subsequently UV-irradiated and incubated for 6 h. Cells were harvested and protein extracts were assayed for luciferase activity. Three independent transfections, each run in triplicate, were performed, and the results are expressed as the means±S.E.M. EMP, pGL3 vector; p300, p300 expression vector; E1A, E1A expression vector; * represent p

    Journal: PLoS ONE

    Article Title: The Role of p300 Histone Acetyltransferase in UV-Induced Histone Modifications and MMP-1 Gene Transcription

    doi: 10.1371/journal.pone.0004864

    Figure Lengend Snippet: p300 overexpression increased MMP-1 promoter activity. A, Schematic structure of MMP-1 promoter constructs. B, HDFs were transiently transfected with a series of 5′ deletion hMMP1/luciferase reporter plasmids (MMP1-1959luc, MMP1-939luc, and MMP1-207luc) together with 200 ng of pCMV-wt-p300 plasmid or pCMV and pCMV-E1A as controls. The cells were subsequently UV-irradiated and incubated for 6 h. Cells were harvested and protein extracts were assayed for luciferase activity. Three independent transfections, each run in triplicate, were performed, and the results are expressed as the means±S.E.M. EMP, pGL3 vector; p300, p300 expression vector; E1A, E1A expression vector; * represent p

    Article Snippet: Plasmid constructs, transient transfection, and luciferase reporter assay The human MMP-1 promoter/luciferase plasmids (MMP-1959luc, MMP1-939luc, MMP1-207luc, mt-MMP1-1959luc and wt-MMP1-1959luc) contained the firefly luciferase gene under the transcriptional control of the human MMP-1 promoter in the pGL3 basic reporter vector (Promega, Madison, WI, USA).

    Techniques: Over Expression, Activity Assay, Construct, Transfection, Luciferase, Plasmid Preparation, Irradiation, Incubation, Expressing

    Activation of KSHV promoters in response to hypoxia and CoCl 2 . Cells were transfected with 1.3 μg of a reporter plasmid encoding the Rta gene promoter (RtaP) (A), the ORF34 promoter (Orf 34P) (B), or the VEGF gene promoter (Vegf P) as a positive control (C). Two hundred nanograms of an internal control plasmid, pSV-β-gal, was cotransfected for normalization of transfection efficiency. After exposure of cells to normoxia (N) or hypoxia (H), in the presence or absence of CoCl 2 for 18 h, luciferase activity was determined and normalized to β-gal activity. Little activity of the pGL3-Basic reporter vector in normoxia or hypoxia was seen in the absence of inserted promoters. In an early representative experiment, for example, the pGL3-Basic reporter vector in normoxia, in hypoxia, and with the ORF34 promoter in normoxia produced 8,230, 7,800, and 90,430 relative light units, respectively. The β-gal activity from the pSV-β-gal internal control plasmid in this experiment yielded 0.148, 0.129, and 0.320 optical density unit, respectively, yielding normalized luciferase values of 5.56 × 10 4 , 6.04 × 10 4 , and 28.2 × 10 4 relative light units/optical density unit, respectively. For each promoter, the fold induction was calculated compared to the normalized value in normoxia alone (N), which was set at unity. All values represent the median of six separate experiments, each done in triplicate. Error bars denote the quartiles. *, P

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Contains Hypoxia Response Elements: Relevance to Lytic Induction by Hypoxia

    doi: 10.1128/JVI.77.12.6761-6768.2003

    Figure Lengend Snippet: Activation of KSHV promoters in response to hypoxia and CoCl 2 . Cells were transfected with 1.3 μg of a reporter plasmid encoding the Rta gene promoter (RtaP) (A), the ORF34 promoter (Orf 34P) (B), or the VEGF gene promoter (Vegf P) as a positive control (C). Two hundred nanograms of an internal control plasmid, pSV-β-gal, was cotransfected for normalization of transfection efficiency. After exposure of cells to normoxia (N) or hypoxia (H), in the presence or absence of CoCl 2 for 18 h, luciferase activity was determined and normalized to β-gal activity. Little activity of the pGL3-Basic reporter vector in normoxia or hypoxia was seen in the absence of inserted promoters. In an early representative experiment, for example, the pGL3-Basic reporter vector in normoxia, in hypoxia, and with the ORF34 promoter in normoxia produced 8,230, 7,800, and 90,430 relative light units, respectively. The β-gal activity from the pSV-β-gal internal control plasmid in this experiment yielded 0.148, 0.129, and 0.320 optical density unit, respectively, yielding normalized luciferase values of 5.56 × 10 4 , 6.04 × 10 4 , and 28.2 × 10 4 relative light units/optical density unit, respectively. For each promoter, the fold induction was calculated compared to the normalized value in normoxia alone (N), which was set at unity. All values represent the median of six separate experiments, each done in triplicate. Error bars denote the quartiles. *, P

    Article Snippet: The PCR fragment was inserted into the corresponding sites of the reporter vector pGL3-Basic (Promega, Madison, Wis.) to generate the reporter RtaP.

    Techniques: Activation Assay, Transfection, Plasmid Preparation, Positive Control, Luciferase, Activity Assay, Produced

    Effect of four PPRs identified in Rbpms gene on transcriptional activity. A schematic shows the four PPRs (white and hatched boxes) that were identified using promoter recognition software with their locations relative to TSS. Expression constructs containing these regulatory regions upstream of the luciferase reporter gene were evaluated in HEK293T cells and the produced luciferase activity is presented relative to that generated from promoter-less control construct. pGL3-B was used as the negative control and arbitrarily set to 1.0. The luciferase reporter gene expression from the − 1691/+ 592 construct containing all four PPRs was ~ 13-fold higher than that obtained from the pGL3-B. Constructs − 1691/− 75, − 1691/− 1416, and − 344/− 75 that do not contain PPRs 3 and 4 produced minimal expression levels, whereas constructs − 344/+ 592 and − 72/+ 592 containing both PPRs 3 and 4 had approximately 23- and 30-fold increase in luciferase activity, respectively, compared to pGL3-B. The data shown are the mean with SD from three independent transfection experiments. Each experiment ran in triplicate. * P

    Journal: Molecular genetics and genomics : MGG

    Article Title: RNA-binding protein Rbpms is represented in human retinas by isoforms A and C and its transcriptional regulation involves Sp1-binding site

    doi: 10.1007/s00438-018-1423-8

    Figure Lengend Snippet: Effect of four PPRs identified in Rbpms gene on transcriptional activity. A schematic shows the four PPRs (white and hatched boxes) that were identified using promoter recognition software with their locations relative to TSS. Expression constructs containing these regulatory regions upstream of the luciferase reporter gene were evaluated in HEK293T cells and the produced luciferase activity is presented relative to that generated from promoter-less control construct. pGL3-B was used as the negative control and arbitrarily set to 1.0. The luciferase reporter gene expression from the − 1691/+ 592 construct containing all four PPRs was ~ 13-fold higher than that obtained from the pGL3-B. Constructs − 1691/− 75, − 1691/− 1416, and − 344/− 75 that do not contain PPRs 3 and 4 produced minimal expression levels, whereas constructs − 344/+ 592 and − 72/+ 592 containing both PPRs 3 and 4 had approximately 23- and 30-fold increase in luciferase activity, respectively, compared to pGL3-B. The data shown are the mean with SD from three independent transfection experiments. Each experiment ran in triplicate. * P

    Article Snippet: PCR fragments were cloned into pGL3-Basic (pGL3-B) vector (Promega, Madison, WI, USA) containing the luciferase reporter gene for subsequent luciferase reporter gene assays.

    Techniques: Activity Assay, Software, Expressing, Construct, Luciferase, Produced, Generated, Negative Control, Transfection

    Identification of vorinostat responsive elements in the IER3 promoter. A, K562 cells were transfected with pGL3-basic vector or a series of IER3 reporters as indicated plus β-galactosidase vector, and then treated with vorinostat (2 µM) or vehicle (Control) for 24 h and reporter activity measured. Results are shown as average fold luciferase/β-galactosidase induction versus control cells transfected with pGL3 ± S.D. from one representative of at least three independent assays done in triplicate using each reporter construct at least from two different clones. B, Scheme of wild type and mutated −124/+32 reporter constructs showing the −91 to −43 nucleotide sequence of IER3 promoter containing the putative TF binding sites present in the −91/−61 region (lane 1) and the mutated nucleotides for the different TF binding sites present in this region (lanes 2 to 6). The mutated nucleotides present in the mutant luciferase reporter plasmids for the different TF binding sites are boldfaced and underlined. +1, denotes transcription start site. C, K562 and HL60 cells were transfected with pGL3-basic vector or with wild type and mutated −124/+32 IER3 reporter constructs for the indicated TF plus β-galactosidase vector and the rest of the procedure was done as in (A).The results are average fold luciferase/β-galactosidase induction versus control cells transfected with pGL3 ± S.D. from one representative assay done in triplicate using mutated plasmids for the same putative TF binding site from different clones, of at least three independent assays performed in both K562 and HL60 cells. Data were analyzed using the ANOVA and the Tukey-Kramer multiple comparison test. *p

    Journal: PLoS ONE

    Article Title: Vorinostat Induces Apoptosis and Differentiation in Myeloid Malignancies: Genetic and Molecular Mechanisms

    doi: 10.1371/journal.pone.0053766

    Figure Lengend Snippet: Identification of vorinostat responsive elements in the IER3 promoter. A, K562 cells were transfected with pGL3-basic vector or a series of IER3 reporters as indicated plus β-galactosidase vector, and then treated with vorinostat (2 µM) or vehicle (Control) for 24 h and reporter activity measured. Results are shown as average fold luciferase/β-galactosidase induction versus control cells transfected with pGL3 ± S.D. from one representative of at least three independent assays done in triplicate using each reporter construct at least from two different clones. B, Scheme of wild type and mutated −124/+32 reporter constructs showing the −91 to −43 nucleotide sequence of IER3 promoter containing the putative TF binding sites present in the −91/−61 region (lane 1) and the mutated nucleotides for the different TF binding sites present in this region (lanes 2 to 6). The mutated nucleotides present in the mutant luciferase reporter plasmids for the different TF binding sites are boldfaced and underlined. +1, denotes transcription start site. C, K562 and HL60 cells were transfected with pGL3-basic vector or with wild type and mutated −124/+32 IER3 reporter constructs for the indicated TF plus β-galactosidase vector and the rest of the procedure was done as in (A).The results are average fold luciferase/β-galactosidase induction versus control cells transfected with pGL3 ± S.D. from one representative assay done in triplicate using mutated plasmids for the same putative TF binding site from different clones, of at least three independent assays performed in both K562 and HL60 cells. Data were analyzed using the ANOVA and the Tukey-Kramer multiple comparison test. *p

    Article Snippet: Reporter Assays K562 and HL60 cells (105 cells/ml well of 24-well plate) were transiently co-transfected with 400 ng indicated wild-type and mutated reporter constructs or with pGL3-basic control vector (Promega) plus 25 ng β-galactosidase reporter control expression vector (Promega) using 4 µl lipofectamine 2000 (Life Technologies) as per manufactureŕs protocol.

    Techniques: Transfection, Plasmid Preparation, Activity Assay, Luciferase, Construct, Clone Assay, Sequencing, Binding Assay, Mutagenesis

    Reporter analysis of novel p53-bound genes. PCR-amplified gene promoters containing peak regions were cloned into a promoterless pGL3 basic vector. p53-null H1299 cells were transiently cotransfected with a p53 expression plasmid (pCMV-p53) and the reporter

    Journal: Physiological Genomics

    Article Title: Genome-wide analysis of the p53 gene regulatory network in the developing mouse kidney

    doi: 10.1152/physiolgenomics.00113.2013

    Figure Lengend Snippet: Reporter analysis of novel p53-bound genes. PCR-amplified gene promoters containing peak regions were cloned into a promoterless pGL3 basic vector. p53-null H1299 cells were transiently cotransfected with a p53 expression plasmid (pCMV-p53) and the reporter

    Article Snippet: A modified pGL3 basic vector was generated by removing the Sma I site from the multicloning site and used in all cloning experiments, because of a reported effect ( ) that the Sma I site is able to interact with p53 and induces luciferase expression of the original pGL3 basic vector (Promega).

    Techniques: Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Expressing

    Regulation of HEY1 promoter activity by NFI. U251 GBM cells were transfected with 10 nM siRNAs, including control (scrambled), NFIA, NFIB, NFIC, NFIX, or combinations of NFI siRNAs. Where indicated (2×), cells underwent two rounds of siRNA transfection. (A) NFIA , NFIB , NFIC , NFIX , and (B) HEY1 mRNA expression was analyzed by qPCR. GAPDH was used as an endogenous control. Similar data were obtained in two separate experiments. (C) U251 GBM cells were transfected with 10 nM siRNAs, including control (scrambled), NFIA, NFIB, NFIC, NFIX, or combinations of NFI siRNAs, followed 24 hours later by transfection with pGL3/HEY1. Cells were harvested 60 hours later, and luciferase activity was quantified. Changes in relative light units (RLU) are relative to RLU obtained in U251 GBM cells transfected with control (scrambled) siRNA and pGL3/HEY1. The data are from three experiments. SEM is indicated by error bars. Statistical significance, determined using the unpaired t test, is indicated by * ( P

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Nuclear Factor I Represses the Notch Effector HEY1 in Glioblastoma

    doi: 10.1016/j.neo.2018.08.007

    Figure Lengend Snippet: Regulation of HEY1 promoter activity by NFI. U251 GBM cells were transfected with 10 nM siRNAs, including control (scrambled), NFIA, NFIB, NFIC, NFIX, or combinations of NFI siRNAs. Where indicated (2×), cells underwent two rounds of siRNA transfection. (A) NFIA , NFIB , NFIC , NFIX , and (B) HEY1 mRNA expression was analyzed by qPCR. GAPDH was used as an endogenous control. Similar data were obtained in two separate experiments. (C) U251 GBM cells were transfected with 10 nM siRNAs, including control (scrambled), NFIA, NFIB, NFIC, NFIX, or combinations of NFI siRNAs, followed 24 hours later by transfection with pGL3/HEY1. Cells were harvested 60 hours later, and luciferase activity was quantified. Changes in relative light units (RLU) are relative to RLU obtained in U251 GBM cells transfected with control (scrambled) siRNA and pGL3/HEY1. The data are from three experiments. SEM is indicated by error bars. Statistical significance, determined using the unpaired t test, is indicated by * ( P

    Article Snippet: The luciferase reporter gene construct was prepared by inserting the 5′ HEY1 flanking DNA from −913 bp to +15 bp into the pGL3-Basic vector (Promega).

    Techniques: Activity Assay, Transfection, Expressing, Real-time Polymerase Chain Reaction, Luciferase

    Mutation of NF-κB or AP-1 binding site attenuated LMP1-increased iE κ activity . (A) Schematic diagram of mutant iE κ constructs were shown. The expansion for NF-κB or AP-1 binding site gave its wild-type sequence and the nucleotides replaced by mutations were underlined. Arrows indicated nucleotides introduced by mutations. (B) Comparison of the activities of iE κ in human nasopharyngeal carcinoma cell lines. Transient transfected the constructs carrying wild-type NF-κB and AP-1 sequences (pα-iE κ wt), mutant NF-κB sequence (pα-iE κ -mtκB), mutant AP-1 sequence (pα-iE κ -mtAP-1), pGL3-α or pGL3-Basic into HNE2 and HNE2-LMP1 cells and luciferase reporter assays were performed as described in Materials and methods. The relative luciferase activity normalized to the value of the internal control plasmid pRL-SV40 activity. Results were expressed as fold induction of pGL3-Basic activity, which was assigned a value of 1. The data represent the mean ± SD of the three independent experiments performed in triplicate. Statistical significance: #P

    Journal: Molecular Cancer

    Article Title: LMP1-augmented kappa intron enhancer activity contributes to upregulation expression of Ig kappa light chain via NF-kappaB and AP-1 pathways in nasopharyngeal carcinoma cells

    doi: 10.1186/1476-4598-8-92

    Figure Lengend Snippet: Mutation of NF-κB or AP-1 binding site attenuated LMP1-increased iE κ activity . (A) Schematic diagram of mutant iE κ constructs were shown. The expansion for NF-κB or AP-1 binding site gave its wild-type sequence and the nucleotides replaced by mutations were underlined. Arrows indicated nucleotides introduced by mutations. (B) Comparison of the activities of iE κ in human nasopharyngeal carcinoma cell lines. Transient transfected the constructs carrying wild-type NF-κB and AP-1 sequences (pα-iE κ wt), mutant NF-κB sequence (pα-iE κ -mtκB), mutant AP-1 sequence (pα-iE κ -mtAP-1), pGL3-α or pGL3-Basic into HNE2 and HNE2-LMP1 cells and luciferase reporter assays were performed as described in Materials and methods. The relative luciferase activity normalized to the value of the internal control plasmid pRL-SV40 activity. Results were expressed as fold induction of pGL3-Basic activity, which was assigned a value of 1. The data represent the mean ± SD of the three independent experiments performed in triplicate. Statistical significance: #P

    Article Snippet: Luciferase reporter assays The pGL3-α, pα-iEκ wt, pα-iEκ -mtκB and pα-iEκ -mtAP-1 luciferase reporter plasmids described above were used in conjunction with the control pGL3-Basic vector (Promega) and the internal control plasmid pRL-SV40 (Promega).

    Techniques: Mutagenesis, Binding Assay, Activity Assay, Construct, Sequencing, Transfection, Luciferase, Plasmid Preparation

    Inhibitors and dominant negative mutants targeting for NF-κB and AP-1 pathways attenuated LMP1-augmented human iE κ activities . (A) Bay11-7082 and SP600125 inhibited the activities of iE κ induced by stable expression LMP1. HNE2 and HNE2-LMP1 cells were transfected with pα-iE κ wt, pGL3-α or pGL3-Basic vector, and pRL-SV40 as an internal control for transfection efficiency. 24hr after transfection, cells were treated with Bay11-7082 (20 μM), SP600125 (20 μM) or 0.1% DMSO for 12hr. Cells were harvested at 36 h after transfection and subjected to the luciferase assay. Statistical significance: * P

    Journal: Molecular Cancer

    Article Title: LMP1-augmented kappa intron enhancer activity contributes to upregulation expression of Ig kappa light chain via NF-kappaB and AP-1 pathways in nasopharyngeal carcinoma cells

    doi: 10.1186/1476-4598-8-92

    Figure Lengend Snippet: Inhibitors and dominant negative mutants targeting for NF-κB and AP-1 pathways attenuated LMP1-augmented human iE κ activities . (A) Bay11-7082 and SP600125 inhibited the activities of iE κ induced by stable expression LMP1. HNE2 and HNE2-LMP1 cells were transfected with pα-iE κ wt, pGL3-α or pGL3-Basic vector, and pRL-SV40 as an internal control for transfection efficiency. 24hr after transfection, cells were treated with Bay11-7082 (20 μM), SP600125 (20 μM) or 0.1% DMSO for 12hr. Cells were harvested at 36 h after transfection and subjected to the luciferase assay. Statistical significance: * P

    Article Snippet: Luciferase reporter assays The pGL3-α, pα-iEκ wt, pα-iEκ -mtκB and pα-iEκ -mtAP-1 luciferase reporter plasmids described above were used in conjunction with the control pGL3-Basic vector (Promega) and the internal control plasmid pRL-SV40 (Promega).

    Techniques: Dominant Negative Mutation, Expressing, Transfection, Plasmid Preparation, Luciferase

    Activation of the iE κ enhancer and enhancement of the iE κ activity by LMP1 in human nasopharyngeal carcinoma cells . (A) Schematic diagram of human iE κ -containing DNA fragment used in these experiments. Position of the iE κ , the NF-κB and the AP-1 binding sites were shown. For simplicity, other protein-binding sites in the iE κ were not shown. (B) Insertion sites for the definite DNA fragment into the pGL3-α plasmid which contains the human immunoglobulin Iα promoter and the firefly luciferase reporter gene. (C) Comparison of the activities of iE κ in human nasopharyngeal carcinoma cell lines. Transient transfected the pα-iE κ wt construct, pGL3-α or pGL3-Basic vector into HNE2 and HNE2-LMP1 cells and luciferase reporter assays were performed as described in Materials and methods. The relative luciferase activity normalized to the value of the internal control plasmid pRL-SV40 activity. Results were expressed as fold induction of pGL3-Basic activity, which was assigned a value of 1. The data represent the mean ± SD of the three independent experiments performed in triplicate. Statistical significance: # P

    Journal: Molecular Cancer

    Article Title: LMP1-augmented kappa intron enhancer activity contributes to upregulation expression of Ig kappa light chain via NF-kappaB and AP-1 pathways in nasopharyngeal carcinoma cells

    doi: 10.1186/1476-4598-8-92

    Figure Lengend Snippet: Activation of the iE κ enhancer and enhancement of the iE κ activity by LMP1 in human nasopharyngeal carcinoma cells . (A) Schematic diagram of human iE κ -containing DNA fragment used in these experiments. Position of the iE κ , the NF-κB and the AP-1 binding sites were shown. For simplicity, other protein-binding sites in the iE κ were not shown. (B) Insertion sites for the definite DNA fragment into the pGL3-α plasmid which contains the human immunoglobulin Iα promoter and the firefly luciferase reporter gene. (C) Comparison of the activities of iE κ in human nasopharyngeal carcinoma cell lines. Transient transfected the pα-iE κ wt construct, pGL3-α or pGL3-Basic vector into HNE2 and HNE2-LMP1 cells and luciferase reporter assays were performed as described in Materials and methods. The relative luciferase activity normalized to the value of the internal control plasmid pRL-SV40 activity. Results were expressed as fold induction of pGL3-Basic activity, which was assigned a value of 1. The data represent the mean ± SD of the three independent experiments performed in triplicate. Statistical significance: # P

    Article Snippet: Luciferase reporter assays The pGL3-α, pα-iEκ wt, pα-iEκ -mtκB and pα-iEκ -mtAP-1 luciferase reporter plasmids described above were used in conjunction with the control pGL3-Basic vector (Promega) and the internal control plasmid pRL-SV40 (Promega).

    Techniques: Activation Assay, Activity Assay, Binding Assay, Protein Binding, Plasmid Preparation, Luciferase, Transfection, Construct

    MALAT1 epigenetically regulates EF1A1. A. p EEF1A1 -luciferase assay. The EEF1A1 promoter (p EEF1A1 ) sequence was cloned into the upstream of luciferase gene. Luciferase reporter assay was performed in CTL group, shNC group and shMALAT1 group by co-transfecting respectively with pGL3-Basic vector or luciferase reporter vector. Data were adjusted over the negative control (CTL) and were represented as means ± SD. ** P

    Journal: American Journal of Cancer Research

    Article Title: Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

    doi:

    Figure Lengend Snippet: MALAT1 epigenetically regulates EF1A1. A. p EEF1A1 -luciferase assay. The EEF1A1 promoter (p EEF1A1 ) sequence was cloned into the upstream of luciferase gene. Luciferase reporter assay was performed in CTL group, shNC group and shMALAT1 group by co-transfecting respectively with pGL3-Basic vector or luciferase reporter vector. Data were adjusted over the negative control (CTL) and were represented as means ± SD. ** P

    Article Snippet: To construct luciferase reporter vectors, the EEF1A1 promoter was amplified from genomic DNA by PCR and subcloned into pGL3-Basic vector (Addgene, #E1751) using KpnI/XhoI (Thermo Fisher Scientific, CA).

    Techniques: Luciferase, Sequencing, Clone Assay, Reporter Assay, CTL Assay, Plasmid Preparation, Negative Control

    PLK1 is a target of miR-100. (A) Western blot analysis of PLK1 protein expression in SKOV-3 cells transfected with pre-miR-100 (pre-miR-NC) or anti-miR-100 (anti-miR-NC). (B) PLK1 3′-UTR and corresponding fragments were inserted into the region immediately downstream of the luciferase gene in pGL3-Basic vector and validated by DNA sequencing. The sequences of the predicted miR-100 binding sites within the PLK1 3′-UTR, including wild-type UTR or UTR segments containing mutant binding site are shown. (C) Detection of luciferase activity. For luciferase reporter assays in 6-well plates, 1-mg luciferase reporter plasmid containing either wild-type (wt) or mutant (mut) PLK1 3′-UTR, and 200 pmol of pre-miR-100, anti-miR-100, pre-miR-NC or anti-miR-100 were transfected. The parental luciferase plasmid was also transfected as a control. At 24 h after transfection, cells were assayed using the Luciferase Gene Reporter Assay kit. The data are presented as the mean ± SD of three experiments. * P

    Journal: Oncology Reports

    Article Title: Prognostic implications of microRNA-100 and its functional roles in human epithelial ovarian cancer

    doi: 10.3892/or.2012.1625

    Figure Lengend Snippet: PLK1 is a target of miR-100. (A) Western blot analysis of PLK1 protein expression in SKOV-3 cells transfected with pre-miR-100 (pre-miR-NC) or anti-miR-100 (anti-miR-NC). (B) PLK1 3′-UTR and corresponding fragments were inserted into the region immediately downstream of the luciferase gene in pGL3-Basic vector and validated by DNA sequencing. The sequences of the predicted miR-100 binding sites within the PLK1 3′-UTR, including wild-type UTR or UTR segments containing mutant binding site are shown. (C) Detection of luciferase activity. For luciferase reporter assays in 6-well plates, 1-mg luciferase reporter plasmid containing either wild-type (wt) or mutant (mut) PLK1 3′-UTR, and 200 pmol of pre-miR-100, anti-miR-100, pre-miR-NC or anti-miR-100 were transfected. The parental luciferase plasmid was also transfected as a control. At 24 h after transfection, cells were assayed using the Luciferase Gene Reporter Assay kit. The data are presented as the mean ± SD of three experiments. * P

    Article Snippet: Luciferase reporter assays The 3′-UTR of human PLK1 (GenBank Accession no. NM_005030) was amplified from human genomic DNA and individually cloned into the pGL3-Basic vector (Ambion) by directional cloning.

    Techniques: Western Blot, Expressing, Transfection, Luciferase, Plasmid Preparation, DNA Sequencing, Binding Assay, Mutagenesis, Activity Assay, Reporter Assay

    Foxo4 negatively regulate MC5R transcription in αMSH inhibited inflammation in mice adipocytes (A) Fragments of MC5R promoter fused to a luciferase reporter plasmid or PGL3-basic (control) were co-transfected into cells together with Renlilla plasmid and pAd-Foxo4 (n=3). Luciferase activity was corrected for Renilla luciferase activity and normalized to control activity (n=3). (B) Chromatin immunoprecipitation (ChIP) analysis of Foxo4 and MC5R interaction. (C, D) After pAd-Foxo4 together with αMSH or pc-MC5R in LPS/saline treatment, MC5R mRNA level was determined in adipocytes (n=3). (E) When adipocytes were treated with pAd-Foxo4 and αMSH in LPS/saline treatment, mRNA levels of Foxo4 , IL-6 , TNFα , MCP-1 and Leptin were analyzed (n=3). Values are means ± SD. vs. control group, * P

    Journal: Oncotarget

    Article Title: αMSH inhibits adipose inflammation via reducing FoxOs transcription and blocking Akt/JNK pathway in mice

    doi: 10.18632/oncotarget.17465

    Figure Lengend Snippet: Foxo4 negatively regulate MC5R transcription in αMSH inhibited inflammation in mice adipocytes (A) Fragments of MC5R promoter fused to a luciferase reporter plasmid or PGL3-basic (control) were co-transfected into cells together with Renlilla plasmid and pAd-Foxo4 (n=3). Luciferase activity was corrected for Renilla luciferase activity and normalized to control activity (n=3). (B) Chromatin immunoprecipitation (ChIP) analysis of Foxo4 and MC5R interaction. (C, D) After pAd-Foxo4 together with αMSH or pc-MC5R in LPS/saline treatment, MC5R mRNA level was determined in adipocytes (n=3). (E) When adipocytes were treated with pAd-Foxo4 and αMSH in LPS/saline treatment, mRNA levels of Foxo4 , IL-6 , TNFα , MCP-1 and Leptin were analyzed (n=3). Values are means ± SD. vs. control group, * P

    Article Snippet: Luciferase reporter assays Four fragments containing MC5R-5’ sequences from -1200 to -210 relative to the transcription initiation site were sub-cloned into pGL3-basic vector (Takara, China).

    Techniques: Mouse Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Chromatin Immunoprecipitation

    Analysis of transcriptional activity of putative intron locus promoter elements. (A) Diagram of genomic location of the viral sequences cloned into pGL3-reporter plasmids. (B) pGL3-reporter constructs were co-transfected into mouse fibroblasts and luciferase induction was assayed 24 h p.i. following either mock co-infection, (C) UV inactivated co-infection, or MCMV co-infection (MOI = 0.05).

    Journal: Virology Journal

    Article Title: Molecular investigation of the 7.2 kb RNA of murine cytomegalovirus

    doi: 10.1186/1743-422X-10-348

    Figure Lengend Snippet: Analysis of transcriptional activity of putative intron locus promoter elements. (A) Diagram of genomic location of the viral sequences cloned into pGL3-reporter plasmids. (B) pGL3-reporter constructs were co-transfected into mouse fibroblasts and luciferase induction was assayed 24 h p.i. following either mock co-infection, (C) UV inactivated co-infection, or MCMV co-infection (MOI = 0.05).

    Article Snippet: After insertion into the pGEM-T-Easy plasmid, the inserts were digested from the plasmid using the flanking EcoRI sequences then subcloned into the pGL3 Basic vector at a newly generated EcoRI site using the site directed mutatgenesis kit (Stratagene).

    Techniques: Activity Assay, Clone Assay, Construct, Transfection, Luciferase, Infection

    USF1 transcriptionally elevates TGF-β1 expression in breast cancer. a The relative luciferase activities were analyzed after co-transfection with various TGF-β1 promoter reporters or the pGL3-basic vector and USF1 or control vector. b Schematic of the location of E-box motif bound by USF1 in TGF-β1 promoter region. c Wild-type (WT) or mutant (Mut) TGF-β1 luciferase reporter vector was co-transfected with USF1 or control vector into MCF-7 and MDA-MB-231 cells, after 48 h of co-transfection, the luciferase activities were tested. d ChIP-qPCR analysis of USF1 binding to the TGF-β1 promoter region in MCF-7 and MDA-MB-231 cells. RNA polymerase II (RNAPII) was used as a positive control. e qRT-PCR (left) and immunoblot (right) analysis of TGF-β1 mRNA expression in USF1-overexpressing MCF-7 cells and USF1 knockdown MDA-MB-231 cells. β-actin was used as a loading control. f The correlation between USF1 and TGF-β1 expression in breast cancer tissues from TCGA database was analyzed by Spearman correlation coefficients ( r = 0.223, n = 1109, p

    Journal: Molecular Cancer

    Article Title: The pro-metastasis effect of circANKS1B in breast cancer

    doi: 10.1186/s12943-018-0914-x

    Figure Lengend Snippet: USF1 transcriptionally elevates TGF-β1 expression in breast cancer. a The relative luciferase activities were analyzed after co-transfection with various TGF-β1 promoter reporters or the pGL3-basic vector and USF1 or control vector. b Schematic of the location of E-box motif bound by USF1 in TGF-β1 promoter region. c Wild-type (WT) or mutant (Mut) TGF-β1 luciferase reporter vector was co-transfected with USF1 or control vector into MCF-7 and MDA-MB-231 cells, after 48 h of co-transfection, the luciferase activities were tested. d ChIP-qPCR analysis of USF1 binding to the TGF-β1 promoter region in MCF-7 and MDA-MB-231 cells. RNA polymerase II (RNAPII) was used as a positive control. e qRT-PCR (left) and immunoblot (right) analysis of TGF-β1 mRNA expression in USF1-overexpressing MCF-7 cells and USF1 knockdown MDA-MB-231 cells. β-actin was used as a loading control. f The correlation between USF1 and TGF-β1 expression in breast cancer tissues from TCGA database was analyzed by Spearman correlation coefficients ( r = 0.223, n = 1109, p

    Article Snippet: For the promoter of TGF-β1 luciferase reporter assay, the wild-type or mutant full-length TGF-β1 promoter construct and six truncation constructs were respectively inserted into pGL3-basic vectors (Promega) between Sacl and Xhol restriction sites, and then cotransfected with USF1 overexpression vector and pRL-TK into breast cancer cells by Lipofectamine 2000.

    Techniques: Expressing, Luciferase, Cotransfection, Plasmid Preparation, Mutagenesis, Transfection, Multiple Displacement Amplification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Positive Control, Quantitative RT-PCR

    Functional analysis of the MTG16 promoter by sequential 5'-deletion . The following 5'deleted reporter constructs were generated from the pGL3 -820-57 (-820) construct and examined after expression in erythroid HEL/TF-1, erythroid/megakaryocytic UT-7 and megakaryocytic MEG-01cells: pGL3 -668-57 (-668), pGL3 -512-57 (-512), pGL3 -359-57 (-359), pGL3 -339-57 (-339), pGL3 -299-57 (-299) and pGL3 -219-57 (-219). Promoterless pGL3/basic and the pGL3/SV40-promoter are used as negative and positive control, respectively. The luciferase activity was normalized against pGL3/SV40- promoter activity. Results are shown for 3 to 5 separate transfections; bars represent the mean and the error bars show SEM.

    Journal: BMC Molecular Biology

    Article Title: The leukemia associated nuclear corepressor ETO homologue genes MTG16 and MTGR1 are regulated differently in hematopoietic cells

    doi: 10.1186/1471-2199-13-11

    Figure Lengend Snippet: Functional analysis of the MTG16 promoter by sequential 5'-deletion . The following 5'deleted reporter constructs were generated from the pGL3 -820-57 (-820) construct and examined after expression in erythroid HEL/TF-1, erythroid/megakaryocytic UT-7 and megakaryocytic MEG-01cells: pGL3 -668-57 (-668), pGL3 -512-57 (-512), pGL3 -359-57 (-359), pGL3 -339-57 (-339), pGL3 -299-57 (-299) and pGL3 -219-57 (-219). Promoterless pGL3/basic and the pGL3/SV40-promoter are used as negative and positive control, respectively. The luciferase activity was normalized against pGL3/SV40- promoter activity. Results are shown for 3 to 5 separate transfections; bars represent the mean and the error bars show SEM.

    Article Snippet: The cloned region was inserted upstream of the luciferase reporter gene in promoterless pGL3/Basic vector to give the pGL3/-989 to +57 plasmid, which was transfected into hematopoietic cell lines and luciferase activity was determined and normalized as described in Methods.

    Techniques: Functional Assay, Construct, Generated, Expressing, Positive Control, Luciferase, Activity Assay, Transfection