pgl3-basic Search Results


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  • 99
    Thermo Fisher pgl3 basic vector
    Pgl3 Basic Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 957 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Promega pgl3 basic vector
    SP110-mediated down-regulation of NF-κB activity is independent of GSK3β. HEK293T cells were co-transfected with <t>pGL3-TNFα</t> promoter-F.Luc, pSV40-R.Luc, and the indicated constructs, and cultured in the absence or presence of GSK3β inhibitor. The relative Luc values for the TNFα promoter were then measured 2 days after co-transfection. The empty vector negative control is shown at the top. The data are presented as the mean ± SD. Statistical significance of the difference between two sample groups was calculated using a two-tailed unpaired t -test. ** P
    Pgl3 Basic Vector, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 28056 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega pgl3 basic
    PXR repression via SMRT. A, transient transfection assays were performed by transfecting <t>pGL3/CYP24A1-3</t> kb with various combinations of pcDNA3.1/mVDR, pCMX/hSMRT, and pCR3/hPXR into Huh7 cells. After 24 h, cells were treated with DMSO, VD 3 (10 nM), and/or Rif (10 μM) for an additional 24 h before being harvested for luciferase assays. Relative luciferase activities were expressed relative to the activity of the DMSO-treated cells transfected with the empty -3-kb reporter plasmid. Bars, mean ± S.D. B, Huh7 cells were transfected with or without pcDNA3.1/mVDR and pCR3/hPXR. After 24 h, transfected cells were treated with DMSO, VD 3 (10 nM), and/or rifampicin (10 μM) for an additional 2 h before being harvested for ChIP assays. Cross-linked chromatin-protein complexes were immunoprecipitated with anti-VDR (αVDR), anti-PXR (αPXR), anti-SMRT (αSMRT), or normal mouse IgG (αIgG). By scanning three independent assays, the relative intensities of the bands were calculated based on the inputs: in the VDR binding, 0.8 ± 0.1, 3.3 ± 0.7, 3.7 ± 1.1, and 3.8 ± 0.9% for VDR plus DMSO, VDR plus VD 3 , VDR plus PXR and VD 3 , and VDR plus PXR, VD 3 , and Rif, respectively; in the PXR binding, 0.2 ± 0.2, 0.3 ± 0.3, 2.7 ± 0.8, and 5.2 ± 1.2% for VDR plus DMSO, VDR plus VD 3 , VDR plus PXR and VD 3 , and VDR plus PXR, VD 3 , and Rif, respectively; in the SMRT binding, 4.7 ± 1.0, 0.0 ± 0.1, 1.3 ± 0.6, and 4.0 ± 0.4% for VDR plus DMSO, VDR plus VD 3 , VDR plus PXR and VD 3 , and VDR plus PXR, VD 3 , and Rif, respectively.
    Pgl3 Basic, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 12584 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega pgl3 basic luciferase reporter vector
    Mutated CCAAT box and SRE reduce transcriptional activity of the zebrafish fads2 promoter. Single or multiple site mutations ( X ) of SRE ( ), CATp (●) and CATd (○) were introduced into the fads2 −244 plasmid. ZFL cells were transiently co-transfected with various mutated fads2 −244 plasmids, nSrebp2 expression plasmid, together with Renilla luciferase reference plasmid pRL-SV40. Luciferase coding region is indicated by shaded box and activity of the fads2 promoter in ZFL cells is expressed as normalised luciferase activity (to Renilla luciferase activity) relative to the empty <t>pGL3-Basic</t> plasmid. Values are means ± SD of three independent experiments (n = 3) (right panel). Groups indicated with different letters are significantly different (Tukey’s test; P
    Pgl3 Basic Luciferase Reporter Vector, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 3609 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 3609 article reviews
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    99
    Promega pgl3 basic luciferase vector
    Mutated CCAAT box and SRE reduce transcriptional activity of the zebrafish fads2 promoter. Single or multiple site mutations ( X ) of SRE ( ), CATp (●) and CATd (○) were introduced into the fads2 −244 plasmid. ZFL cells were transiently co-transfected with various mutated fads2 −244 plasmids, nSrebp2 expression plasmid, together with Renilla luciferase reference plasmid pRL-SV40. Luciferase coding region is indicated by shaded box and activity of the fads2 promoter in ZFL cells is expressed as normalised luciferase activity (to Renilla luciferase activity) relative to the empty <t>pGL3-Basic</t> plasmid. Values are means ± SD of three independent experiments (n = 3) (right panel). Groups indicated with different letters are significantly different (Tukey’s test; P
    Pgl3 Basic Luciferase Vector, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 2072 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega pgl3basic
    Mutation analysis of GATA and ETS sites in the −252/+38 region of the promoter. A . Right; H5V endothelial cells (black bars) and L929 fibroblasts (white bars) were transfected with 80 fmoles of <t>pGL3basic</t> (Ctrl), −8409/+38Luc or a −8409/+38Luc mutant where the GATA (GATAmt) site had been mutated, and with 54 fmoles pCH110 normalization vector. After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Mutation of the GATA site (left, blackened circle) in the originally most active −8409/+38Luc vector resulted in a strong decrease of activity of the reporter in endothelial cells and the endothelial/fibroblasts ratio was decreased by half when compared to −8409/+38Luc. The experiment is representative of a set of three experiments performed in similar conditions. B . Right; H5V (black bars) and L929 (white bars) cells where transfected with 80 fmoles of −8409/+38Luc or the indicated mutated versions in which the predicted EBS (left, numbered circles) were individually mutated by site-directed mutagenesis (left, blackened numbered circles). After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Left; scaled schematic representation of the −8409/+38 wild-type region (top). The location of the GATA (circled G) and EBS (circled numbers) sites is represented to scale along the zoomed region D of the mutants. Mutated sites are represented as blackened circles. Names of the constructs are given according to the cloned 5′ and 3′ end positions relative to the exon-1b transcription start, Luc; luciferase. Results are presented according to Figure 3A . The EBS6/EBS7 (−123/−120) tandem is located in scanD-7 of the linker scanning analysis, EBS5 (−92) is located in scanD-8 and EBS4 (−80), EBS3 (−71), EBS2 (−49) and EBS1 (+28) are all located closer to the transcription start. The experiment is representative of a set of three performed in similar conditions. *** p
    Pgl3basic, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 1160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega pgl3 basic reporter vector
    Mutation analysis of GATA and ETS sites in the −252/+38 region of the promoter. A . Right; H5V endothelial cells (black bars) and L929 fibroblasts (white bars) were transfected with 80 fmoles of <t>pGL3basic</t> (Ctrl), −8409/+38Luc or a −8409/+38Luc mutant where the GATA (GATAmt) site had been mutated, and with 54 fmoles pCH110 normalization vector. After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Mutation of the GATA site (left, blackened circle) in the originally most active −8409/+38Luc vector resulted in a strong decrease of activity of the reporter in endothelial cells and the endothelial/fibroblasts ratio was decreased by half when compared to −8409/+38Luc. The experiment is representative of a set of three experiments performed in similar conditions. B . Right; H5V (black bars) and L929 (white bars) cells where transfected with 80 fmoles of −8409/+38Luc or the indicated mutated versions in which the predicted EBS (left, numbered circles) were individually mutated by site-directed mutagenesis (left, blackened numbered circles). After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Left; scaled schematic representation of the −8409/+38 wild-type region (top). The location of the GATA (circled G) and EBS (circled numbers) sites is represented to scale along the zoomed region D of the mutants. Mutated sites are represented as blackened circles. Names of the constructs are given according to the cloned 5′ and 3′ end positions relative to the exon-1b transcription start, Luc; luciferase. Results are presented according to Figure 3A . The EBS6/EBS7 (−123/−120) tandem is located in scanD-7 of the linker scanning analysis, EBS5 (−92) is located in scanD-8 and EBS4 (−80), EBS3 (−71), EBS2 (−49) and EBS1 (+28) are all located closer to the transcription start. The experiment is representative of a set of three performed in similar conditions. *** p
    Pgl3 Basic Reporter Vector, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 957 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgl3 basic reporter vector/product/Promega
    Average 99 stars, based on 957 article reviews
    Price from $9.99 to $1999.99
    pgl3 basic reporter vector - by Bioz Stars, 2020-11
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    99
    Promega pgl3basic vector
    Mutation analysis of GATA and ETS sites in the −252/+38 region of the promoter. A . Right; H5V endothelial cells (black bars) and L929 fibroblasts (white bars) were transfected with 80 fmoles of <t>pGL3basic</t> (Ctrl), −8409/+38Luc or a −8409/+38Luc mutant where the GATA (GATAmt) site had been mutated, and with 54 fmoles pCH110 normalization vector. After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Mutation of the GATA site (left, blackened circle) in the originally most active −8409/+38Luc vector resulted in a strong decrease of activity of the reporter in endothelial cells and the endothelial/fibroblasts ratio was decreased by half when compared to −8409/+38Luc. The experiment is representative of a set of three experiments performed in similar conditions. B . Right; H5V (black bars) and L929 (white bars) cells where transfected with 80 fmoles of −8409/+38Luc or the indicated mutated versions in which the predicted EBS (left, numbered circles) were individually mutated by site-directed mutagenesis (left, blackened numbered circles). After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Left; scaled schematic representation of the −8409/+38 wild-type region (top). The location of the GATA (circled G) and EBS (circled numbers) sites is represented to scale along the zoomed region D of the mutants. Mutated sites are represented as blackened circles. Names of the constructs are given according to the cloned 5′ and 3′ end positions relative to the exon-1b transcription start, Luc; luciferase. Results are presented according to Figure 3A . The EBS6/EBS7 (−123/−120) tandem is located in scanD-7 of the linker scanning analysis, EBS5 (−92) is located in scanD-8 and EBS4 (−80), EBS3 (−71), EBS2 (−49) and EBS1 (+28) are all located closer to the transcription start. The experiment is representative of a set of three performed in similar conditions. *** p
    Pgl3basic Vector, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 739 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgl3basic vector/product/Promega
    Average 99 stars, based on 739 article reviews
    Price from $9.99 to $1999.99
    pgl3basic vector - by Bioz Stars, 2020-11
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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
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    Average 90 stars, based on 758 article reviews
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    89
    Promega pgl3 basic firefly luciferase reporter 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.
    Pgl3 Basic Firefly Luciferase Reporter Vector, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 295 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Promega pgl3 basic luciferase reporter
    Effects of compounds 1 – 9 (10 μM) on the p16INK4A and GLB1 transcription in the human dermal fibroblasts. Human dermal fibroblasts were transiently co-transfected with the <t>pGL3-p16INK4A</t> ( A ) or pGL3-GLB1 ( B ) promoter with β-galactosidase as a transfection control. Cells were treated overnight with the compounds 1 – 9 (10 μM). The luciferase activity was determined as the ratio of the firefly/Renilla luciferase activities. The activities of the p16Ink4A promoter and the GLB1 promoter in the presence of the compounds 1 – 9 relative to that in the absence of the compounds 1 – 9 are shown. * p
    Pgl3 Basic Luciferase Reporter, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 89 stars, based on 220 article reviews
    Price from $9.99 to $1999.99
    pgl3 basic luciferase reporter - by Bioz Stars, 2020-11
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    Image Search Results


    SP110-mediated down-regulation of NF-κB activity is independent of GSK3β. HEK293T cells were co-transfected with pGL3-TNFα promoter-F.Luc, pSV40-R.Luc, and the indicated constructs, and cultured in the absence or presence of GSK3β inhibitor. The relative Luc values for the TNFα promoter were then measured 2 days after co-transfection. The empty vector negative control is shown at the top. The data are presented as the mean ± SD. Statistical significance of the difference between two sample groups was calculated using a two-tailed unpaired t -test. ** P

    Journal: Journal of Biomedical Science

    Article Title: Functional domains of SP110 that modulate its transcriptional regulatory function and cellular translocation

    doi: 10.1186/s12929-018-0434-4

    Figure Lengend Snippet: SP110-mediated down-regulation of NF-κB activity is independent of GSK3β. HEK293T cells were co-transfected with pGL3-TNFα promoter-F.Luc, pSV40-R.Luc, and the indicated constructs, and cultured in the absence or presence of GSK3β inhibitor. The relative Luc values for the TNFα promoter were then measured 2 days after co-transfection. The empty vector negative control is shown at the top. The data are presented as the mean ± SD. Statistical significance of the difference between two sample groups was calculated using a two-tailed unpaired t -test. ** P

    Article Snippet: For the TNFα promoter activity assay, an approximately 1.9 kb segment of the TNFα 5′-flanking region (− 1786 to + 175), which encompasses the transcriptional start site, was amplified by PCR and inserted into the pGL3-Basic vector (firefly luciferase; Promega) to generate the pGL3-TNFα promoter-F.Luc plasmid (firefly luciferase).

    Techniques: Activity Assay, Transfection, Construct, Cell Culture, Cotransfection, Plasmid Preparation, Negative Control, Two Tailed Test

    SP110 regulates NF-κB-driven transcription. a HEK293T cells were co-transfected with pGL3-p5 × κB-F.Luc, pSV40-R.Luc, and the indicated constructs, and the relative firefly (F.)/ Renilla (R.) luciferase (Luc) values for a basic promoter with 5 × κB sites were measured 2 days after co-transfection. The empty vector negative control is shown on the left. b HEK293T cells were co-transfected with pGL3-TNFα promoter-F.Luc, pSV40-R.Luc, and the indicated constructs, and the relative Luc values for the TNFα promoter were measured 2 days after co-transfection. The empty vector negative control is shown on the left. c and d HEK293T cells were co-transfected with pGL3-TNFα promoter-F.Luc, pSV40-R.Luc, and the indicated constructs, and the relative Luc values for the TNFα promoter were measured 2 days after co-transfection. The empty vector negative control is shown on the top. In ( a and b ), a, b, and c correspond to SP110a, SP110b, and SP110c, respectively. In ( c and d ), schematic representation of SP110 deletion mutants is shown on the left. The data are presented as the mean ± SD. Statistical significance of the difference between two sample groups was calculated using a two-tailed unpaired t -test. * P

    Journal: Journal of Biomedical Science

    Article Title: Functional domains of SP110 that modulate its transcriptional regulatory function and cellular translocation

    doi: 10.1186/s12929-018-0434-4

    Figure Lengend Snippet: SP110 regulates NF-κB-driven transcription. a HEK293T cells were co-transfected with pGL3-p5 × κB-F.Luc, pSV40-R.Luc, and the indicated constructs, and the relative firefly (F.)/ Renilla (R.) luciferase (Luc) values for a basic promoter with 5 × κB sites were measured 2 days after co-transfection. The empty vector negative control is shown on the left. b HEK293T cells were co-transfected with pGL3-TNFα promoter-F.Luc, pSV40-R.Luc, and the indicated constructs, and the relative Luc values for the TNFα promoter were measured 2 days after co-transfection. The empty vector negative control is shown on the left. c and d HEK293T cells were co-transfected with pGL3-TNFα promoter-F.Luc, pSV40-R.Luc, and the indicated constructs, and the relative Luc values for the TNFα promoter were measured 2 days after co-transfection. The empty vector negative control is shown on the top. In ( a and b ), a, b, and c correspond to SP110a, SP110b, and SP110c, respectively. In ( c and d ), schematic representation of SP110 deletion mutants is shown on the left. The data are presented as the mean ± SD. Statistical significance of the difference between two sample groups was calculated using a two-tailed unpaired t -test. * P

    Article Snippet: For the TNFα promoter activity assay, an approximately 1.9 kb segment of the TNFα 5′-flanking region (− 1786 to + 175), which encompasses the transcriptional start site, was amplified by PCR and inserted into the pGL3-Basic vector (firefly luciferase; Promega) to generate the pGL3-TNFα promoter-F.Luc plasmid (firefly luciferase).

    Techniques: Transfection, Construct, Luciferase, Cotransfection, Plasmid Preparation, Negative Control, Two Tailed Test

    Identification of the AQP1 gene upstream promoter region. a , c and e Schematic representation of human AQP1 promoter reporter constructs. Fragments with various lengths of the AQP1 promoter region between − 4000 to 0 bp were cloned downstream of the firefly luciferase reporter. b , d and f Luciferase activity in HEK-293T cells transfected with firefly luciferase reporter plasmids containing various AQP1 upstream regions. The Renilla luciferase reporter was co-transfected with pGL3-basic or a plasmid reporter. The data represent the mean ± SEM. **P

    Journal: BMC Molecular Biology

    Article Title: pH-mediated upregulation of AQP1 gene expression through the Spi-B transcription factor

    doi: 10.1186/s12867-018-0104-9

    Figure Lengend Snippet: Identification of the AQP1 gene upstream promoter region. a , c and e Schematic representation of human AQP1 promoter reporter constructs. Fragments with various lengths of the AQP1 promoter region between − 4000 to 0 bp were cloned downstream of the firefly luciferase reporter. b , d and f Luciferase activity in HEK-293T cells transfected with firefly luciferase reporter plasmids containing various AQP1 upstream regions. The Renilla luciferase reporter was co-transfected with pGL3-basic or a plasmid reporter. The data represent the mean ± SEM. **P

    Article Snippet: The digested DNA fragments were ligated into a pGL3-Basic plasmid (Promega, Madison, USA).

    Techniques: Construct, Clone Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation

    Increased pH elevates AQP1 expression. a AQP1 mRNA levels in HEK-293T cells with various pH treatments. b Luciferase activity in HEK-293T cells transfected with firefly luciferase reporter plasmids containing AQP1 upstream regions. The Renilla luciferase reporter was co-transfected with pGL3-basic or a plasmid reporter. The data represent the mean ± SEM. ns no statistical significance; *P

    Journal: BMC Molecular Biology

    Article Title: pH-mediated upregulation of AQP1 gene expression through the Spi-B transcription factor

    doi: 10.1186/s12867-018-0104-9

    Figure Lengend Snippet: Increased pH elevates AQP1 expression. a AQP1 mRNA levels in HEK-293T cells with various pH treatments. b Luciferase activity in HEK-293T cells transfected with firefly luciferase reporter plasmids containing AQP1 upstream regions. The Renilla luciferase reporter was co-transfected with pGL3-basic or a plasmid reporter. The data represent the mean ± SEM. ns no statistical significance; *P

    Article Snippet: The digested DNA fragments were ligated into a pGL3-Basic plasmid (Promega, Madison, USA).

    Techniques: Expressing, Luciferase, Activity Assay, Transfection, Plasmid Preparation

    Deletion of the 20q-TERRA locus in different human cell lines using the CRISPR-Cas9 system. ( a ) Snapshot depicting, from top to bottom, putative promoter regions (Pr1, Pr2, Pr2 and Pr4), gRNA position, genomic coordinates, RNA-seq from TERRA IP and input 13 , and RNA polymerase 2A (POL2A) and CTCF ChIP-seq data. S2, E1, S2 and E2 are the names of the different gRNAs. ( b ) Graph shows the relative fold increase in firefly luciferase activity seen in the pGL3-containing promoter regions (Pr1-4) relative to the empty vector after normalization to renilla activity. (Mean values±s.e.m., n =3 independent experiments). p21 promoter serves as positive control. One-way Anova with the Dunnett's post test was used for statistical analysis (* P

    Journal: Nature Communications

    Article Title: Telomeric RNAs are essential to maintain telomeres

    doi: 10.1038/ncomms12534

    Figure Lengend Snippet: Deletion of the 20q-TERRA locus in different human cell lines using the CRISPR-Cas9 system. ( a ) Snapshot depicting, from top to bottom, putative promoter regions (Pr1, Pr2, Pr2 and Pr4), gRNA position, genomic coordinates, RNA-seq from TERRA IP and input 13 , and RNA polymerase 2A (POL2A) and CTCF ChIP-seq data. S2, E1, S2 and E2 are the names of the different gRNAs. ( b ) Graph shows the relative fold increase in firefly luciferase activity seen in the pGL3-containing promoter regions (Pr1-4) relative to the empty vector after normalization to renilla activity. (Mean values±s.e.m., n =3 independent experiments). p21 promoter serves as positive control. One-way Anova with the Dunnett's post test was used for statistical analysis (* P

    Article Snippet: Plasmids and reporter assays For construction of the reporter plasmid, PCR products were prepared with primers spanning the promoter regions of interest ( ) and cloned into the plasmid pGL3-basic (Promega).

    Techniques: CRISPR, RNA Sequencing Assay, Chromatin Immunoprecipitation, Luciferase, Activity Assay, Plasmid Preparation, Positive Control

    KLF8 induces VEGFA reporter activity by binding to the CACCC region of the VEGFA promoter and regulating HIF-1α expression. ( a ) The promoter region of VEGF-A (−2068/50 bp) was cloned from human genomic DNA, and the fragment was inserted into pGL3-Basic to construct pGL3-Basic-VEGFA-P plasmid. Dual-Luciferase Reporter Assay System was used to detect luciferase activity, the relative luciferase activity in pcDNA3.1-KLF8 group and pcDNA3.1 group was 3.08 ± 0.04 and 1.49 ± 0.04, respectively. (P

    Journal: Scientific Reports

    Article Title: Krüppel-like factor 8 regulates VEGFA expression and angiogenesis in hepatocellular carcinoma

    doi: 10.1038/s41598-018-35786-6

    Figure Lengend Snippet: KLF8 induces VEGFA reporter activity by binding to the CACCC region of the VEGFA promoter and regulating HIF-1α expression. ( a ) The promoter region of VEGF-A (−2068/50 bp) was cloned from human genomic DNA, and the fragment was inserted into pGL3-Basic to construct pGL3-Basic-VEGFA-P plasmid. Dual-Luciferase Reporter Assay System was used to detect luciferase activity, the relative luciferase activity in pcDNA3.1-KLF8 group and pcDNA3.1 group was 3.08 ± 0.04 and 1.49 ± 0.04, respectively. (P

    Article Snippet: SMMC7721 cells were co-transfected with 0.4 μg of the VEGF-A promoter luciferase reporter constructs, 0.2 μg of pGL3-basic-VEGFA promoter or pGL3-Basic reporter plasmids (Promega), and 0.2 μg of pcDNA3.1 (blank vector as a control) or pcDNA3.1-KLF8.

    Techniques: Activity Assay, Binding Assay, Expressing, Clone Assay, Construct, Plasmid Preparation, Luciferase, Reporter Assay

    PXR repression via SMRT. A, transient transfection assays were performed by transfecting pGL3/CYP24A1-3 kb with various combinations of pcDNA3.1/mVDR, pCMX/hSMRT, and pCR3/hPXR into Huh7 cells. After 24 h, cells were treated with DMSO, VD 3 (10 nM), and/or Rif (10 μM) for an additional 24 h before being harvested for luciferase assays. Relative luciferase activities were expressed relative to the activity of the DMSO-treated cells transfected with the empty -3-kb reporter plasmid. Bars, mean ± S.D. B, Huh7 cells were transfected with or without pcDNA3.1/mVDR and pCR3/hPXR. After 24 h, transfected cells were treated with DMSO, VD 3 (10 nM), and/or rifampicin (10 μM) for an additional 2 h before being harvested for ChIP assays. Cross-linked chromatin-protein complexes were immunoprecipitated with anti-VDR (αVDR), anti-PXR (αPXR), anti-SMRT (αSMRT), or normal mouse IgG (αIgG). By scanning three independent assays, the relative intensities of the bands were calculated based on the inputs: in the VDR binding, 0.8 ± 0.1, 3.3 ± 0.7, 3.7 ± 1.1, and 3.8 ± 0.9% for VDR plus DMSO, VDR plus VD 3 , VDR plus PXR and VD 3 , and VDR plus PXR, VD 3 , and Rif, respectively; in the PXR binding, 0.2 ± 0.2, 0.3 ± 0.3, 2.7 ± 0.8, and 5.2 ± 1.2% for VDR plus DMSO, VDR plus VD 3 , VDR plus PXR and VD 3 , and VDR plus PXR, VD 3 , and Rif, respectively; in the SMRT binding, 4.7 ± 1.0, 0.0 ± 0.1, 1.3 ± 0.6, and 4.0 ± 0.4% for VDR plus DMSO, VDR plus VD 3 , VDR plus PXR and VD 3 , and VDR plus PXR, VD 3 , and Rif, respectively.

    Journal: Molecular Pharmacology

    Article Title: Nuclear Xenobiotic Receptor Pregnane X Receptor Locks Corepressor Silencing Mediator for Retinoid and Thyroid Hormone Receptors (SMRT) onto theCYP24A1 Promoter to Attenuate Vitamin D3 Activation S⃞

    doi: 10.1124/mol.108.051904

    Figure Lengend Snippet: PXR repression via SMRT. A, transient transfection assays were performed by transfecting pGL3/CYP24A1-3 kb with various combinations of pcDNA3.1/mVDR, pCMX/hSMRT, and pCR3/hPXR into Huh7 cells. After 24 h, cells were treated with DMSO, VD 3 (10 nM), and/or Rif (10 μM) for an additional 24 h before being harvested for luciferase assays. Relative luciferase activities were expressed relative to the activity of the DMSO-treated cells transfected with the empty -3-kb reporter plasmid. Bars, mean ± S.D. B, Huh7 cells were transfected with or without pcDNA3.1/mVDR and pCR3/hPXR. After 24 h, transfected cells were treated with DMSO, VD 3 (10 nM), and/or rifampicin (10 μM) for an additional 2 h before being harvested for ChIP assays. Cross-linked chromatin-protein complexes were immunoprecipitated with anti-VDR (αVDR), anti-PXR (αPXR), anti-SMRT (αSMRT), or normal mouse IgG (αIgG). By scanning three independent assays, the relative intensities of the bands were calculated based on the inputs: in the VDR binding, 0.8 ± 0.1, 3.3 ± 0.7, 3.7 ± 1.1, and 3.8 ± 0.9% for VDR plus DMSO, VDR plus VD 3 , VDR plus PXR and VD 3 , and VDR plus PXR, VD 3 , and Rif, respectively; in the PXR binding, 0.2 ± 0.2, 0.3 ± 0.3, 2.7 ± 0.8, and 5.2 ± 1.2% for VDR plus DMSO, VDR plus VD 3 , VDR plus PXR and VD 3 , and VDR plus PXR, VD 3 , and Rif, respectively; in the SMRT binding, 4.7 ± 1.0, 0.0 ± 0.1, 1.3 ± 0.6, and 4.0 ± 0.4% for VDR plus DMSO, VDR plus VD 3 , VDR plus PXR and VD 3 , and VDR plus PXR, VD 3 , and Rif, respectively.

    Article Snippet: To construct the reporter plasmids pGL3/ CYP24A1 -5 kb (-5003/+40) and pGL3/ CYP24A1 -3 kb (-2870/+40), amplified sequences from human genomic DNA was cloned into the KpnI and XhoI sites of pGL3-basic from Promega (Madison, WI).

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

    CAR repression of vitamin D 3 activation via SMRT. A, Huh7 cells were transfected with or without pcDNA3.1/mVDR and pCR3/hCAR. After 24 h, cells were treated with DMSO, VD 3 (10 nM), CITCO (250 nM), and/or PK11195 (10 μM) for an additional 24 h before being harvested for qRT-PCR. Relative CYP24A1 mRNA levels were expressed by taking the level of the DMSO-treated cells transfected with empty plasmid as 1. Bars, mean ± S.D. B, pGL3/CYP24A1-3 kb was transfected with or without pcDNA3.1/mVDR and pCR3/hCAR into Huh7 cells. After 24 h, cells were treated with DMSO, VD 3 (10 nM), CITCO (25, 75, and 250 nM), and PK11195 (1, 3, and 10 μM) for an additional 24 h before being harvested for luciferase assays. Relative luciferase activities were expressed relative to the activity of the DMSO-treated cells transfected with empty plasmid as 1. Bars, mean ± S.D. C, Huh7 cells were transfected with pcDNA3.1/mVDR and pcDNA3.1/mVDR plus pCR3/hCAR. After 24 h, cells were treated with DMSO, VD 3 (10 nM), CITCO (250 nM), and/or PK11195 (10 μM) for an additional 2 h before being harvested for ChIP assays. Cross-linked chromatin-protein complexes were immunoprecipitated with anti-VDR (αVDR), anti-CAR (αCAR), anti-SMRT (αSMRT), or normal mouse IgG (αIgG). By scanning three independent assays, the relative intensities of the bands were calculated based the inputs: in the VDR binding, 3.4 ± 0.8, 3.3 ± 1.0, 3.4 ± 1.3, and 3.4 ± 1.2% for VDR plus VD 3 , VDR plus CAR and VD 3 , VDR plus CAR, VD 3 , and CITCO, and VDR plus CAR, VD 3 , and PK11195, respectively; in the CAR binding, 0.1 ± 0.3, 1.0 ± 0.3, 2.9 ± 0.5, and 3.7 ± 1.2% for VDR plus VD 3 , VDR plus CAR and VD 3 , VDR plus CAR, VD 3 , and CITCO, and VDR plus CAR, VD 3 , and PK11195, respectively; in the SMRT binding, 0.0 ± 0.0, 0.8 ± 0.1, 2.0 ± 0.5, and 2.1 ± 0.3% for VDR plus VD 3 , VDR plus CAR and VD 3 , VDR plus CAR, VD 3 , and CITCO, and VDR plus CAR, VD 3 , and PK11195, respectively.

    Journal: Molecular Pharmacology

    Article Title: Nuclear Xenobiotic Receptor Pregnane X Receptor Locks Corepressor Silencing Mediator for Retinoid and Thyroid Hormone Receptors (SMRT) onto theCYP24A1 Promoter to Attenuate Vitamin D3 Activation S⃞

    doi: 10.1124/mol.108.051904

    Figure Lengend Snippet: CAR repression of vitamin D 3 activation via SMRT. A, Huh7 cells were transfected with or without pcDNA3.1/mVDR and pCR3/hCAR. After 24 h, cells were treated with DMSO, VD 3 (10 nM), CITCO (250 nM), and/or PK11195 (10 μM) for an additional 24 h before being harvested for qRT-PCR. Relative CYP24A1 mRNA levels were expressed by taking the level of the DMSO-treated cells transfected with empty plasmid as 1. Bars, mean ± S.D. B, pGL3/CYP24A1-3 kb was transfected with or without pcDNA3.1/mVDR and pCR3/hCAR into Huh7 cells. After 24 h, cells were treated with DMSO, VD 3 (10 nM), CITCO (25, 75, and 250 nM), and PK11195 (1, 3, and 10 μM) for an additional 24 h before being harvested for luciferase assays. Relative luciferase activities were expressed relative to the activity of the DMSO-treated cells transfected with empty plasmid as 1. Bars, mean ± S.D. C, Huh7 cells were transfected with pcDNA3.1/mVDR and pcDNA3.1/mVDR plus pCR3/hCAR. After 24 h, cells were treated with DMSO, VD 3 (10 nM), CITCO (250 nM), and/or PK11195 (10 μM) for an additional 2 h before being harvested for ChIP assays. Cross-linked chromatin-protein complexes were immunoprecipitated with anti-VDR (αVDR), anti-CAR (αCAR), anti-SMRT (αSMRT), or normal mouse IgG (αIgG). By scanning three independent assays, the relative intensities of the bands were calculated based the inputs: in the VDR binding, 3.4 ± 0.8, 3.3 ± 1.0, 3.4 ± 1.3, and 3.4 ± 1.2% for VDR plus VD 3 , VDR plus CAR and VD 3 , VDR plus CAR, VD 3 , and CITCO, and VDR plus CAR, VD 3 , and PK11195, respectively; in the CAR binding, 0.1 ± 0.3, 1.0 ± 0.3, 2.9 ± 0.5, and 3.7 ± 1.2% for VDR plus VD 3 , VDR plus CAR and VD 3 , VDR plus CAR, VD 3 , and CITCO, and VDR plus CAR, VD 3 , and PK11195, respectively; in the SMRT binding, 0.0 ± 0.0, 0.8 ± 0.1, 2.0 ± 0.5, and 2.1 ± 0.3% for VDR plus VD 3 , VDR plus CAR and VD 3 , VDR plus CAR, VD 3 , and CITCO, and VDR plus CAR, VD 3 , and PK11195, respectively.

    Article Snippet: To construct the reporter plasmids pGL3/ CYP24A1 -5 kb (-5003/+40) and pGL3/ CYP24A1 -3 kb (-2870/+40), amplified sequences from human genomic DNA was cloned into the KpnI and XhoI sites of pGL3-basic from Promega (Madison, WI).

    Techniques: Activation Assay, Transfection, Quantitative RT-PCR, Plasmid Preparation, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Binding Assay

    PXR repression depending on vitamin D 3 concentration. A, pGL3/CYP24A1-3 kb was cotransfected with pcDNA3.1/mVDR and pCR3/hPXR into Huh7 cells. After 24 h, cells were treated with DMSO and rifampicin and with the increased concentrations of VD 3 for an additional 24 h before being harvested for luciferase assays. Relative luciferase activities were expressed relative to the activity of the cells treated with DMSO alone as 1. Bars, mean ± S.D. B, Huh7 cells were transfected with pcDNA3.1/mVDR and pCR3/hPXR. At 24 h after the transfection, cells were treated with Rif (1 and 10 μM) and VD 3 (1, 10, and 100 nM) at their various concentrations for an additional 2 h before being harvested for ChIP assays. Cross-linked chromatin-protein complexes were immunoprecipitated with anti-SMRT (αSMRT) or normal mouse IgG (αIgG). By scanning three independent assays, the relative intensities of the bands were calculated based on the inputs: 4.2 ± 1.0, 2.5 ± 0.7, 1.4 ± 0.5, 5.7 ± 1.1, 4.6 ± 1.1, and 3.8 ± 0.9% for the bars from left to right.

    Journal: Molecular Pharmacology

    Article Title: Nuclear Xenobiotic Receptor Pregnane X Receptor Locks Corepressor Silencing Mediator for Retinoid and Thyroid Hormone Receptors (SMRT) onto theCYP24A1 Promoter to Attenuate Vitamin D3 Activation S⃞

    doi: 10.1124/mol.108.051904

    Figure Lengend Snippet: PXR repression depending on vitamin D 3 concentration. A, pGL3/CYP24A1-3 kb was cotransfected with pcDNA3.1/mVDR and pCR3/hPXR into Huh7 cells. After 24 h, cells were treated with DMSO and rifampicin and with the increased concentrations of VD 3 for an additional 24 h before being harvested for luciferase assays. Relative luciferase activities were expressed relative to the activity of the cells treated with DMSO alone as 1. Bars, mean ± S.D. B, Huh7 cells were transfected with pcDNA3.1/mVDR and pCR3/hPXR. At 24 h after the transfection, cells were treated with Rif (1 and 10 μM) and VD 3 (1, 10, and 100 nM) at their various concentrations for an additional 2 h before being harvested for ChIP assays. Cross-linked chromatin-protein complexes were immunoprecipitated with anti-SMRT (αSMRT) or normal mouse IgG (αIgG). By scanning three independent assays, the relative intensities of the bands were calculated based on the inputs: 4.2 ± 1.0, 2.5 ± 0.7, 1.4 ± 0.5, 5.7 ± 1.1, 4.6 ± 1.1, and 3.8 ± 0.9% for the bars from left to right.

    Article Snippet: To construct the reporter plasmids pGL3/ CYP24A1 -5 kb (-5003/+40) and pGL3/ CYP24A1 -3 kb (-2870/+40), amplified sequences from human genomic DNA was cloned into the KpnI and XhoI sites of pGL3-basic from Promega (Madison, WI).

    Techniques: Concentration Assay, Luciferase, Activity Assay, Transfection, Chromatin Immunoprecipitation, Immunoprecipitation

    PXR repression of vitamin D 3 -activated CYP24A1 promoter. pCR3/hPXR was transfected with or without pcDNA3.1/mVDR into Huh7 cells. After 24 h, cells were treated with DMSO, VD 3 (10 nM), and Rif (10 μM) for an additional 24 h before being harvested for qRT-PCR. Relative CYP24A1 mRNA levels were expressed by taking the level of the DMSO-treated cells transfected without VDR and PXR as one. Bars, mean ± S.D. B, transient transfection assays were performed by transfecting pGL3/CYP24A1-3 kb with or without pcDNA3.1/mVDR and pCR3/hPXR into Huh7 cells. After 24 h, cells were treated with DMSO, VD 3 (10 nM), and Rif (10 μM) for an additional 24 h before being harvested for luciferase assays. C, various pGL3 constructs of the CYP24A1 promoter-luciferase reporter gene were transfected with or without pcDNA3.1/mVDR and pCR3/hPXR into Huh7 cells. After 24 h, cells were treated with DMSO, VD 3 (10 nM), and rifampicin (10 μM) for an additional 24 h before being harvested for transient transfection assays. Relative luciferase activities in both B and C are expressed relative to the activity of the DMSO-treated cells transfected with the empty plasmid. Bars, mean ± S.D.

    Journal: Molecular Pharmacology

    Article Title: Nuclear Xenobiotic Receptor Pregnane X Receptor Locks Corepressor Silencing Mediator for Retinoid and Thyroid Hormone Receptors (SMRT) onto theCYP24A1 Promoter to Attenuate Vitamin D3 Activation S⃞

    doi: 10.1124/mol.108.051904

    Figure Lengend Snippet: PXR repression of vitamin D 3 -activated CYP24A1 promoter. pCR3/hPXR was transfected with or without pcDNA3.1/mVDR into Huh7 cells. After 24 h, cells were treated with DMSO, VD 3 (10 nM), and Rif (10 μM) for an additional 24 h before being harvested for qRT-PCR. Relative CYP24A1 mRNA levels were expressed by taking the level of the DMSO-treated cells transfected without VDR and PXR as one. Bars, mean ± S.D. B, transient transfection assays were performed by transfecting pGL3/CYP24A1-3 kb with or without pcDNA3.1/mVDR and pCR3/hPXR into Huh7 cells. After 24 h, cells were treated with DMSO, VD 3 (10 nM), and Rif (10 μM) for an additional 24 h before being harvested for luciferase assays. C, various pGL3 constructs of the CYP24A1 promoter-luciferase reporter gene were transfected with or without pcDNA3.1/mVDR and pCR3/hPXR into Huh7 cells. After 24 h, cells were treated with DMSO, VD 3 (10 nM), and rifampicin (10 μM) for an additional 24 h before being harvested for transient transfection assays. Relative luciferase activities in both B and C are expressed relative to the activity of the DMSO-treated cells transfected with the empty plasmid. Bars, mean ± S.D.

    Article Snippet: To construct the reporter plasmids pGL3/ CYP24A1 -5 kb (-5003/+40) and pGL3/ CYP24A1 -3 kb (-2870/+40), amplified sequences from human genomic DNA was cloned into the KpnI and XhoI sites of pGL3-basic from Promega (Madison, WI).

    Techniques: Transfection, Quantitative RT-PCR, Luciferase, Construct, Activity Assay, Plasmid Preparation

    PXR activation of VDRE. A, transient transfection assays were performed by transfecting pCR3/hPXR with various pGL3 constructs of the CYP24A1 promoter-luciferase reporter gene in Huh7 cells. After 24 h, cells were treated with DMSO or rifampicin (Rif, 10 μM) for an additional 24 h before being harvested for luciferase assays. Relative luciferase activities were expressed relative to the activity of the DMSO-treated cells transfected with the empty plasmid and pGL3/CYP24A1-5 kb reporter plasmid. Bars, mean ± S.D. B, PXR binding to VDRE. Gel-shift assays were performed by incubating radiolabeled VDRE1 probe with in vitro transcribed and translated proteins: PXR, RXR, PXR/RXR, VDR, or VDR/RXR as described under Materials and Methods . C, chromatin binding of PXR. ChIP assays were performed using nuclear extracts prepared from pCR3/hPXR-transfected Huh7 cells. Cells were transfected for 24 h and subsequently treated with DMSO or rifampicin (10 μM) as indicated and incubated for an additional 2 h. By scanning three independent assays, the relative intensities of the bands were calculated based on the inputs: 0.2 ± 0.2, 1.6 ± 0.4, and 5.0 ± 0.9 for DMSO, PXR plus DMSO, and PXR plus Rif, respectively.

    Journal: Molecular Pharmacology

    Article Title: Nuclear Xenobiotic Receptor Pregnane X Receptor Locks Corepressor Silencing Mediator for Retinoid and Thyroid Hormone Receptors (SMRT) onto theCYP24A1 Promoter to Attenuate Vitamin D3 Activation S⃞

    doi: 10.1124/mol.108.051904

    Figure Lengend Snippet: PXR activation of VDRE. A, transient transfection assays were performed by transfecting pCR3/hPXR with various pGL3 constructs of the CYP24A1 promoter-luciferase reporter gene in Huh7 cells. After 24 h, cells were treated with DMSO or rifampicin (Rif, 10 μM) for an additional 24 h before being harvested for luciferase assays. Relative luciferase activities were expressed relative to the activity of the DMSO-treated cells transfected with the empty plasmid and pGL3/CYP24A1-5 kb reporter plasmid. Bars, mean ± S.D. B, PXR binding to VDRE. Gel-shift assays were performed by incubating radiolabeled VDRE1 probe with in vitro transcribed and translated proteins: PXR, RXR, PXR/RXR, VDR, or VDR/RXR as described under Materials and Methods . C, chromatin binding of PXR. ChIP assays were performed using nuclear extracts prepared from pCR3/hPXR-transfected Huh7 cells. Cells were transfected for 24 h and subsequently treated with DMSO or rifampicin (10 μM) as indicated and incubated for an additional 2 h. By scanning three independent assays, the relative intensities of the bands were calculated based on the inputs: 0.2 ± 0.2, 1.6 ± 0.4, and 5.0 ± 0.9 for DMSO, PXR plus DMSO, and PXR plus Rif, respectively.

    Article Snippet: To construct the reporter plasmids pGL3/ CYP24A1 -5 kb (-5003/+40) and pGL3/ CYP24A1 -3 kb (-2870/+40), amplified sequences from human genomic DNA was cloned into the KpnI and XhoI sites of pGL3-basic from Promega (Madison, WI).

    Techniques: Activation Assay, Transfection, Construct, Luciferase, Activity Assay, Plasmid Preparation, Binding Assay, Electrophoretic Mobility Shift Assay, In Vitro, Chromatin Immunoprecipitation, Incubation

    Analysis of putative PPAR binding sites by site-directed mutagenesis. Site-mutation constructs are presented in the left panel. Promoter activity of constructs is presented in the middle. Promoter activity treated with agonist was presented in the right panel. ( A ) site-mutations of PPARα binding sites on pGl3-CPTIα1b-2276 and pGl3-CPTIα2a-2041 vectors ( B ) site-mutation of PPARγ binding sites on pGl3-CPTIα1b-2276 and pGl3-CPTIα2a-1304 vectors. Values represent the ratio between firefly and renilla luciferase activities, normalized to the control plasmid pGL3-Basic. Bars are the mean ± SEM of three independent experiments (Student’s t -test, * p

    Journal: International Journal of Molecular Sciences

    Article Title: Structure and Functional Analysis of Promoters from Two Liver Isoforms of CPT I in Grass Carp Ctenopharyngodon idella

    doi: 10.3390/ijms18112405

    Figure Lengend Snippet: Analysis of putative PPAR binding sites by site-directed mutagenesis. Site-mutation constructs are presented in the left panel. Promoter activity of constructs is presented in the middle. Promoter activity treated with agonist was presented in the right panel. ( A ) site-mutations of PPARα binding sites on pGl3-CPTIα1b-2276 and pGl3-CPTIα2a-2041 vectors ( B ) site-mutation of PPARγ binding sites on pGl3-CPTIα1b-2276 and pGl3-CPTIα2a-1304 vectors. Values represent the ratio between firefly and renilla luciferase activities, normalized to the control plasmid pGL3-Basic. Bars are the mean ± SEM of three independent experiments (Student’s t -test, * p

    Article Snippet: For the generation of the luciferase reporter constructs, the PCR product and pGl3-Basic vectors (Promega, Madison, WI, USA) were purified and digested using corresponding endonucleases, and then products were ligated using ClonExpress™ II One Step Cloning Kit (Vazyme, Piscataway, NJ, USA).

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

    5′ Unidirectional deletion analysis of the CPT Iα1b and CPT Iα2a promoter regions for grass carp. Schematic diagrams of truncated promoters were shown at the left panel. The corresponding luciferase reporter assay results were shown in the right panel. Promoter activity of constructs is presented with the values of relative light unit. A series of plasmids containing 5′ unidirectional deletions of the CPT Iα1b promoter region fused in frame to the luciferase gene were transfected into HepG2 cells ( A ) and HEK293 cells ( B ), and a series of plasmids containing 5′ unidirectional deletions of the CPT Iα2a promoter region were transfected into HepG2 cells ( C ) and HEK293 cells ( D ). Values represent the ratio between firefly and renilla luciferase activities, normalized to the control plasmid pGl3-Basic. Results were expressed as the mean ± SEM of three independent experiments (Student’s t -test, * p

    Journal: International Journal of Molecular Sciences

    Article Title: Structure and Functional Analysis of Promoters from Two Liver Isoforms of CPT I in Grass Carp Ctenopharyngodon idella

    doi: 10.3390/ijms18112405

    Figure Lengend Snippet: 5′ Unidirectional deletion analysis of the CPT Iα1b and CPT Iα2a promoter regions for grass carp. Schematic diagrams of truncated promoters were shown at the left panel. The corresponding luciferase reporter assay results were shown in the right panel. Promoter activity of constructs is presented with the values of relative light unit. A series of plasmids containing 5′ unidirectional deletions of the CPT Iα1b promoter region fused in frame to the luciferase gene were transfected into HepG2 cells ( A ) and HEK293 cells ( B ), and a series of plasmids containing 5′ unidirectional deletions of the CPT Iα2a promoter region were transfected into HepG2 cells ( C ) and HEK293 cells ( D ). Values represent the ratio between firefly and renilla luciferase activities, normalized to the control plasmid pGl3-Basic. Results were expressed as the mean ± SEM of three independent experiments (Student’s t -test, * p

    Article Snippet: For the generation of the luciferase reporter constructs, the PCR product and pGl3-Basic vectors (Promega, Madison, WI, USA) were purified and digested using corresponding endonucleases, and then products were ligated using ClonExpress™ II One Step Cloning Kit (Vazyme, Piscataway, NJ, USA).

    Techniques: Cycling Probe Technology, Luciferase, Reporter Assay, Activity Assay, Construct, Transfection, Plasmid Preparation

    Mutated CCAAT box and SRE reduce transcriptional activity of the zebrafish fads2 promoter. Single or multiple site mutations ( X ) of SRE ( ), CATp (●) and CATd (○) were introduced into the fads2 −244 plasmid. ZFL cells were transiently co-transfected with various mutated fads2 −244 plasmids, nSrebp2 expression plasmid, together with Renilla luciferase reference plasmid pRL-SV40. Luciferase coding region is indicated by shaded box and activity of the fads2 promoter in ZFL cells is expressed as normalised luciferase activity (to Renilla luciferase activity) relative to the empty pGL3-Basic plasmid. Values are means ± SD of three independent experiments (n = 3) (right panel). Groups indicated with different letters are significantly different (Tukey’s test; P

    Journal: Scientific Reports

    Article Title: Transcriptional activation of zebrafish fads2 promoter and its transient transgene expression in yolk syncytial layer of zebrafish embryos

    doi: 10.1038/s41598-018-22157-4

    Figure Lengend Snippet: Mutated CCAAT box and SRE reduce transcriptional activity of the zebrafish fads2 promoter. Single or multiple site mutations ( X ) of SRE ( ), CATp (●) and CATd (○) were introduced into the fads2 −244 plasmid. ZFL cells were transiently co-transfected with various mutated fads2 −244 plasmids, nSrebp2 expression plasmid, together with Renilla luciferase reference plasmid pRL-SV40. Luciferase coding region is indicated by shaded box and activity of the fads2 promoter in ZFL cells is expressed as normalised luciferase activity (to Renilla luciferase activity) relative to the empty pGL3-Basic plasmid. Values are means ± SD of three independent experiments (n = 3) (right panel). Groups indicated with different letters are significantly different (Tukey’s test; P

    Article Snippet: The resulted PCR products were gel purified, cloned into the pGL3-Basic luciferase reporter vector (Promega, USA) and sequenced.

    Techniques: Activity Assay, Plasmid Preparation, Transfection, Expressing, Luciferase

    5′-deletion analysis of the zebrafish fads2 promoter. ZFL cells were transiently co-transfected with the fads2 5′-deletion promoter-luciferase reporter plasmids, nSrebp2 expression plasmid together with Renilla luciferase reference plasmid pRL - SV40. 5′-deletion plasmids were named according to respective position in relation to TSS (+1) and are represented by horizontal line on the left. Non-coding exon is indicated with open boxes. Luciferase activity of fads2 promoter in ZFL cells is expressed as normalised luciferase activity (to Renilla luciferase activity) relative to empty pGL3-Basic plasmid. Values are means ± S.D. (n = 3) (right panel). Groups indicated with different letters are significantly different (Tukey’s test; P

    Journal: Scientific Reports

    Article Title: Transcriptional activation of zebrafish fads2 promoter and its transient transgene expression in yolk syncytial layer of zebrafish embryos

    doi: 10.1038/s41598-018-22157-4

    Figure Lengend Snippet: 5′-deletion analysis of the zebrafish fads2 promoter. ZFL cells were transiently co-transfected with the fads2 5′-deletion promoter-luciferase reporter plasmids, nSrebp2 expression plasmid together with Renilla luciferase reference plasmid pRL - SV40. 5′-deletion plasmids were named according to respective position in relation to TSS (+1) and are represented by horizontal line on the left. Non-coding exon is indicated with open boxes. Luciferase activity of fads2 promoter in ZFL cells is expressed as normalised luciferase activity (to Renilla luciferase activity) relative to empty pGL3-Basic plasmid. Values are means ± S.D. (n = 3) (right panel). Groups indicated with different letters are significantly different (Tukey’s test; P

    Article Snippet: The resulted PCR products were gel purified, cloned into the pGL3-Basic luciferase reporter vector (Promega, USA) and sequenced.

    Techniques: Transfection, Luciferase, Expressing, Plasmid Preparation, Activity Assay

    Effects of nSrebp proteins on the zebrafish fads2 promoter activation. ZFL cells were co-transfected with nSrebp1 or nSrebp2 expression plasmid (empty pcDNA3.1 expression plasmid as control), and pGL3 promoter-luciferase reporter plasmid fused with the zebrafish fads2 desaturase promoter (−1735/+19 bp). Results represent normalised luciferase activity (to Renilla luciferase activity) of fads2 promoter in the ZFL cells relative to empty pGL3-Basic plasmid. Values are means ± SD (n = 3); P

    Journal: Scientific Reports

    Article Title: Transcriptional activation of zebrafish fads2 promoter and its transient transgene expression in yolk syncytial layer of zebrafish embryos

    doi: 10.1038/s41598-018-22157-4

    Figure Lengend Snippet: Effects of nSrebp proteins on the zebrafish fads2 promoter activation. ZFL cells were co-transfected with nSrebp1 or nSrebp2 expression plasmid (empty pcDNA3.1 expression plasmid as control), and pGL3 promoter-luciferase reporter plasmid fused with the zebrafish fads2 desaturase promoter (−1735/+19 bp). Results represent normalised luciferase activity (to Renilla luciferase activity) of fads2 promoter in the ZFL cells relative to empty pGL3-Basic plasmid. Values are means ± SD (n = 3); P

    Article Snippet: The resulted PCR products were gel purified, cloned into the pGL3-Basic luciferase reporter vector (Promega, USA) and sequenced.

    Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay

    PAX7 promoter activity modulated by the 10-bp indel polymorphism. ( A ) Luciferase activity in C2C12 cells transfected with recombinant plasmids containing serial promoter fragments (pGL3-pro1, pGL3-pro2, pGL3-pro3, pGL3-pro4, and pGL3-pro5) of the Pax7 promoter. The backbone vector pGL3-Basic was used as a negative control. P pGL3-pro2 vs. pGL3-pro1 = 0.0118, P pGL3-pro2 vs. pGL3-pro3 = 0.0042, P pGL3-pro2 vs. pGL3-pro4 = 0.0495, P pGL3-pro2 vs. pGL3-pro5 = 0.0241. ( B ) Luciferase activity in C2C12 cells transfected with recombinant plasmids containing the Ins-Ins or Del-Del genotype. pcDNA3.1-ZNF219 was transiently co-transfected into C2C12 cells together with the reporter vectors. Each experiment was repeated at least three times. The data were mean ± standard error (S.E.) of the normalized luciferase activity. * P

    Journal: Scientific Reports

    Article Title: A novel PAX7 10-bp indel variant modulates promoter activity, gene expression and contributes to different phenotypes of Chinese cattle

    doi: 10.1038/s41598-018-20177-8

    Figure Lengend Snippet: PAX7 promoter activity modulated by the 10-bp indel polymorphism. ( A ) Luciferase activity in C2C12 cells transfected with recombinant plasmids containing serial promoter fragments (pGL3-pro1, pGL3-pro2, pGL3-pro3, pGL3-pro4, and pGL3-pro5) of the Pax7 promoter. The backbone vector pGL3-Basic was used as a negative control. P pGL3-pro2 vs. pGL3-pro1 = 0.0118, P pGL3-pro2 vs. pGL3-pro3 = 0.0042, P pGL3-pro2 vs. pGL3-pro4 = 0.0495, P pGL3-pro2 vs. pGL3-pro5 = 0.0241. ( B ) Luciferase activity in C2C12 cells transfected with recombinant plasmids containing the Ins-Ins or Del-Del genotype. pcDNA3.1-ZNF219 was transiently co-transfected into C2C12 cells together with the reporter vectors. Each experiment was repeated at least three times. The data were mean ± standard error (S.E.) of the normalized luciferase activity. * P

    Article Snippet: For luciferase reporter construction, the insert was released by Nhe I and Hin dIII digestion, and then subcloned into the luciferase reporter vector pGL3-Basic (Promega, Madison, WI).

    Techniques: Activity Assay, Luciferase, Transfection, Recombinant, Plasmid Preparation, Negative Control

    Mutation analysis of GATA and ETS sites in the −252/+38 region of the promoter. A . Right; H5V endothelial cells (black bars) and L929 fibroblasts (white bars) were transfected with 80 fmoles of pGL3basic (Ctrl), −8409/+38Luc or a −8409/+38Luc mutant where the GATA (GATAmt) site had been mutated, and with 54 fmoles pCH110 normalization vector. After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Mutation of the GATA site (left, blackened circle) in the originally most active −8409/+38Luc vector resulted in a strong decrease of activity of the reporter in endothelial cells and the endothelial/fibroblasts ratio was decreased by half when compared to −8409/+38Luc. The experiment is representative of a set of three experiments performed in similar conditions. B . Right; H5V (black bars) and L929 (white bars) cells where transfected with 80 fmoles of −8409/+38Luc or the indicated mutated versions in which the predicted EBS (left, numbered circles) were individually mutated by site-directed mutagenesis (left, blackened numbered circles). After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Left; scaled schematic representation of the −8409/+38 wild-type region (top). The location of the GATA (circled G) and EBS (circled numbers) sites is represented to scale along the zoomed region D of the mutants. Mutated sites are represented as blackened circles. Names of the constructs are given according to the cloned 5′ and 3′ end positions relative to the exon-1b transcription start, Luc; luciferase. Results are presented according to Figure 3A . The EBS6/EBS7 (−123/−120) tandem is located in scanD-7 of the linker scanning analysis, EBS5 (−92) is located in scanD-8 and EBS4 (−80), EBS3 (−71), EBS2 (−49) and EBS1 (+28) are all located closer to the transcription start. The experiment is representative of a set of three performed in similar conditions. *** p

    Journal: PLoS ONE

    Article Title: VE-statin/egfl7 Expression in Endothelial Cells Is Regulated by a Distal Enhancer and a Proximal Promoter under the Direct Control of Erg and GATA-2

    doi: 10.1371/journal.pone.0012156

    Figure Lengend Snippet: Mutation analysis of GATA and ETS sites in the −252/+38 region of the promoter. A . Right; H5V endothelial cells (black bars) and L929 fibroblasts (white bars) were transfected with 80 fmoles of pGL3basic (Ctrl), −8409/+38Luc or a −8409/+38Luc mutant where the GATA (GATAmt) site had been mutated, and with 54 fmoles pCH110 normalization vector. After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Mutation of the GATA site (left, blackened circle) in the originally most active −8409/+38Luc vector resulted in a strong decrease of activity of the reporter in endothelial cells and the endothelial/fibroblasts ratio was decreased by half when compared to −8409/+38Luc. The experiment is representative of a set of three experiments performed in similar conditions. B . Right; H5V (black bars) and L929 (white bars) cells where transfected with 80 fmoles of −8409/+38Luc or the indicated mutated versions in which the predicted EBS (left, numbered circles) were individually mutated by site-directed mutagenesis (left, blackened numbered circles). After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Left; scaled schematic representation of the −8409/+38 wild-type region (top). The location of the GATA (circled G) and EBS (circled numbers) sites is represented to scale along the zoomed region D of the mutants. Mutated sites are represented as blackened circles. Names of the constructs are given according to the cloned 5′ and 3′ end positions relative to the exon-1b transcription start, Luc; luciferase. Results are presented according to Figure 3A . The EBS6/EBS7 (−123/−120) tandem is located in scanD-7 of the linker scanning analysis, EBS5 (−92) is located in scanD-8 and EBS4 (−80), EBS3 (−71), EBS2 (−49) and EBS1 (+28) are all located closer to the transcription start. The experiment is representative of a set of three performed in similar conditions. *** p

    Article Snippet: A 13.0kb genomic fragment corresponding to the −12969/+38 sequence of the VE-statin/egfl7 promoter was recovered by NheI/SalI digestion of a mouse genomic clone and inserted into the NheI/XhoI restriction sites of pGL3basic (Promega).

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

    Region D is necessary for expression of the gene in endothelial cells. A . Right: H5V endothelial cells (black bars) and L929 fibroblasts (white bars) were transfected with the pGL3basic vector (Ctrl) or successive 5′-deletion mutants of the −1768/+38Luc vector cloned into pGL3basic (80 fmoles) and with 54 fmoles of pCH110 normalization vector. After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Results are presented as in Figure 3A . The experiment is representative of a set of three performed in similar conditions. *** p

    Journal: PLoS ONE

    Article Title: VE-statin/egfl7 Expression in Endothelial Cells Is Regulated by a Distal Enhancer and a Proximal Promoter under the Direct Control of Erg and GATA-2

    doi: 10.1371/journal.pone.0012156

    Figure Lengend Snippet: Region D is necessary for expression of the gene in endothelial cells. A . Right: H5V endothelial cells (black bars) and L929 fibroblasts (white bars) were transfected with the pGL3basic vector (Ctrl) or successive 5′-deletion mutants of the −1768/+38Luc vector cloned into pGL3basic (80 fmoles) and with 54 fmoles of pCH110 normalization vector. After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Results are presented as in Figure 3A . The experiment is representative of a set of three performed in similar conditions. *** p

    Article Snippet: A 13.0kb genomic fragment corresponding to the −12969/+38 sequence of the VE-statin/egfl7 promoter was recovered by NheI/SalI digestion of a mouse genomic clone and inserted into the NheI/XhoI restriction sites of pGL3basic (Promega).

    Techniques: Expressing, Transfection, Plasmid Preparation, Clone Assay, Luciferase, Activity Assay

    Identification of important sequences in region D. A . H5V endothelial cells (black bars) and L929 fibroblasts (white bars) were transfected with 80 fmoles of either pGL3basic (Ctrl), the non-mutated −252/+38Luc construct, or various mutated versions of it (scan D-2 to 14) which corresponded to the successive exchange of 20bp of wild-type sequence for a 20bp transactivation-null cassette (black box), and with 54 fmoles of pCH110 normalization vector. After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over the Ctrl mean value set to 1. Results are displayed as in Figure 3A . The experiment is representative of a set of three experiments performed in similar conditions. *** p

    Journal: PLoS ONE

    Article Title: VE-statin/egfl7 Expression in Endothelial Cells Is Regulated by a Distal Enhancer and a Proximal Promoter under the Direct Control of Erg and GATA-2

    doi: 10.1371/journal.pone.0012156

    Figure Lengend Snippet: Identification of important sequences in region D. A . H5V endothelial cells (black bars) and L929 fibroblasts (white bars) were transfected with 80 fmoles of either pGL3basic (Ctrl), the non-mutated −252/+38Luc construct, or various mutated versions of it (scan D-2 to 14) which corresponded to the successive exchange of 20bp of wild-type sequence for a 20bp transactivation-null cassette (black box), and with 54 fmoles of pCH110 normalization vector. After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over the Ctrl mean value set to 1. Results are displayed as in Figure 3A . The experiment is representative of a set of three experiments performed in similar conditions. *** p

    Article Snippet: A 13.0kb genomic fragment corresponding to the −12969/+38 sequence of the VE-statin/egfl7 promoter was recovered by NheI/SalI digestion of a mouse genomic clone and inserted into the NheI/XhoI restriction sites of pGL3basic (Promega).

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

    5′-deletion study of the VE-statin/egfl7 promoter region. A . H5V endothelial (black bars) and L929 fibroblast (right, white bars) were transfected with the pGL3basic luciferase reporter vector (Ctrl) or pGL3basic in which −12969/+38 VE-statin/egfl7 promoter region or 5′ deletions of it were inserted. These reporters (80 fmoles) were transfected together with 54 fmoles pCH110 normalization vector. After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Numbers associated with the black bars represent the calculated endothelial/fibroblast ratio of activity for the corresponding construct. Left: scaled schematic representation of the constructs, names are given according to the cloned 5′ and 3′ end positions relative to the exon-1b transcription start, Luc; luciferase. The experiment is representative of a set of at least three experiments performed in similar conditions. Constructions were designed so that the luciferase gene was placed within exon-1b because initial experiments showed that placing it where the coding sequence starts in exon-3 resulted in no detectable activity (not shown). ** p

    Journal: PLoS ONE

    Article Title: VE-statin/egfl7 Expression in Endothelial Cells Is Regulated by a Distal Enhancer and a Proximal Promoter under the Direct Control of Erg and GATA-2

    doi: 10.1371/journal.pone.0012156

    Figure Lengend Snippet: 5′-deletion study of the VE-statin/egfl7 promoter region. A . H5V endothelial (black bars) and L929 fibroblast (right, white bars) were transfected with the pGL3basic luciferase reporter vector (Ctrl) or pGL3basic in which −12969/+38 VE-statin/egfl7 promoter region or 5′ deletions of it were inserted. These reporters (80 fmoles) were transfected together with 54 fmoles pCH110 normalization vector. After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Numbers associated with the black bars represent the calculated endothelial/fibroblast ratio of activity for the corresponding construct. Left: scaled schematic representation of the constructs, names are given according to the cloned 5′ and 3′ end positions relative to the exon-1b transcription start, Luc; luciferase. The experiment is representative of a set of at least three experiments performed in similar conditions. Constructions were designed so that the luciferase gene was placed within exon-1b because initial experiments showed that placing it where the coding sequence starts in exon-3 resulted in no detectable activity (not shown). ** p

    Article Snippet: A 13.0kb genomic fragment corresponding to the −12969/+38 sequence of the VE-statin/egfl7 promoter was recovered by NheI/SalI digestion of a mouse genomic clone and inserted into the NheI/XhoI restriction sites of pGL3basic (Promega).

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

    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

    Effects of compounds 1 – 9 (10 μM) on the p16INK4A and GLB1 transcription in the human dermal fibroblasts. Human dermal fibroblasts were transiently co-transfected with the pGL3-p16INK4A ( A ) or pGL3-GLB1 ( B ) promoter with β-galactosidase as a transfection control. Cells were treated overnight with the compounds 1 – 9 (10 μM). The luciferase activity was determined as the ratio of the firefly/Renilla luciferase activities. The activities of the p16Ink4A promoter and the GLB1 promoter in the presence of the compounds 1 – 9 relative to that in the absence of the compounds 1 – 9 are shown. * p

    Journal: Nutrients

    Article Title: Metabolite Profiling of Rambutan (Nephelium lappaceum L.) Seeds Using UPLC-qTOF-MS/MS and Senomorphic Effects in Aged Human Dermal Fibroblasts

    doi: 10.3390/nu12051430

    Figure Lengend Snippet: Effects of compounds 1 – 9 (10 μM) on the p16INK4A and GLB1 transcription in the human dermal fibroblasts. Human dermal fibroblasts were transiently co-transfected with the pGL3-p16INK4A ( A ) or pGL3-GLB1 ( B ) promoter with β-galactosidase as a transfection control. Cells were treated overnight with the compounds 1 – 9 (10 μM). The luciferase activity was determined as the ratio of the firefly/Renilla luciferase activities. The activities of the p16Ink4A promoter and the GLB1 promoter in the presence of the compounds 1 – 9 relative to that in the absence of the compounds 1 – 9 are shown. * p

    Article Snippet: The PCR product cleaved with KpnI and XhoI was cloned into the pGL3-basic luciferase reporter and digested with KpnI and XhoI to generate the pGL3_GLB1 promoter.

    Techniques: Transfection, Luciferase, Activity Assay