Structured Review

Promega pgl3 basic vector
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 <t>pGL3-Basic-AKR1C1-P</t> 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.
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Images

1) Product Images from "Hepatitis B Virus X Protein Up-Regulates AKR1C1 Expression Through Nuclear Factor-Y in Human Hepatocarcinoma Cells"

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

Journal: Hepatitis Monthly

doi: 10.5812/hepatmon.8792

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.
Figure Legend 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.

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

2) Product Images from "TCF/?-catenin plays an important role in HCCR-1 oncogene expression"

Article Title: TCF/?-catenin plays an important role in HCCR-1 oncogene expression

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-10-42

Mutational analysis of the HCCR-1 promoter . The diagrams depict locations of the predicted transcriptional elements in the HCCR-1 promoter. When mutated, the diagrams are indicated by a cross. 4 per construct). " title="... 100%) displayed by cells co-transfected with pRL-CMV and pGL3-control ( n > 4 per construct). " property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: Mutational analysis of the HCCR-1 promoter . The diagrams depict locations of the predicted transcriptional elements in the HCCR-1 promoter. When mutated, the diagrams are indicated by a cross. "Tcf" and "My" indicate the consensus sequences for the TCF and C-Myb elements, respectively. Each luciferase reporter construct was co-transfected in K562 cells with pRL-CMV. The horizontal axis shows the ratio of luciferase (LUC) to renilla (REN) activity normalized by LUC/REN (= 100%) displayed by cells co-transfected with pRL-CMV and pGL3-control ( n > 4 per construct).

Techniques Used: Luciferase, Construct, Transfection, Activity Assay

Expression of the truncated HCCR-1 promoter in K562, HEK/293, or A549 cells . The DNA constructs containing various lengths of the HCCR-1 promoter region were cloned into upstream of the firefly luciferase (LUC) reporter gene. Each luciferase reporter construct shown in the diagram was co-transfected in K562 ( A and B ; filled bars), HEK293 ( A ; open bars), and A549 ( A ; shadow bars) cells with the pRL-CMV normalizing reporter plasmid encoding with the renilla (REN) gene. The horizontal axis shows the ratio of luciferase to renilla activity normalized by LUC/REN (= 100%) displayed by cells co-transfected with pRL-CMV and pGL3-control. We designated each recombinant vector as pGL3X~Y, where X is the first base and Y the last base of each truncated promoter ( n > 5 per construct).
Figure Legend Snippet: Expression of the truncated HCCR-1 promoter in K562, HEK/293, or A549 cells . The DNA constructs containing various lengths of the HCCR-1 promoter region were cloned into upstream of the firefly luciferase (LUC) reporter gene. Each luciferase reporter construct shown in the diagram was co-transfected in K562 ( A and B ; filled bars), HEK293 ( A ; open bars), and A549 ( A ; shadow bars) cells with the pRL-CMV normalizing reporter plasmid encoding with the renilla (REN) gene. The horizontal axis shows the ratio of luciferase to renilla activity normalized by LUC/REN (= 100%) displayed by cells co-transfected with pRL-CMV and pGL3-control. We designated each recombinant vector as pGL3X~Y, where X is the first base and Y the last base of each truncated promoter ( n > 5 per construct).

Techniques Used: Expressing, Construct, Clone Assay, Luciferase, Transfection, Plasmid Preparation, Activity Assay, Recombinant

3) Product Images from "VEGF Promotes the Transcription of the Human PRL-3 Gene in HUVEC through Transcription Factor MEF2C"

Article Title: VEGF Promotes the Transcription of the Human PRL-3 Gene in HUVEC through Transcription Factor MEF2C

Journal: PLoS ONE

doi: 10.1371/journal.pone.0027165

MEF2 binding sites are critical for the promoter activity of PRL-3 . ( A ) The region between −158 and −33 is important for the transcriptional activity of the PRL-3 promoter, as measured by luciferase activity. ( B ) The effect of site-directed mutations in the MEF2 binding sites, as measured by luciferase activity. All luciferase activity were normalized to β-galactosidase activity and standardized to the normalized activity of pGL3-Basic. Each column represents the mean ± SD of three independent experiments. ** significantly different from the control at P
Figure Legend Snippet: MEF2 binding sites are critical for the promoter activity of PRL-3 . ( A ) The region between −158 and −33 is important for the transcriptional activity of the PRL-3 promoter, as measured by luciferase activity. ( B ) The effect of site-directed mutations in the MEF2 binding sites, as measured by luciferase activity. All luciferase activity were normalized to β-galactosidase activity and standardized to the normalized activity of pGL3-Basic. Each column represents the mean ± SD of three independent experiments. ** significantly different from the control at P

Techniques Used: Binding Assay, Activity Assay, Luciferase

4) Product Images from "Transcriptional regulation of the potential tumor suppressor ABI3 gene in thyroid carcinomas: interplay between methylation and NKX2-1 availability"

Article Title: Transcriptional regulation of the potential tumor suppressor ABI3 gene in thyroid carcinomas: interplay between methylation and NKX2-1 availability

Journal: Oncotarget

doi: 10.18632/oncotarget.8416

( A ) Relationship between NKX2-1 and R1 methylation in the ABI3 expression in 5-aza-dC treated cells. ABI3 expression was restored in follicular carcinoma cell lines (FTC 238, FTC 236, FTC 133 and WRO) when NKX2-1 was present and R1 demethylated. In melanoma cells (NPA) the treatment with 5-aza-dC did not restore the expression of the ABI3 in NPA cells. ( B ) Transient transfection of NKX2-1 into NPA cells restored ABI3 expression. ( C ) Luciferase reporter assays showing the regulatory effect of R1 region of ABI3 promoter (white bars) compared to the pGL3-Control empty vector (black bars). Luciferase activity was increased after transfection of FTC 238 cells with pGL-control-R1 plasmid (white bars) as compared with cells transfected with pGL-control vector (black bars). The transcriptional activity was significantly increased when the cells were co-transfected with NKX2-1. NTC: no-template control.
Figure Legend Snippet: ( A ) Relationship between NKX2-1 and R1 methylation in the ABI3 expression in 5-aza-dC treated cells. ABI3 expression was restored in follicular carcinoma cell lines (FTC 238, FTC 236, FTC 133 and WRO) when NKX2-1 was present and R1 demethylated. In melanoma cells (NPA) the treatment with 5-aza-dC did not restore the expression of the ABI3 in NPA cells. ( B ) Transient transfection of NKX2-1 into NPA cells restored ABI3 expression. ( C ) Luciferase reporter assays showing the regulatory effect of R1 region of ABI3 promoter (white bars) compared to the pGL3-Control empty vector (black bars). Luciferase activity was increased after transfection of FTC 238 cells with pGL-control-R1 plasmid (white bars) as compared with cells transfected with pGL-control vector (black bars). The transcriptional activity was significantly increased when the cells were co-transfected with NKX2-1. NTC: no-template control.

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

5) Product Images from "Synergistic impact of mutations in Hepatitis B Virus genome contribute to its occult phenotype in chronic Hepatitis C Virus carriers"

Article Title: Synergistic impact of mutations in Hepatitis B Virus genome contribute to its occult phenotype in chronic Hepatitis C Virus carriers

Journal: Scientific Reports

doi: 10.1038/s41598-017-09965-w

Effects of substitutions in Enh-II on enhancer activity, pgRNA expression and HBV-DNA level. ( A ) Relative luciferase activities of the constructs pGL3-P-wt-Enh-II and pGL3-P-mt-Enh-II carrying wild-type and mutant Enh-II elements respectively in pGL3-promoter vector. Relative expression of pgRNA ( B ), intracellular HBV-DNA level ( C ) and extracellular HBV-DNA level ( D ), as measured by quantitative real-time PCR, following transfection of the full-length, wild-type HBV/D (HBV/D-wt) and corresponding Enh-II-mutated HBV [HBV/D-mt(Enh-II)] in Huh7 cells. Paired t-test p values; *p
Figure Legend Snippet: Effects of substitutions in Enh-II on enhancer activity, pgRNA expression and HBV-DNA level. ( A ) Relative luciferase activities of the constructs pGL3-P-wt-Enh-II and pGL3-P-mt-Enh-II carrying wild-type and mutant Enh-II elements respectively in pGL3-promoter vector. Relative expression of pgRNA ( B ), intracellular HBV-DNA level ( C ) and extracellular HBV-DNA level ( D ), as measured by quantitative real-time PCR, following transfection of the full-length, wild-type HBV/D (HBV/D-wt) and corresponding Enh-II-mutated HBV [HBV/D-mt(Enh-II)] in Huh7 cells. Paired t-test p values; *p

Techniques Used: Activity Assay, Expressing, Luciferase, Construct, Mutagenesis, Plasmid Preparation, Real-time Polymerase Chain Reaction, Transfection

Effects of substitutions in X-promoter on promoter activity, pgRNA expression and HBV-DNA level. ( A ) Relative luciferase activities of the constructs pGL3-B-HBx-promoter-wt and pGL3-B-HBx-promoter-mt carrying wild-type and mutated X-promoter respectively cloned in pGL3-Basic vector. Relative expression of pgRNA ( B ) and intracellular HBV-DNA level ( C ) as measured by quantitative real-time PCR, following transfection of the full-length, wild-type HBV/D (HBV/D-wt) and corresponding X-promoter-mutated HBV [HBV/D-mt(X-prom)] in Huh7 cells. Paired t-test p values; *p
Figure Legend Snippet: Effects of substitutions in X-promoter on promoter activity, pgRNA expression and HBV-DNA level. ( A ) Relative luciferase activities of the constructs pGL3-B-HBx-promoter-wt and pGL3-B-HBx-promoter-mt carrying wild-type and mutated X-promoter respectively cloned in pGL3-Basic vector. Relative expression of pgRNA ( B ) and intracellular HBV-DNA level ( C ) as measured by quantitative real-time PCR, following transfection of the full-length, wild-type HBV/D (HBV/D-wt) and corresponding X-promoter-mutated HBV [HBV/D-mt(X-prom)] in Huh7 cells. Paired t-test p values; *p

Techniques Used: Activity Assay, Expressing, Luciferase, Construct, Clone Assay, Plasmid Preparation, Real-time Polymerase Chain Reaction, Transfection

Effects of mutations in PreS2 and X-ORFs as well as in Enh-II on generation of ER stress by analyzing GRP78-promoter activity. The full-length, wild-type HBV (HBV/D-wt) along with the mutant variants harbouring mutations in (i) PreS2 and X-ORF [HBV/D-mt(PreS2 + HBX)] and (ii) Enh-II element [HBV/D-mt(Enh-II)] were co-transfected with pGL3-GRP78-promoter construct separately in Huh7 cells and the relative luciferase activities were determined after 48 hrs. Paired t-test p values; *p
Figure Legend Snippet: Effects of mutations in PreS2 and X-ORFs as well as in Enh-II on generation of ER stress by analyzing GRP78-promoter activity. The full-length, wild-type HBV (HBV/D-wt) along with the mutant variants harbouring mutations in (i) PreS2 and X-ORF [HBV/D-mt(PreS2 + HBX)] and (ii) Enh-II element [HBV/D-mt(Enh-II)] were co-transfected with pGL3-GRP78-promoter construct separately in Huh7 cells and the relative luciferase activities were determined after 48 hrs. Paired t-test p values; *p

Techniques Used: Activity Assay, Mutagenesis, Transfection, Construct, Luciferase

6) Product Images from "The expression of AURKA is androgen regulated in castration-resistant prostate cancer"

Article Title: The expression of AURKA is androgen regulated in castration-resistant prostate cancer

Journal: Scientific Reports

doi: 10.1038/s41598-017-18210-3

Activity of ARBS in promoter and intronic regions of AURKA . A luciferase assay was performed in LNCaP-ARhi cells with different DHT concentrations (0, 10 or 100 nM). Cells were transfected with pGL3-basic-LUC (LUC), pGL3-PSA5.8-LUC (PSA), pGL3-AURKApromoter-LUC (AURKA) and pGL3-AURKAenhancer-LUC (AURKA enh.), and luciferase activities were normalized to the renilla control plasmid (LUC). ( a ) Activity of AR binding to the AURKA promoter and positive (PSA) as well as negative control regions with different androgen stimulation. ( b ) Activity of AR binding to the AURKA enhancer and promoter with enhancer region and positive (PSA) and negative control (pGL3) regions with different androgen stimulation. * P
Figure Legend Snippet: Activity of ARBS in promoter and intronic regions of AURKA . A luciferase assay was performed in LNCaP-ARhi cells with different DHT concentrations (0, 10 or 100 nM). Cells were transfected with pGL3-basic-LUC (LUC), pGL3-PSA5.8-LUC (PSA), pGL3-AURKApromoter-LUC (AURKA) and pGL3-AURKAenhancer-LUC (AURKA enh.), and luciferase activities were normalized to the renilla control plasmid (LUC). ( a ) Activity of AR binding to the AURKA promoter and positive (PSA) as well as negative control regions with different androgen stimulation. ( b ) Activity of AR binding to the AURKA enhancer and promoter with enhancer region and positive (PSA) and negative control (pGL3) regions with different androgen stimulation. * P

Techniques Used: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Binding Assay, Negative Control

7) Product Images from "Molecular mechanisms governing microRNA-125a expression in human hepatocellular carcinoma cells"

Article Title: Molecular mechanisms governing microRNA-125a expression in human hepatocellular carcinoma cells

Journal: Scientific Reports

doi: 10.1038/s41598-017-11418-3

Isolation of miR-125a promoter. ( A ) Two major transcripts of SPACA6 gene and map of its genomic locus; exons and microRNAs are indicated by black boxes and loops, respectively; the first base of pre-miR-99b was assigned as nucleotide 1 ( B ) Five genomic DNA segments (grey bars in the left side of the panel) spanning nucleotides -36 to -3875 were cloned in the luciferase reporter plasmid pGL3-basic and assayed for transcription promoter activity in HepG2 cells. The reporter constructs are named according to the size of the cloned genomic fragment and their activity is reported in the adjacent plot; 869mut construct carries two point mutations (marked by white dots) eliminating putative translation start sites. Assays were performed at least in triplicate and expressed as mean ± SD.
Figure Legend Snippet: Isolation of miR-125a promoter. ( A ) Two major transcripts of SPACA6 gene and map of its genomic locus; exons and microRNAs are indicated by black boxes and loops, respectively; the first base of pre-miR-99b was assigned as nucleotide 1 ( B ) Five genomic DNA segments (grey bars in the left side of the panel) spanning nucleotides -36 to -3875 were cloned in the luciferase reporter plasmid pGL3-basic and assayed for transcription promoter activity in HepG2 cells. The reporter constructs are named according to the size of the cloned genomic fragment and their activity is reported in the adjacent plot; 869mut construct carries two point mutations (marked by white dots) eliminating putative translation start sites. Assays were performed at least in triplicate and expressed as mean ± SD.

Techniques Used: Isolation, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay, Construct

8) Product Images from "Cloning and Transcriptional Activity Analysis of the Porcine Abcb1 Gene Promoter: Transcription Factor Sp1 Regulates the Expression of Porcine Abcb1"

Article Title: Cloning and Transcriptional Activity Analysis of the Porcine Abcb1 Gene Promoter: Transcription Factor Sp1 Regulates the Expression of Porcine Abcb1

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00373

The effect of Sp1 knockdown on pig Abcb1 promoter activity and endogenous expression of pig P-gp expression in IPEC-J2 cells. (A) Downregulation of Sp1 protein level by specific siRNAs in IPEC-J2 cells was confirmed by Western blot, as compared to the negative control (NC) siRNA. (B) Semi-quantification of Western blot. (C) IPEC-J2 cells were co-transfected with pGL3-D4 construct and Sp1 siRNA or NC siRNA for luciferase assays. The Renilla luciferase reporter plasmid pRL-TK (Promega) was used as an internal control to estimate the transfection efficiency. ∗∗ p
Figure Legend Snippet: The effect of Sp1 knockdown on pig Abcb1 promoter activity and endogenous expression of pig P-gp expression in IPEC-J2 cells. (A) Downregulation of Sp1 protein level by specific siRNAs in IPEC-J2 cells was confirmed by Western blot, as compared to the negative control (NC) siRNA. (B) Semi-quantification of Western blot. (C) IPEC-J2 cells were co-transfected with pGL3-D4 construct and Sp1 siRNA or NC siRNA for luciferase assays. The Renilla luciferase reporter plasmid pRL-TK (Promega) was used as an internal control to estimate the transfection efficiency. ∗∗ p

Techniques Used: Activity Assay, Expressing, Western Blot, Negative Control, Transfection, Construct, Luciferase, Plasmid Preparation

9) Product Images from "A Functional Polymorphism (rs937283) in the MDM2 Promoter Region is Associated with Poor Prognosis of Retinoblastoma in Chinese Han Population"

Article Title: A Functional Polymorphism (rs937283) in the MDM2 Promoter Region is Associated with Poor Prognosis of Retinoblastoma in Chinese Han Population

Journal: Scientific Reports

doi: 10.1038/srep31240

Effect of MDM2 rs937283 polymorphism in the MDM2 promoter activity. Schematic representation of reporter plasmids containing A or G allele at rs937283, which was inserted into upstream of the luciferase reporter gene in the pGL3 Basic plasmid. pRL-SV40 were cotransfected into Y79, WERI-Rb1, HeLa cells as the internal control of Renilla luciferase. Columns, mean from three independent experiments; bars, standard deviation. *P
Figure Legend Snippet: Effect of MDM2 rs937283 polymorphism in the MDM2 promoter activity. Schematic representation of reporter plasmids containing A or G allele at rs937283, which was inserted into upstream of the luciferase reporter gene in the pGL3 Basic plasmid. pRL-SV40 were cotransfected into Y79, WERI-Rb1, HeLa cells as the internal control of Renilla luciferase. Columns, mean from three independent experiments; bars, standard deviation. *P

Techniques Used: Activity Assay, Luciferase, Plasmid Preparation, Standard Deviation

10) Product Images from "Agrin Influences Botulinum Neurotoxin A-Induced Nerve Sprouting via miR-144-agrin-MuSK Signaling"

Article Title: Agrin Influences Botulinum Neurotoxin A-Induced Nerve Sprouting via miR-144-agrin-MuSK Signaling

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00015

miR-144 regulates agrin expression by directly targeting the 3′ UTR of agrin mRNA. (A) Schematic representation of three putative miRNA-binding sites on the agrin 3′ UTR sequence. (B) The 3′ UTR fragment of agrin was cloned into the Xbal restriction site downstream of the firefly luciferase gene of the pGL3-Basic vector. Luciferase activity of each sample was measured 48h after transfection and normalized to Renilla luciferase activity. pGL3-Basic vector was used as control. Graph error bars indicate SD calculated from at least three independent experiments. * P
Figure Legend Snippet: miR-144 regulates agrin expression by directly targeting the 3′ UTR of agrin mRNA. (A) Schematic representation of three putative miRNA-binding sites on the agrin 3′ UTR sequence. (B) The 3′ UTR fragment of agrin was cloned into the Xbal restriction site downstream of the firefly luciferase gene of the pGL3-Basic vector. Luciferase activity of each sample was measured 48h after transfection and normalized to Renilla luciferase activity. pGL3-Basic vector was used as control. Graph error bars indicate SD calculated from at least three independent experiments. * P

Techniques Used: Expressing, Binding Assay, Sequencing, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay, Transfection

11) Product Images from "Cellular and Viral Factors Regulate the Varicella-Zoster Virus gE Promoter during Viral Replication ▿"

Article Title: Cellular and Viral Factors Regulate the Varicella-Zoster Virus gE Promoter during Viral Replication ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00553-07

Elements of the gE promoter region required for basal activity. (A) Schematic representation of the gI-gE intergenic region. The three deletions ΔI, ΔII, ΔIII, the two Sp1 binding sites (Sp1-A and Sp1-B), and the putative TATA-box (in boldface and italics) are indicated. The arrow indicates the transcriptional start site, and the rectangle encloses the gE ATG. (B) Effect of stepwise deletions on the transcriptional activity of the gE promoter. The bars indicate the means ± the standard deviations of three independent transfections done in duplicate. ut, untransfected cells; pGL3, cells transfected with the luciferase vector without any promoter.
Figure Legend Snippet: Elements of the gE promoter region required for basal activity. (A) Schematic representation of the gI-gE intergenic region. The three deletions ΔI, ΔII, ΔIII, the two Sp1 binding sites (Sp1-A and Sp1-B), and the putative TATA-box (in boldface and italics) are indicated. The arrow indicates the transcriptional start site, and the rectangle encloses the gE ATG. (B) Effect of stepwise deletions on the transcriptional activity of the gE promoter. The bars indicate the means ± the standard deviations of three independent transfections done in duplicate. ut, untransfected cells; pGL3, cells transfected with the luciferase vector without any promoter.

Techniques Used: Activity Assay, Binding Assay, Transfection, Luciferase, Plasmid Preparation

12) Product Images from "Regulation of a Novel Androgen Receptor Target Gene, the Cyclin B1 Gene, through Androgen-Dependent E2F Family Member Switching"

Article Title: Regulation of a Novel Androgen Receptor Target Gene, the Cyclin B1 Gene, through Androgen-Dependent E2F Family Member Switching

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.06663-11

Interaction between AR and E2F1 facilitates nuclear E2F4 binding to E2F binding site. (A) AR interacts with E2F1 only in the presence of androgen by co-IP. Using AR as a pulldown antibody and E2F1 as detection antibody (lane 1) or using E2F1 as the pulldown antibody and AR as the detection antibody (lane 2), AR interacts with E2F1 only in the presence of androgen (10 nM). (B) AR interacts with E2F4 only in the absence of androgen by co-IP. Interaction between AR and E2F4 was not detected (lanes 3 and 4) in the presence of androgen and was present only in the absence of androgen. (B to D) Recruitment of E2F1 and E2F4 on the E2F site and the ARE region was detected by reporter ChIP with 130bp-LUC (containing an E2F site but without AREs) and 70bp-LUC (including AREs but excluding an E2F site). pGL34XAREE4 (containing four AREs upstream of the E4 promoter) and pGL3-basic served as positive and negative controls, respectively. (B) With AR immunoprecipitation, the 70bp-LUC gave positive signal only in the presence of androgen, while the 130bp-LUC showed no signal regardless of androgen. With E2F1 immunoprecipitation, the 70bp-LUC gave signal in the presence of androgen as that from AR. (C) Meanwhile, E2F1 showed dramatically decreased recruitment on the 130bp-LUC in the presence of androgen. (D) With E2F4 immunoprecipitation, the 130bp-LUC gave a positive signal, while the 70bp-LUC showed no signal in the presence of androgen. (E) Cell fraction showed nuclear (N) and cytoplasmic (C) localization of AR (nuclear localization in the presence of androgen and cytoplasmic localization in the absence of androgen; lane 1), E2F1 (nuclear localization in the presence and absence of androgen; lane 2), and E2F4 (nuclear localization in the presence of androgen and cytoplasmic localization in the absence of androgen; lane 3) by cell fractionation. H4 serves as a control for nuclear protein, and β-actin serves as a control for cytoplasmic protein. (F) E2F4 shows interaction with SMRT by co-IP regardless of androgen, using SMRT as the pulldown antibody and E2F4 as detection antibody (lane 1) or using E2F4 as pulldown antibody and SMRT as the detection antibody (lane 2). The interaction between E2F4 and SMRT appears to be stronger in the presence of androgen. SMRT shows interaction with AR in the absence of androgen, either using SMRT as the pulldown antibody and AR as detection antibody (lane 3) or using AR as the pulldown antibody and SMRT as the detection antibody (lane 4). E2F4 shows interaction with HDAC3 by co-IP regardless of androgen status, using HDAC3 as the pulldown antibody and E2F4 as the detection antibody (lane 5) or using E2F4 as the pulldown antibody and HDAC3 as the detection antibody (lane 6). (G) SMRT and HDAC3 were recruited to the E2F binding site in the presence of androgen by ChIP with primers for E2F site amplification. (H and I) HDAC inhibitor (SAHA) reverses the androgen-mediated transcription repression of cyclin B1 promoter in PShTertAR cells (I) that is seen in PShTert cells (H). (J) SMRT serves as a corepressor for E2F4 repression in the luciferase assay with increased SMRT. The 130-bp cyclin B1 promoter reporter (only containing the E2F binding site and lacking ARE) was used in the assay. (K) SMRT knockdown by siRNA released the transcriptional repression by E2F4. (L and M) ChIP assay showed that the recruitment of acetyl-histone H3, acetyl-histone H4, and PolII to the cyclin B1 promoter region was dramatically reduced in the presence of androgen. UTR, untranslated region.
Figure Legend Snippet: Interaction between AR and E2F1 facilitates nuclear E2F4 binding to E2F binding site. (A) AR interacts with E2F1 only in the presence of androgen by co-IP. Using AR as a pulldown antibody and E2F1 as detection antibody (lane 1) or using E2F1 as the pulldown antibody and AR as the detection antibody (lane 2), AR interacts with E2F1 only in the presence of androgen (10 nM). (B) AR interacts with E2F4 only in the absence of androgen by co-IP. Interaction between AR and E2F4 was not detected (lanes 3 and 4) in the presence of androgen and was present only in the absence of androgen. (B to D) Recruitment of E2F1 and E2F4 on the E2F site and the ARE region was detected by reporter ChIP with 130bp-LUC (containing an E2F site but without AREs) and 70bp-LUC (including AREs but excluding an E2F site). pGL34XAREE4 (containing four AREs upstream of the E4 promoter) and pGL3-basic served as positive and negative controls, respectively. (B) With AR immunoprecipitation, the 70bp-LUC gave positive signal only in the presence of androgen, while the 130bp-LUC showed no signal regardless of androgen. With E2F1 immunoprecipitation, the 70bp-LUC gave signal in the presence of androgen as that from AR. (C) Meanwhile, E2F1 showed dramatically decreased recruitment on the 130bp-LUC in the presence of androgen. (D) With E2F4 immunoprecipitation, the 130bp-LUC gave a positive signal, while the 70bp-LUC showed no signal in the presence of androgen. (E) Cell fraction showed nuclear (N) and cytoplasmic (C) localization of AR (nuclear localization in the presence of androgen and cytoplasmic localization in the absence of androgen; lane 1), E2F1 (nuclear localization in the presence and absence of androgen; lane 2), and E2F4 (nuclear localization in the presence of androgen and cytoplasmic localization in the absence of androgen; lane 3) by cell fractionation. H4 serves as a control for nuclear protein, and β-actin serves as a control for cytoplasmic protein. (F) E2F4 shows interaction with SMRT by co-IP regardless of androgen, using SMRT as the pulldown antibody and E2F4 as detection antibody (lane 1) or using E2F4 as pulldown antibody and SMRT as the detection antibody (lane 2). The interaction between E2F4 and SMRT appears to be stronger in the presence of androgen. SMRT shows interaction with AR in the absence of androgen, either using SMRT as the pulldown antibody and AR as detection antibody (lane 3) or using AR as the pulldown antibody and SMRT as the detection antibody (lane 4). E2F4 shows interaction with HDAC3 by co-IP regardless of androgen status, using HDAC3 as the pulldown antibody and E2F4 as the detection antibody (lane 5) or using E2F4 as the pulldown antibody and HDAC3 as the detection antibody (lane 6). (G) SMRT and HDAC3 were recruited to the E2F binding site in the presence of androgen by ChIP with primers for E2F site amplification. (H and I) HDAC inhibitor (SAHA) reverses the androgen-mediated transcription repression of cyclin B1 promoter in PShTertAR cells (I) that is seen in PShTert cells (H). (J) SMRT serves as a corepressor for E2F4 repression in the luciferase assay with increased SMRT. The 130-bp cyclin B1 promoter reporter (only containing the E2F binding site and lacking ARE) was used in the assay. (K) SMRT knockdown by siRNA released the transcriptional repression by E2F4. (L and M) ChIP assay showed that the recruitment of acetyl-histone H3, acetyl-histone H4, and PolII to the cyclin B1 promoter region was dramatically reduced in the presence of androgen. UTR, untranslated region.

Techniques Used: Binding Assay, Co-Immunoprecipitation Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Cell Fractionation, Amplification, Luciferase

Novel androgen-responsive element identified on cyclin B1 promoter. (A) Promoter occupancy of AR on cyclin B1 promoter by ChIP. Immunoprecipitation was performed with 2 μg of anti-AR antibody. The p21 promoter region was selected as a positive control for AR recruitment, and the ß-actin promoter region served as a negative control. AR was recruited to the promoter of cyclin B1 and p21 in the presence of androgen, while no recruitment to the promoter region of ß-actin upon androgen treatment was seen. (B) Dual-luciferase assays with 1kb-LUC, 0.8 kb-LUC, 0.6 kb-LUC, 0.4 kb-LUC, and 0.2 kb-LUC reporter constructs showed transcriptional repression by AR in the presence of androgen. (C) Schematic representation of a putative binding site for transcription factor, ARE sequences on the cyclin B1 promoter, and construction of luciferase reporter (200bp-LUC, 130bp-LUC, 70bp-LUC) with fragments of the cyclin B1 promoter. (D) Luciferase assay for reporter constructions 200bp-LUC and 130bp-LUC. One hundred ng 200bp-LUC or 130bp-LUC was transfected into PShTertAR cells in the absence or presence of ligand (R1881; 0.1, 1, and 10 nM). 200bp-LUC showed induction, while the 130bp-LUC luciferase reporter lost induction upon treatment with R1881. (E) Reporter ChIP was performed with the 200bp-LUC, 130bp-LUC, and 70bp-LUC luciferase reporter plasmids. The luciferase reporter pGL4AREE4 (containing four AREs upstream of the E4 promoter) served as a positive control and the empty luciferase pGL3 basic as a negative control. The results show that AR binds to the 200-bp (left) and the proximal 70-bp (left) but not the distal 130-bp cyclin B1 promoter in the presence of androgen. (F) Luciferase assay was performed with wild-type 200bp-LUC and mutated 200bp-LUC AREs. Two mutations (TT to AA) in the putative ARE abolish the AR-mediated transcriptional repression of the cyclin B1 promoter.
Figure Legend Snippet: Novel androgen-responsive element identified on cyclin B1 promoter. (A) Promoter occupancy of AR on cyclin B1 promoter by ChIP. Immunoprecipitation was performed with 2 μg of anti-AR antibody. The p21 promoter region was selected as a positive control for AR recruitment, and the ß-actin promoter region served as a negative control. AR was recruited to the promoter of cyclin B1 and p21 in the presence of androgen, while no recruitment to the promoter region of ß-actin upon androgen treatment was seen. (B) Dual-luciferase assays with 1kb-LUC, 0.8 kb-LUC, 0.6 kb-LUC, 0.4 kb-LUC, and 0.2 kb-LUC reporter constructs showed transcriptional repression by AR in the presence of androgen. (C) Schematic representation of a putative binding site for transcription factor, ARE sequences on the cyclin B1 promoter, and construction of luciferase reporter (200bp-LUC, 130bp-LUC, 70bp-LUC) with fragments of the cyclin B1 promoter. (D) Luciferase assay for reporter constructions 200bp-LUC and 130bp-LUC. One hundred ng 200bp-LUC or 130bp-LUC was transfected into PShTertAR cells in the absence or presence of ligand (R1881; 0.1, 1, and 10 nM). 200bp-LUC showed induction, while the 130bp-LUC luciferase reporter lost induction upon treatment with R1881. (E) Reporter ChIP was performed with the 200bp-LUC, 130bp-LUC, and 70bp-LUC luciferase reporter plasmids. The luciferase reporter pGL4AREE4 (containing four AREs upstream of the E4 promoter) served as a positive control and the empty luciferase pGL3 basic as a negative control. The results show that AR binds to the 200-bp (left) and the proximal 70-bp (left) but not the distal 130-bp cyclin B1 promoter in the presence of androgen. (F) Luciferase assay was performed with wild-type 200bp-LUC and mutated 200bp-LUC AREs. Two mutations (TT to AA) in the putative ARE abolish the AR-mediated transcriptional repression of the cyclin B1 promoter.

Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Positive Control, Negative Control, Luciferase, Construct, Binding Assay, Transfection

13) Product Images from "Reversal of Xenopus Oct25 Function by Disruption of the POU Domain Structure *"

Article Title: Reversal of Xenopus Oct25 Function by Disruption of the POU Domain Structure *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.064386

Analysis of the Oct25ΔPOU(273–301) effect on gene transcription. A , injection of 1 ng of Oct25ΔPOU(273–301) RNA leads to an up-regulation of genes that induce mesoderm and endoderm and dorsalize body axis but does not generate an appreciable effect on the genes in the BMP pathway ( BMP4 , Xvent1 , and Xvent2 ). B , injection of 1 ng of Oct25ΔPOU(273–301) RNA also increases the transcription of mesendoderm inducing genes and germ layer dorsalizing genes in animal caps. C , Oct25ΔPOU(273–301) stimulates luciferase reporter activity for the promoters of Gsc (GscLuc(−1500)), Xnr1 (Xnr1Luc(−907)), Xnr3 (Xnr3Luc), Sia (SiaLuc(−802)), the artificial promoter composed of six repeats of the distal element on Gsc promoter ( 6 × DE ), and the Wnt-responsive artificial promoter reporter TopFlash. For control, Oct25ΔPOU(273–301) does not have any strong effect on the pGL3-basic vector or on FopFlash, the negative control reporter for TopFlash. In each luciferase assay, 40 pg of reporter plasmid and 400 pg of RNA were injected. D–F , induction of genes by Oct25ΔPOU(273–301) is severely compromised after blocking the activities of VegT, activin/nodal, and Wnt/β-catenin signaling pathways. D , specific knockdown of VegT by 40 ng of an antisense morpholino (VegTMO) leads to reduced transcription of Gsc , Chd , Xnr1 , Xnr2 , and Sia . Injection of 1 ng of Oct25ΔPOU(273–301) RNA results in strong up-regulation of these genes. Up-regulation is lost when the RNA is co-injected with VegTMO. E , blocking of the nodal/activin signaling pathway by injection of 800 pg of a dominant-negative activin receptor I (dnXAR1) reduces the transcription of genes, like Gsc , Chd , Xnr1 , and Xnr2 , although injection of 1 ng of Oct25ΔPOU(273–301) RNA stimulates these genes. This stimulation is weakened by co-injection of dnXAR1 and Oct25ΔPOU(273–301) RNAs. F , when the Wnt/β-catenin signaling pathway is inhibited by injection of 400 pg of dominant-negative TCF3 (dnTCF3) RNA, transcription of Chd , Gsc , Xnrs , and Sia is dramatically decreased. Consequently, co-injection of 1 ng of Oct25ΔPOU(273–301) cannot stimulate the transcription of these genes anymore.
Figure Legend Snippet: Analysis of the Oct25ΔPOU(273–301) effect on gene transcription. A , injection of 1 ng of Oct25ΔPOU(273–301) RNA leads to an up-regulation of genes that induce mesoderm and endoderm and dorsalize body axis but does not generate an appreciable effect on the genes in the BMP pathway ( BMP4 , Xvent1 , and Xvent2 ). B , injection of 1 ng of Oct25ΔPOU(273–301) RNA also increases the transcription of mesendoderm inducing genes and germ layer dorsalizing genes in animal caps. C , Oct25ΔPOU(273–301) stimulates luciferase reporter activity for the promoters of Gsc (GscLuc(−1500)), Xnr1 (Xnr1Luc(−907)), Xnr3 (Xnr3Luc), Sia (SiaLuc(−802)), the artificial promoter composed of six repeats of the distal element on Gsc promoter ( 6 × DE ), and the Wnt-responsive artificial promoter reporter TopFlash. For control, Oct25ΔPOU(273–301) does not have any strong effect on the pGL3-basic vector or on FopFlash, the negative control reporter for TopFlash. In each luciferase assay, 40 pg of reporter plasmid and 400 pg of RNA were injected. D–F , induction of genes by Oct25ΔPOU(273–301) is severely compromised after blocking the activities of VegT, activin/nodal, and Wnt/β-catenin signaling pathways. D , specific knockdown of VegT by 40 ng of an antisense morpholino (VegTMO) leads to reduced transcription of Gsc , Chd , Xnr1 , Xnr2 , and Sia . Injection of 1 ng of Oct25ΔPOU(273–301) RNA results in strong up-regulation of these genes. Up-regulation is lost when the RNA is co-injected with VegTMO. E , blocking of the nodal/activin signaling pathway by injection of 800 pg of a dominant-negative activin receptor I (dnXAR1) reduces the transcription of genes, like Gsc , Chd , Xnr1 , and Xnr2 , although injection of 1 ng of Oct25ΔPOU(273–301) RNA stimulates these genes. This stimulation is weakened by co-injection of dnXAR1 and Oct25ΔPOU(273–301) RNAs. F , when the Wnt/β-catenin signaling pathway is inhibited by injection of 400 pg of dominant-negative TCF3 (dnTCF3) RNA, transcription of Chd , Gsc , Xnrs , and Sia is dramatically decreased. Consequently, co-injection of 1 ng of Oct25ΔPOU(273–301) cannot stimulate the transcription of these genes anymore.

Techniques Used: Injection, Luciferase, Activity Assay, Plasmid Preparation, Negative Control, Blocking Assay, Dominant Negative Mutation

14) Product Images from "Expression profile and promoter analysis of HEPIS"

Article Title: Expression profile and promoter analysis of HEPIS

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2017.5374

Cloning and activity of pHEPIS. (A) Six different truncated pHEPISs were cloned into a pGL3-basic LUC expression vector. These plasmids were designated as pHEPIS-1.7K, pHEPIS-1.6K, pHEPIS-1.4K, pHEPIS-1.3K, pHEPIS-1.1K and pHEPIS-0.6K. (B) Dual LUC activity assays of six pHEPIS constructs. Six recombinant vectors containing pHEPISs of different lengths and pRL-TK were cotransfected into 293T cells. *P
Figure Legend Snippet: Cloning and activity of pHEPIS. (A) Six different truncated pHEPISs were cloned into a pGL3-basic LUC expression vector. These plasmids were designated as pHEPIS-1.7K, pHEPIS-1.6K, pHEPIS-1.4K, pHEPIS-1.3K, pHEPIS-1.1K and pHEPIS-0.6K. (B) Dual LUC activity assays of six pHEPIS constructs. Six recombinant vectors containing pHEPISs of different lengths and pRL-TK were cotransfected into 293T cells. *P

Techniques Used: Clone Assay, Activity Assay, Expressing, Plasmid Preparation, Construct, Recombinant

15) Product Images from "AC and AG dinucleotide repeats in the PAX6 P1 promoter are associated with high myopia"

Article Title: AC and AG dinucleotide repeats in the PAX6 P1 promoter are associated with high myopia

Journal: Molecular Vision

doi:

Transcriptional activity of dinucleotide repeats in the PAX6 P1 promoter. A 1,851 bp genomic fragment (from –1278 to +573) containing the PAX6 P1 promoter with different dinucleotide repeats was cloned into an empty pGL3-Basic vector (pGL3) and transfected into ARPE-19 cells. The activity of each allelic construct is expressed relative to the construct (AC) 20 (AG) 6 . Data are represented as mean±SD for five independent experiments. A and B: Immunoblotting results and a bar chart show relative luciferase activity for grouped (AC) m repeats with a stable (AG) 6 . C and D : Immunoblotting results and a bar chart show relative luciferase activity for (AG) n repeats with (AC) 21 . E and F : Immunoblotting results and a bar chart show relative luciferase activity for combined (AC) m (AG) n repeats.
Figure Legend Snippet: Transcriptional activity of dinucleotide repeats in the PAX6 P1 promoter. A 1,851 bp genomic fragment (from –1278 to +573) containing the PAX6 P1 promoter with different dinucleotide repeats was cloned into an empty pGL3-Basic vector (pGL3) and transfected into ARPE-19 cells. The activity of each allelic construct is expressed relative to the construct (AC) 20 (AG) 6 . Data are represented as mean±SD for five independent experiments. A and B: Immunoblotting results and a bar chart show relative luciferase activity for grouped (AC) m repeats with a stable (AG) 6 . C and D : Immunoblotting results and a bar chart show relative luciferase activity for (AG) n repeats with (AC) 21 . E and F : Immunoblotting results and a bar chart show relative luciferase activity for combined (AC) m (AG) n repeats.

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

16) Product Images from "Functional analysis of the C-reactive protein (CRP) gene -717A > G polymorphism associated with coronary heart disease"

Article Title: Functional analysis of the C-reactive protein (CRP) gene -717A > G polymorphism associated with coronary heart disease

Journal: BMC Medical Genetics

doi: 10.1186/1471-2350-10-73

Allelic change from G (nonsusceptible allele) to A (susceptible allele) at the site of rs2794521 located in the promoter of CRP gene enhanced transcriptional activity of the promoter of CRP in HepG2 cells . A , schematic representation of the plasmid constructs. 915 bp fragment of CRP promoter (-858/+57) containing either the A or G allele (i.e. A and G promoter, respectively) was inserted in the pGL3-basic luciferase expression vector. Empty vector: pGL3-basic vector. B , HepG2 cells were contransfected with individual firefly luciferase (FLuc)expressing plasmid (A promoter, G promoter, empty vector) and pRL-TK as described in Materials and Methods. Cells were harvested after 24 hours, and relative luciferase activity (FLuc/Renilla luciferase) was determined. Fold luciferase activity of the variant A construct was 1- to 2-fold higher than that of the common G construct (P
Figure Legend Snippet: Allelic change from G (nonsusceptible allele) to A (susceptible allele) at the site of rs2794521 located in the promoter of CRP gene enhanced transcriptional activity of the promoter of CRP in HepG2 cells . A , schematic representation of the plasmid constructs. 915 bp fragment of CRP promoter (-858/+57) containing either the A or G allele (i.e. A and G promoter, respectively) was inserted in the pGL3-basic luciferase expression vector. Empty vector: pGL3-basic vector. B , HepG2 cells were contransfected with individual firefly luciferase (FLuc)expressing plasmid (A promoter, G promoter, empty vector) and pRL-TK as described in Materials and Methods. Cells were harvested after 24 hours, and relative luciferase activity (FLuc/Renilla luciferase) was determined. Fold luciferase activity of the variant A construct was 1- to 2-fold higher than that of the common G construct (P

Techniques Used: Activity Assay, Plasmid Preparation, Construct, Luciferase, Expressing, Variant Assay

17) Product Images from "Up-regulation of human cervical cancer proto-oncogene contributes to hepatitis B virus-induced malignant transformation of hepatocyte by down-regulating E-cadherin"

Article Title: Up-regulation of human cervical cancer proto-oncogene contributes to hepatitis B virus-induced malignant transformation of hepatocyte by down-regulating E-cadherin

Journal: Oncotarget

doi:

HCCR represses the expression of E-cadherin through its promoter A, B. Relative expression of HCCR and E-cadherin mRNA and protein in Huh-7 cells 48 h after transfection with pcDNA3.1-HCCR or a control vector. C, D. Relative expression of HCCR and E-cadherin mRNA and protein in Huh-7 cells 48 h after transfection with pSilencer-HCCR or a control vector. E. E-cadherin promoter activity in Huh-7 cells transfected with pGL3-E-cadherinP plus pcDNA3.1-HCCR/pcDNA3.1, or with pSilencer-HCCR/pSilencer. Relative expression of HCCR and E-cadherin mRNA was normalized to that of GAPDH. Assays were performed in triplicate. Data are expressed as the mean ± SEM. * P
Figure Legend Snippet: HCCR represses the expression of E-cadherin through its promoter A, B. Relative expression of HCCR and E-cadherin mRNA and protein in Huh-7 cells 48 h after transfection with pcDNA3.1-HCCR or a control vector. C, D. Relative expression of HCCR and E-cadherin mRNA and protein in Huh-7 cells 48 h after transfection with pSilencer-HCCR or a control vector. E. E-cadherin promoter activity in Huh-7 cells transfected with pGL3-E-cadherinP plus pcDNA3.1-HCCR/pcDNA3.1, or with pSilencer-HCCR/pSilencer. Relative expression of HCCR and E-cadherin mRNA was normalized to that of GAPDH. Assays were performed in triplicate. Data are expressed as the mean ± SEM. * P

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

18) Product Images from "Regulation of the Hsp90-binding immunophilin, cyclophilin 40, is mediated by multiple sites for GA-binding protein (GABP)"

Article Title: Regulation of the Hsp90-binding immunophilin, cyclophilin 40, is mediated by multiple sites for GA-binding protein (GABP)

Journal: Cell Stress & Chaperones

doi:

Fig 2. Functional analysis of the human CyP40 5′-flanking region in different cell lines. A schematic representation of the CyP40 promoter constructs with putative transcription factor binding sites is indicated above the histograms. Progressive 5′ deletions of the CyP40 promoter were cloned into the luciferase reporter vector pGL3-Basic and tested for promoter activity by transient transfections of MCF-7, BT-20, HeLa, and A549 cells cotransfected with pcDNA CAT control vector for normalization. Mean luciferase activity of each construct is given relative to that of the pGL3-Basic vector set at a value of 1. The data represent the mean of triplicate (± SD) determinations. The results shown are representative of 2 separate experiments
Figure Legend Snippet: Fig 2. Functional analysis of the human CyP40 5′-flanking region in different cell lines. A schematic representation of the CyP40 promoter constructs with putative transcription factor binding sites is indicated above the histograms. Progressive 5′ deletions of the CyP40 promoter were cloned into the luciferase reporter vector pGL3-Basic and tested for promoter activity by transient transfections of MCF-7, BT-20, HeLa, and A549 cells cotransfected with pcDNA CAT control vector for normalization. Mean luciferase activity of each construct is given relative to that of the pGL3-Basic vector set at a value of 1. The data represent the mean of triplicate (± SD) determinations. The results shown are representative of 2 separate experiments

Techniques Used: Functional Assay, Construct, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay, Transfection

19) Product Images from "Nuclear retention of importin α coordinates cell fate through changes in gene expression"

Article Title: Nuclear retention of importin α coordinates cell fate through changes in gene expression

Journal: The EMBO Journal

doi: 10.1038/emboj.2011.360

Importin α2 can access to a promoter region of STK35 . ( A ) Genomic DNA sequences of Stk35 described were constructed into the pGL3-Basic luciferase vector and were transfected into GC2 cells with an internal control plasmid pRL-TK. After 24 h incubation, the relative luciferase activity was determined by the ratio of the activities of firefly luciferase and Renilla luciferase. Values are means±s.e.m. ( n =3 each). * P
Figure Legend Snippet: Importin α2 can access to a promoter region of STK35 . ( A ) Genomic DNA sequences of Stk35 described were constructed into the pGL3-Basic luciferase vector and were transfected into GC2 cells with an internal control plasmid pRL-TK. After 24 h incubation, the relative luciferase activity was determined by the ratio of the activities of firefly luciferase and Renilla luciferase. Values are means±s.e.m. ( n =3 each). * P

Techniques Used: Construct, Luciferase, Plasmid Preparation, Transfection, Incubation, Activity Assay

20) Product Images from "Regulation of FADS2 transcription by SREBP-1 and PPAR-α influences LC-PUFA biosynthesis in fish"

Article Title: Regulation of FADS2 transcription by SREBP-1 and PPAR-α influences LC-PUFA biosynthesis in fish

Journal: Scientific Reports

doi: 10.1038/srep40024

Relative gene transcription levels in the livers of experimental fishes ( A–C ) and dual-luciferase detection results ( D–F ). Gene transcription levels and luciferase activity are presented as the mean ± SEM (n = 3). The plasmid transfection groups are as follows: NIC: PGL3-Basic + PCS2+ + PRL-CMV; R: PGL-RF + PCS2+ + PRL-CMV; R-S: PGL-RF + PCS-RS + PRL-CMV; R-P1: PGL-RF + PCS-RP1 + PRL-CMV; R-P2: PGL-FR + PCS-RP2 + PRL-CMV; J: PGL-JF + PCS2+ + PRL-CMV; J-S: PGL-JF + PCS-JS + PRL-CMV; J-P1: PGL-JF + PCS-JP1 + PRL-CMV; J-P2: PGL-JF + PCS-JP2 + PRL-CMV; Y: PGL-YF + PCS2+ + PRL-CMV; Y-S: PGL-YF + PCS-YS + PRL-CMV; Y-P: PGL-YF + PCS-YP + PRL-CMV. Different letters above the bars denote significant differences between diet groups at the P
Figure Legend Snippet: Relative gene transcription levels in the livers of experimental fishes ( A–C ) and dual-luciferase detection results ( D–F ). Gene transcription levels and luciferase activity are presented as the mean ± SEM (n = 3). The plasmid transfection groups are as follows: NIC: PGL3-Basic + PCS2+ + PRL-CMV; R: PGL-RF + PCS2+ + PRL-CMV; R-S: PGL-RF + PCS-RS + PRL-CMV; R-P1: PGL-RF + PCS-RP1 + PRL-CMV; R-P2: PGL-FR + PCS-RP2 + PRL-CMV; J: PGL-JF + PCS2+ + PRL-CMV; J-S: PGL-JF + PCS-JS + PRL-CMV; J-P1: PGL-JF + PCS-JP1 + PRL-CMV; J-P2: PGL-JF + PCS-JP2 + PRL-CMV; Y: PGL-YF + PCS2+ + PRL-CMV; Y-S: PGL-YF + PCS-YS + PRL-CMV; Y-P: PGL-YF + PCS-YP + PRL-CMV. Different letters above the bars denote significant differences between diet groups at the P

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

21) Product Images from "Characterisation of the androgen regulation of glycine N-methyltransferase in prostate cancer cells"

Article Title: Characterisation of the androgen regulation of glycine N-methyltransferase in prostate cancer cells

Journal: Journal of Molecular Endocrinology

doi: 10.1530/JME-13-0169

Site-directed mutagenesis analysis of the three predicted AREs. (A) The wild-type GNMT promoter reporter construct was generated by cloning a 1.2 kb region of the GNMT gene into a pGL3-basic luciferase vector. This construct was used as a template for site-directed mutagenesis of ARE-I (ARE-I*), ARE-II (ARE-II*) and ARE-III (ARE-III*). The mutants were generated by replacing part of the ARE sequence with the MluI restriction site sequence (5′-acggct-3′). (B) The activity of the GNMT ARE mutants was tested using the luciferase reporter assay. Firefly luciferase activities were normalised for transfection efficiency against the Renilla luciferase activities. Results are shown as mean of three independent experiments, each performed in triplicate. Error bars represent the s.e.m. Statistical significance was calculated by unpaired two-tailed Student's t -test: ** P
Figure Legend Snippet: Site-directed mutagenesis analysis of the three predicted AREs. (A) The wild-type GNMT promoter reporter construct was generated by cloning a 1.2 kb region of the GNMT gene into a pGL3-basic luciferase vector. This construct was used as a template for site-directed mutagenesis of ARE-I (ARE-I*), ARE-II (ARE-II*) and ARE-III (ARE-III*). The mutants were generated by replacing part of the ARE sequence with the MluI restriction site sequence (5′-acggct-3′). (B) The activity of the GNMT ARE mutants was tested using the luciferase reporter assay. Firefly luciferase activities were normalised for transfection efficiency against the Renilla luciferase activities. Results are shown as mean of three independent experiments, each performed in triplicate. Error bars represent the s.e.m. Statistical significance was calculated by unpaired two-tailed Student's t -test: ** P

Techniques Used: Mutagenesis, Construct, Generated, Clone Assay, Luciferase, Plasmid Preparation, Sequencing, Activity Assay, Reporter Assay, Transfection, Two Tailed Test

22) Product Images from "Identification of a secondary promoter of CASP8 and its related transcription factor PUR?"

Article Title: Identification of a secondary promoter of CASP8 and its related transcription factor PUR?

Journal: International Journal of Oncology

doi: 10.3892/ijo.2014.2436

Identification of the MAX fragment on chromosome 2 that shows transcriptional activity. (A) The black boxes in the upper portion represent the hypothetical promoter regions predicted by the programs Promoter 2.0 ( 64 ), NNPP ( 65 ), and TSSW ( 66 ). The bars in the lower portion indicate potential binding regions for the indicated transcription factors in the fragment of interest based on the ENCODE ChIP-seq database. The degree of shading of the bars indicates the signal intensity. (B) Luciferase reporter assay in KYSE510 cells. The MAX fragment (1547 bp) was introduced into the promoter-deficit pGL3-Basic vector and transiently transfected into KYSE510 cells. The original transcription activities of the fragment were calculated as the ratio to the intensity value of pRL-TK internal control vector. To compare the ratios to that of the promoter-deficit pGL3-Basic vector, we normalized all the ratios by that of pGL3-Basic vector. Values represent the mean ± standard deviation of three independent experiments. The P-value was obtained by t-test of two independent samples.
Figure Legend Snippet: Identification of the MAX fragment on chromosome 2 that shows transcriptional activity. (A) The black boxes in the upper portion represent the hypothetical promoter regions predicted by the programs Promoter 2.0 ( 64 ), NNPP ( 65 ), and TSSW ( 66 ). The bars in the lower portion indicate potential binding regions for the indicated transcription factors in the fragment of interest based on the ENCODE ChIP-seq database. The degree of shading of the bars indicates the signal intensity. (B) Luciferase reporter assay in KYSE510 cells. The MAX fragment (1547 bp) was introduced into the promoter-deficit pGL3-Basic vector and transiently transfected into KYSE510 cells. The original transcription activities of the fragment were calculated as the ratio to the intensity value of pRL-TK internal control vector. To compare the ratios to that of the promoter-deficit pGL3-Basic vector, we normalized all the ratios by that of pGL3-Basic vector. Values represent the mean ± standard deviation of three independent experiments. The P-value was obtained by t-test of two independent samples.

Techniques Used: Activity Assay, Binding Assay, Chromatin Immunoprecipitation, Luciferase, Reporter Assay, Plasmid Preparation, Transfection, Standard Deviation

The novel promoter activity and mRNA expression of CASP8 isoform G require PURα expression. (A) After transfection with siRNA, the knockdown of PURα was confirmed by western blotting. The Ctrl lane is the negative control group transfected with random control siRNAs. (B) The promoter activity of the construct pGL3-M5N3 was examined in KYSE510 cells transfected with negative control siRNA or PURα-specific siRNA by the luciferase reporter assay. (C) The upper panel is the relative position of primary promoter and secondary promoter within gene CASP8 . The two transcripts stand for two kinds transcripts of CASP8 based on their transcription starting sites. The rectangles encompass the two promoter regions. The lower panel shows the quantitative RT-PCR results of the KYSE510 and EC0156 cell lines, knocking down PURα by siRNA (Si) or not (NC). The y-axis are values of 2 −ΔΔCt , and ΔCt equals the gene average Ct minus the internal control (GAPDH) average Ct.
Figure Legend Snippet: The novel promoter activity and mRNA expression of CASP8 isoform G require PURα expression. (A) After transfection with siRNA, the knockdown of PURα was confirmed by western blotting. The Ctrl lane is the negative control group transfected with random control siRNAs. (B) The promoter activity of the construct pGL3-M5N3 was examined in KYSE510 cells transfected with negative control siRNA or PURα-specific siRNA by the luciferase reporter assay. (C) The upper panel is the relative position of primary promoter and secondary promoter within gene CASP8 . The two transcripts stand for two kinds transcripts of CASP8 based on their transcription starting sites. The rectangles encompass the two promoter regions. The lower panel shows the quantitative RT-PCR results of the KYSE510 and EC0156 cell lines, knocking down PURα by siRNA (Si) or not (NC). The y-axis are values of 2 −ΔΔCt , and ΔCt equals the gene average Ct minus the internal control (GAPDH) average Ct.

Techniques Used: Activity Assay, Expressing, Transfection, Western Blot, Negative Control, Construct, Luciferase, Reporter Assay, Quantitative RT-PCR

23) Product Images from "Nrf2-regulated glutathione recycling independent of biosynthesis is critical for cell survival during oxidative stress"

Article Title: Nrf2-regulated glutathione recycling independent of biosynthesis is critical for cell survival during oxidative stress

Journal: Free radical biology & medicine

doi: 10.1016/j.freeradbiomed.2008.10.040

Nrf2 mediates transcriptional regulation of the GSR gene via the ARE. Functional analysis of three AREs in the GSR promoter by transient transfection in Nrf2 −/− , Nrf2 +/+ , and Keap −/− MEFs. (A) A 2-kb portion 5′ upstream of the translation start site of the GSR gene was analyzed for the presence of AREs. The three AREs identified are highlighted in red with orientation indicated by the black arrows. The transcription start site is indicated in yellow. (B) The sequence alignment of the individual AREs and the NQO1 ARE is shown along with the consensus sequence. (C) Fragments of the GSR promoter containing all three AREs and deletion constructs containing ARE1 and ARE2 or only ARE1 were cloned into the pGL3 Basic luciferase reporter vector. These constructs were transiently transfected into Nrf2 −/− and Keap −/− MEFs and luciferase activity was recorded after 48 h. (D) The full-length promoter region and individual AREs were cloned into the pTAL luciferase reporter vector with minimal promoter (ARE3, −1703 to −997; ARE2, −857 to −725; and ARE1, − 143 to +14). The putative AREs in individual constructs were subjected to site-directed mutagenesis using the primers described under Materials and methods. These constructs (individual and mu-AREs) were transiently transfected into Nrf2 −/− MEFs (no Nrf2 activity) and Keap −/− MEFs (high Nrf2 activity) and luciferase activity was measured after 48 h. Luciferase activity was normalized by measuring the Renilla luciferase activity from a cotransfected reporter vector. Values are means±SE from three different experiments ( p
Figure Legend Snippet: Nrf2 mediates transcriptional regulation of the GSR gene via the ARE. Functional analysis of three AREs in the GSR promoter by transient transfection in Nrf2 −/− , Nrf2 +/+ , and Keap −/− MEFs. (A) A 2-kb portion 5′ upstream of the translation start site of the GSR gene was analyzed for the presence of AREs. The three AREs identified are highlighted in red with orientation indicated by the black arrows. The transcription start site is indicated in yellow. (B) The sequence alignment of the individual AREs and the NQO1 ARE is shown along with the consensus sequence. (C) Fragments of the GSR promoter containing all three AREs and deletion constructs containing ARE1 and ARE2 or only ARE1 were cloned into the pGL3 Basic luciferase reporter vector. These constructs were transiently transfected into Nrf2 −/− and Keap −/− MEFs and luciferase activity was recorded after 48 h. (D) The full-length promoter region and individual AREs were cloned into the pTAL luciferase reporter vector with minimal promoter (ARE3, −1703 to −997; ARE2, −857 to −725; and ARE1, − 143 to +14). The putative AREs in individual constructs were subjected to site-directed mutagenesis using the primers described under Materials and methods. These constructs (individual and mu-AREs) were transiently transfected into Nrf2 −/− MEFs (no Nrf2 activity) and Keap −/− MEFs (high Nrf2 activity) and luciferase activity was measured after 48 h. Luciferase activity was normalized by measuring the Renilla luciferase activity from a cotransfected reporter vector. Values are means±SE from three different experiments ( p

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

24) Product Images from "Genetic Polymorphisms in the Apoptosis-Associated Gene CASP3 and the Risk of Lung Cancer in Chinese Population"

Article Title: Genetic Polymorphisms in the Apoptosis-Associated Gene CASP3 and the Risk of Lung Cancer in Chinese Population

Journal: PLoS ONE

doi: 10.1371/journal.pone.0164358

Transcription activity analysis of the CASP3 829 A > C variant in A549 and NCI-H1975 cells. Luciferase activity profiles were assayed following transfection of the constructs into A549 and NCI-H1975. pGL3-829A and pGL3-829C denote caspase-3 promoter constructs containing the 829A or 829C allele, respectively. All of the constructs were cotransfected with pRL-SV40 to standardize the transfection efficiency. Values were means±SD from more than 3 separate experiments that were each performed in triplicate. **P
Figure Legend Snippet: Transcription activity analysis of the CASP3 829 A > C variant in A549 and NCI-H1975 cells. Luciferase activity profiles were assayed following transfection of the constructs into A549 and NCI-H1975. pGL3-829A and pGL3-829C denote caspase-3 promoter constructs containing the 829A or 829C allele, respectively. All of the constructs were cotransfected with pRL-SV40 to standardize the transfection efficiency. Values were means±SD from more than 3 separate experiments that were each performed in triplicate. **P

Techniques Used: Activity Assay, Variant Assay, Luciferase, Transfection, Construct

25) Product Images from "Characterization of the Chromosomal Binding Sites and Dimerization Partners of the Viral Oncoprotein Meq in Marek's Disease Virus-Transformed T Cells"

Article Title: Characterization of the Chromosomal Binding Sites and Dimerization Partners of the Viral Oncoprotein Meq in Marek's Disease Virus-Transformed T Cells

Journal: Journal of Virology

doi: 10.1128/JVI.77.23.12841-12851.2003

Regulation of MDV gene promoter activity by Meq and c-Jun. (A) Activation of the meq promoter. The top diagram illustrates the meq promoter-driven luciferase (Luc) reporter in the pGL3-Basic vector. Luciferase activation (bottom panel) by transient expression of Meq (M) and/or c-Jun (J) constructs in transfected cells. RLU, relative luciferase units. (B) Repression of the bidirectional pp14 and pp38 promoters by Meq. The top diagram depicts the MDV origin of replication and the flanking bidirectional promoters of pp14 and pp38. Boxed areas indicate UL9 binding sites. The underlined sequence is the CA repeat binding site for Meq. The dose-dependent repression of the pp14 and pp38 promoter-driven luciferase activities by transient expression of Meq constructs in transfected cells is shown in the bottom left and right panels, respectively.
Figure Legend Snippet: Regulation of MDV gene promoter activity by Meq and c-Jun. (A) Activation of the meq promoter. The top diagram illustrates the meq promoter-driven luciferase (Luc) reporter in the pGL3-Basic vector. Luciferase activation (bottom panel) by transient expression of Meq (M) and/or c-Jun (J) constructs in transfected cells. RLU, relative luciferase units. (B) Repression of the bidirectional pp14 and pp38 promoters by Meq. The top diagram depicts the MDV origin of replication and the flanking bidirectional promoters of pp14 and pp38. Boxed areas indicate UL9 binding sites. The underlined sequence is the CA repeat binding site for Meq. The dose-dependent repression of the pp14 and pp38 promoter-driven luciferase activities by transient expression of Meq constructs in transfected cells is shown in the bottom left and right panels, respectively.

Techniques Used: Activity Assay, Activation Assay, Luciferase, Plasmid Preparation, Expressing, Construct, Transfection, Binding Assay, Sequencing

26) Product Images from "A macrophage-specific synthetic promoter for therapeutic application of adiponectin"

Article Title: A macrophage-specific synthetic promoter for therapeutic application of adiponectin

Journal: Gene Therapy

doi: 10.1038/gt.2014.3

Constructs of plasmid vectors for APN expression. ( a ) SP146-C1 was selected to develop a therapeutic vector. The original luciferase gene in the pGL3-basic plasmid was replaced by the APN gene. ( b ) Cells were transfected and mRNA expression of APN with CMV-APN and SP146-C1-APN (SP-APN) was detected 24 h later by RT-PCR in 293 T and RAW264.7 cells. CMV-APN was used as a positive control. Densities of bands were measured using Alpha easy FC software and normalized to that obtained with actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ( c ) Concentrations of APN in the media were measured 48 h later using a human APN ELISA Kit in 293 T and RAW264.7 cells. Data shown are means±s.d.'s. ( d ) The ratio of expressions of SP-APN and CMV-APN was calculated and expressed in graphs. Both mRNA level of APN and released APN showed high ratio in RAW264.7 cells. ** P
Figure Legend Snippet: Constructs of plasmid vectors for APN expression. ( a ) SP146-C1 was selected to develop a therapeutic vector. The original luciferase gene in the pGL3-basic plasmid was replaced by the APN gene. ( b ) Cells were transfected and mRNA expression of APN with CMV-APN and SP146-C1-APN (SP-APN) was detected 24 h later by RT-PCR in 293 T and RAW264.7 cells. CMV-APN was used as a positive control. Densities of bands were measured using Alpha easy FC software and normalized to that obtained with actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ( c ) Concentrations of APN in the media were measured 48 h later using a human APN ELISA Kit in 293 T and RAW264.7 cells. Data shown are means±s.d.'s. ( d ) The ratio of expressions of SP-APN and CMV-APN was calculated and expressed in graphs. Both mRNA level of APN and released APN showed high ratio in RAW264.7 cells. ** P

Techniques Used: Construct, Plasmid Preparation, Expressing, Luciferase, Transfection, Reverse Transcription Polymerase Chain Reaction, Positive Control, Software, Enzyme-linked Immunosorbent Assay

Designs of the promoter constructs and comparisons of luciferase activity among macrophage-specific SPs. ( a ) Constructs of SPs. SP107 and SP146 were inserted in front of p47 phox elements containing the PU.1 site (C1, C2 and C3). All promoters were constructed on the pGL3-basic plasmid. ( b ) The non-macrophage cell lines 293 T and HeLa and the macrophage cell line RAW264.7 were transfected, and promoter activities were measured 48 h later by dual-luciferase assay. Renilla luciferase vector was co-transfected with SPs for control of the transfection efficiency. Luciferase activity was further normalized to that obtained with the pGL3-basic vector. ( c ) To evaluate macrophage specificity, promoter activities were represented as the ratios of RAW264.7/293 T and RAW264.7/HeLa. Data shown are means±s.d.'s.
Figure Legend Snippet: Designs of the promoter constructs and comparisons of luciferase activity among macrophage-specific SPs. ( a ) Constructs of SPs. SP107 and SP146 were inserted in front of p47 phox elements containing the PU.1 site (C1, C2 and C3). All promoters were constructed on the pGL3-basic plasmid. ( b ) The non-macrophage cell lines 293 T and HeLa and the macrophage cell line RAW264.7 were transfected, and promoter activities were measured 48 h later by dual-luciferase assay. Renilla luciferase vector was co-transfected with SPs for control of the transfection efficiency. Luciferase activity was further normalized to that obtained with the pGL3-basic vector. ( c ) To evaluate macrophage specificity, promoter activities were represented as the ratios of RAW264.7/293 T and RAW264.7/HeLa. Data shown are means±s.d.'s.

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

27) Product Images from "Differential regulation of mouse and human Mu opioid receptor gene depends on the single stranded DNA structure of its promoter and α-complex protein 1"

Article Title: Differential regulation of mouse and human Mu opioid receptor gene depends on the single stranded DNA structure of its promoter and α-complex protein 1

Journal: Biomedical Reports

doi: 10.3892/br.2017.877

S1 nuclease sensitivity of PPy/u motif-containing plasmids. (A) The PPy/u motif-containing promoters fused with the promoterless pGL3-basic vector (p322/292 and p336/306) and pGL3-basic (control) were treated with vehicle (lane 2 in each panel) and increasing quantities of S1 nuclease (lanes 3 and 4 in each panel) followed by digestion with the XbaI restriction enzyme. Lane 1, molecular size markers (1-kb ladder). The XbaI-linearized plasmid (denoted by the single asterisk) and the 3.2- and 1.8-kb fragments are indicated by arrows. (B) The luciferase activity of p322/292 and p336/306 plasmids. Activities of luciferase reporter were expressed as n-fold relative to the activity of each corresponding luciferase reporter transfected with vector p322/292, which was assigned an activity value of 1.0. Transfection efficiencies were normalized to β-galactosidase activity. Data are presented as the mean of three independent experiments with at least two different plasmid preparations. Standard deviation is indicated by the error bars. **P
Figure Legend Snippet: S1 nuclease sensitivity of PPy/u motif-containing plasmids. (A) The PPy/u motif-containing promoters fused with the promoterless pGL3-basic vector (p322/292 and p336/306) and pGL3-basic (control) were treated with vehicle (lane 2 in each panel) and increasing quantities of S1 nuclease (lanes 3 and 4 in each panel) followed by digestion with the XbaI restriction enzyme. Lane 1, molecular size markers (1-kb ladder). The XbaI-linearized plasmid (denoted by the single asterisk) and the 3.2- and 1.8-kb fragments are indicated by arrows. (B) The luciferase activity of p322/292 and p336/306 plasmids. Activities of luciferase reporter were expressed as n-fold relative to the activity of each corresponding luciferase reporter transfected with vector p322/292, which was assigned an activity value of 1.0. Transfection efficiencies were normalized to β-galactosidase activity. Data are presented as the mean of three independent experiments with at least two different plasmid preparations. Standard deviation is indicated by the error bars. **P

Techniques Used: Plasmid Preparation, Luciferase, Activity Assay, Transfection, Standard Deviation

28) Product Images from "Genetic polymorphisms in glycolytic pathway are associated with the prognosis of patients with early stage non-small cell lung cancer"

Article Title: Genetic polymorphisms in glycolytic pathway are associated with the prognosis of patients with early stage non-small cell lung cancer

Journal: Scientific Reports

doi: 10.1038/srep35603

Functional analysis of the ENO1 rs2274971A > G by dual luciferase reporter assay. NSCLC cells (A549, H1299, and H1703) were transfected with pRL-SV40 and pGL3-basic- ENO1 rs2274971A or G allele-containing constructs ( A ). At 24 h posttransfection, A549 cells were exposed to hypoxia for 24 h ( B ). For HIF-1α overexpression, we cotransfected pRL-SV40 and pGL3-basic- ENO1 constructs into A549 cells with pEGFP-HIF-1α ( C ). Luciferase activity was measured using Dual-Luciferase ® Reporter Assay System. Firefly luciferase activity was normalized by pRL-SV40 Renilla luciferase activity. Each bar represents mean ± S.E.M. from three independent experiments carried out in quadruplicate. P value, Student’s t-test.
Figure Legend Snippet: Functional analysis of the ENO1 rs2274971A > G by dual luciferase reporter assay. NSCLC cells (A549, H1299, and H1703) were transfected with pRL-SV40 and pGL3-basic- ENO1 rs2274971A or G allele-containing constructs ( A ). At 24 h posttransfection, A549 cells were exposed to hypoxia for 24 h ( B ). For HIF-1α overexpression, we cotransfected pRL-SV40 and pGL3-basic- ENO1 constructs into A549 cells with pEGFP-HIF-1α ( C ). Luciferase activity was measured using Dual-Luciferase ® Reporter Assay System. Firefly luciferase activity was normalized by pRL-SV40 Renilla luciferase activity. Each bar represents mean ± S.E.M. from three independent experiments carried out in quadruplicate. P value, Student’s t-test.

Techniques Used: Functional Assay, Luciferase, Reporter Assay, Transfection, Construct, Over Expression, Activity Assay

29) Product Images from "The promoter of IL-18 binding protein: Activation by an IFN-?-induced complex of IFN regulatory factor 1 and CCAAT/enhancer binding protein ?"

Article Title: The promoter of IL-18 binding protein: Activation by an IFN-?-induced complex of IFN regulatory factor 1 and CCAAT/enhancer binding protein ?

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

doi: 10.1073/pnas.262663399

Basal and IFN-γ-induced activity of luciferase reporter vectors carrying the human IL-18BP promoter. Insert size, extending upstream of the transcription start site (+1), is shown in parentheses. Circled numbers represent the various response elements: 1, GAS; 2, IRF-E; 3, C/EBP - E (two sites); 4, silencer; 5, distal enhancer. Filled squares depict mutation in a specific response element. Hep G2 cells were cotransfected with the indicated reporter vector and pSV40 β-galactosidase. All luciferase values were normalized to β-galactosidase activity. ( A ) Luciferase activity in extracts of uninduced cells relative to that of cells transfected with pGL3-basic vector. ( B ) Luciferase activity in cells transfected with the indicated vectors and induced with IFN-γ. Fold induction is over basal activity as given in A .
Figure Legend Snippet: Basal and IFN-γ-induced activity of luciferase reporter vectors carrying the human IL-18BP promoter. Insert size, extending upstream of the transcription start site (+1), is shown in parentheses. Circled numbers represent the various response elements: 1, GAS; 2, IRF-E; 3, C/EBP - E (two sites); 4, silencer; 5, distal enhancer. Filled squares depict mutation in a specific response element. Hep G2 cells were cotransfected with the indicated reporter vector and pSV40 β-galactosidase. All luciferase values were normalized to β-galactosidase activity. ( A ) Luciferase activity in extracts of uninduced cells relative to that of cells transfected with pGL3-basic vector. ( B ) Luciferase activity in cells transfected with the indicated vectors and induced with IFN-γ. Fold induction is over basal activity as given in A .

Techniques Used: Activity Assay, Luciferase, Mutagenesis, Plasmid Preparation, Transfection

30) Product Images from "Late SV40 Factor (LSF) Enhances Angiogenesis by Transcriptionally Up-regulating Matrix Metalloproteinase-9 (MMP-9) *"

Article Title: Late SV40 Factor (LSF) Enhances Angiogenesis by Transcriptionally Up-regulating Matrix Metalloproteinase-9 (MMP-9) *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.298976

LSF transcriptionally up-regulated MMP-9. Control-8, LSF-1, and LSF-17 clones of HepG3 cells ( A ) and Control-1, LSFdn-8, and LSFdn-15 clones of QGY-7703 cells ( B ) were transfected with pGL3-basic vector or MMP-9-Prom-luc along with Renilla luciferase expression vector. Luciferase assay was performed 2 days later, and firefly luciferase activity was normalized by Renilla luciferase activity. Data represent mean ± S.E. *, p
Figure Legend Snippet: LSF transcriptionally up-regulated MMP-9. Control-8, LSF-1, and LSF-17 clones of HepG3 cells ( A ) and Control-1, LSFdn-8, and LSFdn-15 clones of QGY-7703 cells ( B ) were transfected with pGL3-basic vector or MMP-9-Prom-luc along with Renilla luciferase expression vector. Luciferase assay was performed 2 days later, and firefly luciferase activity was normalized by Renilla luciferase activity. Data represent mean ± S.E. *, p

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

31) Product Images from "Homodimerization Is Essential for the Receptor for Advanced Glycation End Products (RAGE)-mediated Signal Transduction *"

Article Title: Homodimerization Is Essential for the Receptor for Advanced Glycation End Products (RAGE)-mediated Signal Transduction *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.133827

Soluble RAGE and RAGE V peptide inhibit S100B/RAGE-induced NF-κB pathway. A , HEK293T cells were maintained in 2% FCS containing DMEM and mock-transfected or transfected with 2 μg of pRL and 200 μg of pGL3 basic vector or pNFκB-Luc.
Figure Legend Snippet: Soluble RAGE and RAGE V peptide inhibit S100B/RAGE-induced NF-κB pathway. A , HEK293T cells were maintained in 2% FCS containing DMEM and mock-transfected or transfected with 2 μg of pRL and 200 μg of pGL3 basic vector or pNFκB-Luc.

Techniques Used: Transfection, Plasmid Preparation

32) Product Images from "Interferon Regulatory Factor-1 (IRF-1) Is Involved in the Induction of Phosphatidylserine Receptor (PSR) in Response to dsRNA Virus Infection and Contributes to Apoptotic Cell Clearance in CHSE-214 Cell"

Article Title: Interferon Regulatory Factor-1 (IRF-1) Is Involved in the Induction of Phosphatidylserine Receptor (PSR) in Response to dsRNA Virus Infection and Contributes to Apoptotic Cell Clearance in CHSE-214 Cell

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms151019281

The PSR promoter can be induced by rIFN-α or IPNV in CHSE-214 cells. ( A ) CHSE-214 cells were transfected with the pPSRP:EGFP construct, stimulated with either rIFN-α (0.5 μg/mL) or IPNV (MOI = 1) for 24 h, and then visualized by fluorescent microscopy. The PSR promoter was activated by the stimuli (fluorescent green pointed by arrows; scale bar = 100 μm); ( B ) PSR promoter-activated cells (green) had the potential to engulf apoptotic cells (pointed triangle) in both rIFN-α or IPNV-stimulated cells (scale bar = 25 μm); ( C ) CHSE-214 cells were transfected with the pGL3-basic-PSRP construct, stimulated with either rIFN-α or IPNV for 24 and 48 h, and then evaluated with a luciferase assay. PSR promoter activity was expressed as fold induction of relative luciferase activity (RLU) compared to the promoterless vector and then compared with the values at 1 h. The data indicated that the PSR promoter was activated by the stimuli (* p
Figure Legend Snippet: The PSR promoter can be induced by rIFN-α or IPNV in CHSE-214 cells. ( A ) CHSE-214 cells were transfected with the pPSRP:EGFP construct, stimulated with either rIFN-α (0.5 μg/mL) or IPNV (MOI = 1) for 24 h, and then visualized by fluorescent microscopy. The PSR promoter was activated by the stimuli (fluorescent green pointed by arrows; scale bar = 100 μm); ( B ) PSR promoter-activated cells (green) had the potential to engulf apoptotic cells (pointed triangle) in both rIFN-α or IPNV-stimulated cells (scale bar = 25 μm); ( C ) CHSE-214 cells were transfected with the pGL3-basic-PSRP construct, stimulated with either rIFN-α or IPNV for 24 and 48 h, and then evaluated with a luciferase assay. PSR promoter activity was expressed as fold induction of relative luciferase activity (RLU) compared to the promoterless vector and then compared with the values at 1 h. The data indicated that the PSR promoter was activated by the stimuli (* p

Techniques Used: Transfection, Construct, Microscopy, Luciferase, Activity Assay, Plasmid Preparation

33) Product Images from "Annexin A2 contributes to cisplatin resistance by activation of JNK-p53 pathway in non-small cell lung cancer cells"

Article Title: Annexin A2 contributes to cisplatin resistance by activation of JNK-p53 pathway in non-small cell lung cancer cells

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-017-0594-1

Annexin A2 inhibited p53 expression through c-Jun-mediated suppression of p53 promoter activity. a A549/DDP cells were transfected with Annexin A2 siRNA, c-Jun protein expression was analyzed by Western blot. b A549 cells were transfected with pCMV6-Annexin A2, and then treated with SP600125, c-Jun protein expression was analyzed by Western blot. c A549/DDP cells were transfected with Annexin A2 siRNA for 48 h, the binding capacity of c-Jun to p53 promoter were determined by chromatin immunoprecipitation. The enrichment of the p53 promoter in immunoprecipitated (IP) c-Jun and the input of c-Jun are shown. IgG was used as a negative control. One representative data set of three individual experiments is shown. d - e HEK293T cells ( d ) and A549 cells ( e ) were co-transfected with pGL3-p53-Luc, or mut-pGL3-p53 and pCMV-Annexin A2 for 48 h, cells were harvested and assayed by the Dual-Luciferase Reporter Assay System. *P
Figure Legend Snippet: Annexin A2 inhibited p53 expression through c-Jun-mediated suppression of p53 promoter activity. a A549/DDP cells were transfected with Annexin A2 siRNA, c-Jun protein expression was analyzed by Western blot. b A549 cells were transfected with pCMV6-Annexin A2, and then treated with SP600125, c-Jun protein expression was analyzed by Western blot. c A549/DDP cells were transfected with Annexin A2 siRNA for 48 h, the binding capacity of c-Jun to p53 promoter were determined by chromatin immunoprecipitation. The enrichment of the p53 promoter in immunoprecipitated (IP) c-Jun and the input of c-Jun are shown. IgG was used as a negative control. One representative data set of three individual experiments is shown. d - e HEK293T cells ( d ) and A549 cells ( e ) were co-transfected with pGL3-p53-Luc, or mut-pGL3-p53 and pCMV-Annexin A2 for 48 h, cells were harvested and assayed by the Dual-Luciferase Reporter Assay System. *P

Techniques Used: Expressing, Activity Assay, Transfection, Western Blot, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control, Luciferase, Reporter Assay

34) Product Images from "Intestine specific regulation of pig cytidine-5′-monophospho-N-acetylneuraminic acid hydroxylase gene for N-glycolylneuraminic acid biosynthesis"

Article Title: Intestine specific regulation of pig cytidine-5′-monophospho-N-acetylneuraminic acid hydroxylase gene for N-glycolylneuraminic acid biosynthesis

Journal: Scientific Reports

doi: 10.1038/s41598-019-40522-9

Identification of intestine specific promoter (Pi) of the pcmah gene. ( a ) Genomic structure of pcmah . Ubiquitous housekeeping promoter region of pcmah plays a role in the expression of 5′ pcmah -2. Putative promoter region of the intestine specific splicing variant of pcmah (5′ pcmah -1) is indicated by ‘Pi (intestine specific promoter)’. Shaded boxes indicate the coding exons, while open boxes indicate untranslated exons. Two splicing variants of pcmah share a common ORF region (shaded arrow boxes). ( b ) The relative levels of 5′ pcmah -1 expression were measured and analyzed by RT-PCR in three different cells of PK15, IPI-2I, and MYP30. β -Actin was used as a positive control. ( c ) The relative reporter activities of the luciferase reporter plasmids with the proximal region of exon 1a in PK15, IPI-2I, and MYP30 cells. DNA fragments containing various lengths of the Pi promoter region, which is the proximal region of exon 1a, were subcloned into a PGL3-basic vector (pGL3), and each generated plasmid was transiently transfected into the three different pig cells of PK15, IPI-2I, and MYP30. Two enzyme activities of luciferase and β -Gal in each of the transfected cells were assayed as described in the ‘Materials and Methods’ section. In each transfected cell, the luciferase enzyme activity was normalized with the measured β-Gal activity. The relative fold value was then calculated from the ratio of the normalized activity and the activity in the empty pGL3-basic vector-transfected cells. Statistic bars represent the mean ± SE of three independent determinations. Differences in the fold value of luciferase enzyme activities were statistically analyzed using the Student t -test: * p
Figure Legend Snippet: Identification of intestine specific promoter (Pi) of the pcmah gene. ( a ) Genomic structure of pcmah . Ubiquitous housekeeping promoter region of pcmah plays a role in the expression of 5′ pcmah -2. Putative promoter region of the intestine specific splicing variant of pcmah (5′ pcmah -1) is indicated by ‘Pi (intestine specific promoter)’. Shaded boxes indicate the coding exons, while open boxes indicate untranslated exons. Two splicing variants of pcmah share a common ORF region (shaded arrow boxes). ( b ) The relative levels of 5′ pcmah -1 expression were measured and analyzed by RT-PCR in three different cells of PK15, IPI-2I, and MYP30. β -Actin was used as a positive control. ( c ) The relative reporter activities of the luciferase reporter plasmids with the proximal region of exon 1a in PK15, IPI-2I, and MYP30 cells. DNA fragments containing various lengths of the Pi promoter region, which is the proximal region of exon 1a, were subcloned into a PGL3-basic vector (pGL3), and each generated plasmid was transiently transfected into the three different pig cells of PK15, IPI-2I, and MYP30. Two enzyme activities of luciferase and β -Gal in each of the transfected cells were assayed as described in the ‘Materials and Methods’ section. In each transfected cell, the luciferase enzyme activity was normalized with the measured β-Gal activity. The relative fold value was then calculated from the ratio of the normalized activity and the activity in the empty pGL3-basic vector-transfected cells. Statistic bars represent the mean ± SE of three independent determinations. Differences in the fold value of luciferase enzyme activities were statistically analyzed using the Student t -test: * p

Techniques Used: Expressing, Variant Assay, Reverse Transcription Polymerase Chain Reaction, Positive Control, Luciferase, Plasmid Preparation, Generated, Transfection, Activity Assay

35) Product Images from "miR-1 inhibits the proliferation of breast cancer stem cells by targeting EVI-1"

Article Title: miR-1 inhibits the proliferation of breast cancer stem cells by targeting EVI-1

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S188836

miR-1 negatively regulates the expression of EVI-1 in BCSCs. Notes: ( A ) Real-time PCR analysis of miR-1 expression in breast cancer and normal tissues. ( B ) Real-time PCR analysis of EVI-1 expression in breast cancer and normal tissues. ( C , D ) Expression of miR-1 was increased or reduced in BCSCs transfected with miR-1 agomir or miR-1 antagomir. ( E ) Real-time PCR and Western blotting were performed to detect the expression of EVI-1 in BCSCs transfected with pcDNA3.1-EVI-1 or pSilencer2.1-EVI-1. ( F ) Real-time PCR and Western blotting were performed to detect the EVI-1 expression level in BCSCs transfected with different vectors. ( G ) The effect of miR-1 on reporters of pGL3-EVI-1-WT and pGL3-EVI-1-MUT in BCSCs was measured by luciferase reporter gene assays. * P
Figure Legend Snippet: miR-1 negatively regulates the expression of EVI-1 in BCSCs. Notes: ( A ) Real-time PCR analysis of miR-1 expression in breast cancer and normal tissues. ( B ) Real-time PCR analysis of EVI-1 expression in breast cancer and normal tissues. ( C , D ) Expression of miR-1 was increased or reduced in BCSCs transfected with miR-1 agomir or miR-1 antagomir. ( E ) Real-time PCR and Western blotting were performed to detect the expression of EVI-1 in BCSCs transfected with pcDNA3.1-EVI-1 or pSilencer2.1-EVI-1. ( F ) Real-time PCR and Western blotting were performed to detect the EVI-1 expression level in BCSCs transfected with different vectors. ( G ) The effect of miR-1 on reporters of pGL3-EVI-1-WT and pGL3-EVI-1-MUT in BCSCs was measured by luciferase reporter gene assays. * P

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

36) Product Images from "A SNP uncoupling Mina expression from the TGFβ signaling pathway"

Article Title: A SNP uncoupling Mina expression from the TGFβ signaling pathway

Journal: Immunity, Inflammation and Disease

doi: 10.1002/iid3.191

Reporter activity of allelic versions of E3 and E4. (A) Shown is a schematic of the Mina locus region −1588/+3755 (thick horizontal line) depicting exons 1 and 2 (black boxes above the thick horizontal line), DHSs 2–6 (black downward arrowheads) and SNPs 1–17 (gray upward triangles). Shown below are the locations of fragments containing the Mina promoter (gray box), E3 (magenta box), SNPs 6–10 (white box), and E4 (yellow box). (B) Dual luciferase reporter analysis of allelic versions of E3 and E4. PGL3 basic vector containing the indicated color‐coded Mina locus fragments were transfected into EL4 cells and analyzed 48 h later for the ratio of firefly over renilla luciferase activity (FL/RL). Data are representative of three independent experiments with similar results.
Figure Legend Snippet: Reporter activity of allelic versions of E3 and E4. (A) Shown is a schematic of the Mina locus region −1588/+3755 (thick horizontal line) depicting exons 1 and 2 (black boxes above the thick horizontal line), DHSs 2–6 (black downward arrowheads) and SNPs 1–17 (gray upward triangles). Shown below are the locations of fragments containing the Mina promoter (gray box), E3 (magenta box), SNPs 6–10 (white box), and E4 (yellow box). (B) Dual luciferase reporter analysis of allelic versions of E3 and E4. PGL3 basic vector containing the indicated color‐coded Mina locus fragments were transfected into EL4 cells and analyzed 48 h later for the ratio of firefly over renilla luciferase activity (FL/RL). Data are representative of three independent experiments with similar results.

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

Mina enhancer 1 (E1) located in Mina promoter region +80/+150. (A) Shown is a schematic of the Mina locus (thick horizontal line) depicting exons 1–10 (black boxes above the thick horizontal line), the Mina transcription unit (horizontal dotted arrow) and SNPs 1–21 (gray upward triangles), an enlargement of region −1588/+1201 (encompassed by blue lines) containing the Mina promoter, exon1, and the proximal part of intron 1, showing fragment −1588/+354 labeled “promoter region” (light horizontal gray line) employed as the promoter for reporter assays in Figures 3 – 5 and 8 and region −64/+354 (encompassed by red lines), the focus of the reporter assay analysis in part B of this figure. (B) Analysis of Mina promoter elements by dual luciferase assay. PGL3 basic vector containing the indicated Mina promoter fragments were transfected into EL4 cells and analyzed 48 h later. FL/RL is the ratio of firefly over renilla luciferase activity. Numbering throughout is with respect to the transcriptional start site in P1. Data are representative of 3 independent experiments with similar results.
Figure Legend Snippet: Mina enhancer 1 (E1) located in Mina promoter region +80/+150. (A) Shown is a schematic of the Mina locus (thick horizontal line) depicting exons 1–10 (black boxes above the thick horizontal line), the Mina transcription unit (horizontal dotted arrow) and SNPs 1–21 (gray upward triangles), an enlargement of region −1588/+1201 (encompassed by blue lines) containing the Mina promoter, exon1, and the proximal part of intron 1, showing fragment −1588/+354 labeled “promoter region” (light horizontal gray line) employed as the promoter for reporter assays in Figures 3 – 5 and 8 and region −64/+354 (encompassed by red lines), the focus of the reporter assay analysis in part B of this figure. (B) Analysis of Mina promoter elements by dual luciferase assay. PGL3 basic vector containing the indicated Mina promoter fragments were transfected into EL4 cells and analyzed 48 h later. FL/RL is the ratio of firefly over renilla luciferase activity. Numbering throughout is with respect to the transcriptional start site in P1. Data are representative of 3 independent experiments with similar results.

Techniques Used: Labeling, Reporter Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay

Identification of Mina enhancers E2, E3, and E4. (A) Shown is a schematic of the Mina locus region −1588/+3755 (thick horizontal line) depicting exons 1 and 2 (black boxes above the thick horizontal line), DHSs 2–6 (black downward arrowheads) and SNPs 1–17 (gray upward triangles). Shown below are the locations of fragments containing the Mina promoter (gray box), E2 (red box), E3 (magenta box), SNPs 6–10 (white box), and E4 (yellow box). (B) Dual luciferase reporter analysis of Mina locus fragments spanning DHSs 4, 5, and 6. PGL3 basic vector containing the indicated color‐coded Mina locus fragments were transfected into EL4 cells and analyzed 48 h later for the ratio of firefly over renilla luciferase activity (FL/RL). Data are representative of three independent experiments with similar results.
Figure Legend Snippet: Identification of Mina enhancers E2, E3, and E4. (A) Shown is a schematic of the Mina locus region −1588/+3755 (thick horizontal line) depicting exons 1 and 2 (black boxes above the thick horizontal line), DHSs 2–6 (black downward arrowheads) and SNPs 1–17 (gray upward triangles). Shown below are the locations of fragments containing the Mina promoter (gray box), E2 (red box), E3 (magenta box), SNPs 6–10 (white box), and E4 (yellow box). (B) Dual luciferase reporter analysis of Mina locus fragments spanning DHSs 4, 5, and 6. PGL3 basic vector containing the indicated color‐coded Mina locus fragments were transfected into EL4 cells and analyzed 48 h later for the ratio of firefly over renilla luciferase activity (FL/RL). Data are representative of three independent experiments with similar results.

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

Effect of rs4191790, TGFβ, and the Smad3 inhibitor SIS3 on Mina E2 reporter activity. TGFβ enhances the reporter activity of the rs4191790 G but not the rs4191790 A allelic version of E2 and this enhancement is abolished by the Smad3 inhibitor SIS3. PGL3 basic vector containing the Mina promoter and the E2 fragment +354/+1201 was transfected into EL4 cells for 24 h and then treated with 5 ng/ml TGFβ alone or together with 0.5 SIS3 for 24 h before harvesting for luciferase analysis. FL/RL, the ratio of firefly over renilla luciferase activity. Data are representative of two independent experiments with similar results.
Figure Legend Snippet: Effect of rs4191790, TGFβ, and the Smad3 inhibitor SIS3 on Mina E2 reporter activity. TGFβ enhances the reporter activity of the rs4191790 G but not the rs4191790 A allelic version of E2 and this enhancement is abolished by the Smad3 inhibitor SIS3. PGL3 basic vector containing the Mina promoter and the E2 fragment +354/+1201 was transfected into EL4 cells for 24 h and then treated with 5 ng/ml TGFβ alone or together with 0.5 SIS3 for 24 h before harvesting for luciferase analysis. FL/RL, the ratio of firefly over renilla luciferase activity. Data are representative of two independent experiments with similar results.

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

Allelic activity of E2 maps to SNP17 (rs4191790). (A) Shown is a schematic of the Mina locus region −1588/+3755 (thick horizontal line) depicting exons 1 and 2 (black boxes above the thick horizontal line), DHSs 2–4 (black downward arrowheads), and SNPs 15–17 (gray upward triangles). Shown below are the locations of fragments containing the Mina promoter (gray box) and E2 (red box). Vertical lines represent the locations of SNPs 15–17. (B) Dual luciferase reporter analysis of C57BL/6 and BALB/c allelic versions of E2. PGL3 basic vector containing the indicated color‐coded Mina locus fragments were transfected into EL4 cells and analyzed 48 h later for the ratio of firefly over renilla luciferase activity (FL/RL). (C) Dual luciferase reporter analysis of allelic versions of E2 differing only at SNP17 (rs4191790). PGL3 basic vector containing the indicated color‐coded Mina locus fragments were transfected into EL4 cells and analyzed 48 h later for the ratio of firefly over renilla luciferase activity (FL/RL). Data are the mean and SEM from three independent experiments. Statistical significance was determined by Student's t test.
Figure Legend Snippet: Allelic activity of E2 maps to SNP17 (rs4191790). (A) Shown is a schematic of the Mina locus region −1588/+3755 (thick horizontal line) depicting exons 1 and 2 (black boxes above the thick horizontal line), DHSs 2–4 (black downward arrowheads), and SNPs 15–17 (gray upward triangles). Shown below are the locations of fragments containing the Mina promoter (gray box) and E2 (red box). Vertical lines represent the locations of SNPs 15–17. (B) Dual luciferase reporter analysis of C57BL/6 and BALB/c allelic versions of E2. PGL3 basic vector containing the indicated color‐coded Mina locus fragments were transfected into EL4 cells and analyzed 48 h later for the ratio of firefly over renilla luciferase activity (FL/RL). (C) Dual luciferase reporter analysis of allelic versions of E2 differing only at SNP17 (rs4191790). PGL3 basic vector containing the indicated color‐coded Mina locus fragments were transfected into EL4 cells and analyzed 48 h later for the ratio of firefly over renilla luciferase activity (FL/RL). Data are the mean and SEM from three independent experiments. Statistical significance was determined by Student's t test.

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

37) Product Images from "Overexpression of long non-coding RNA SOX2OT promotes esophageal squamous cell carcinoma growth"

Article Title: Overexpression of long non-coding RNA SOX2OT promotes esophageal squamous cell carcinoma growth

Journal: Cancer Cell International

doi: 10.1186/s12935-018-0570-7

Ectopic expression of SOX2 promoted SOX2OT expression at transcriptional level. a , c qPCR was used to detect SOX2 mRNA and SOX2OT expression. Overexpression of SOX2 promoted SOX2OT expression in EC109 and EC9706 cells. b , d Luciferase reporter assay showed that SOX2 could increase the luciferase activity of SOX2OT-pGL3/Basic. ** P
Figure Legend Snippet: Ectopic expression of SOX2 promoted SOX2OT expression at transcriptional level. a , c qPCR was used to detect SOX2 mRNA and SOX2OT expression. Overexpression of SOX2 promoted SOX2OT expression in EC109 and EC9706 cells. b , d Luciferase reporter assay showed that SOX2 could increase the luciferase activity of SOX2OT-pGL3/Basic. ** P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Over Expression, Luciferase, Reporter Assay, Activity Assay

38) Product Images from "SIP30 Is Regulated by ERK in Peripheral Nerve Injury-induced Neuropathic Pain *"

Article Title: SIP30 Is Regulated by ERK in Peripheral Nerve Injury-induced Neuropathic Pain *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.036756

ERK is involved in the regulation of sip30 promoter activity. A , rat sip30 gene promoter-reporter construct. The underlined 2.4-kb fragment was cloned into pGL3-basic vector ( 5′UTR-SIP30-Luc ) by KpnI and NheI double digestion and used to check
Figure Legend Snippet: ERK is involved in the regulation of sip30 promoter activity. A , rat sip30 gene promoter-reporter construct. The underlined 2.4-kb fragment was cloned into pGL3-basic vector ( 5′UTR-SIP30-Luc ) by KpnI and NheI double digestion and used to check

Techniques Used: Activity Assay, Construct, Clone Assay, Plasmid Preparation

39) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Induction by Hypoxia and Hypoxia-Inducible Factors"

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Induction by Hypoxia and Hypoxia-Inducible Factors

Journal: Journal of Virology

doi: 10.1128/JVI.05167-11

HRE4R can mediate much of the activation of the LTi LANA promoter by HIFs. (A) Schematic diagram of the LTi promoter luciferase reporter constructs with progressive deletions of HRE4R [pGL3-LANA(i)p-luc(D1)] and 5R [pGL3-LANA(i)p-luc(D2)]. The wild-type
Figure Legend Snippet: HRE4R can mediate much of the activation of the LTi LANA promoter by HIFs. (A) Schematic diagram of the LTi promoter luciferase reporter constructs with progressive deletions of HRE4R [pGL3-LANA(i)p-luc(D1)] and 5R [pGL3-LANA(i)p-luc(D2)]. The wild-type

Techniques Used: Activation Assay, Luciferase, Construct

40) Product Images from "Oct4/Sox2-Regulated miR-302 Targets Cyclin D1 in Human Embryonic Stem Cells ▿Oct4/Sox2-Regulated miR-302 Targets Cyclin D1 in Human Embryonic Stem Cells ▿ †"

Article Title: Oct4/Sox2-Regulated miR-302 Targets Cyclin D1 in Human Embryonic Stem Cells ▿Oct4/Sox2-Regulated miR-302 Targets Cyclin D1 in Human Embryonic Stem Cells ▿ †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00359-08

Oct4 and Sox2 transcription factors are required for expression of miR-302 miRNAs. (A) BG-01 cells were transfected with 200 nM nonspecific control (NS) or 100 nM each of Oct4 and Sox2 (O/S) siRNA oligonucleotides as described in Materials and Methods, and whole-cell extracts were made. Oct4, Sox2, and β-actin protein expression levels were analyzed by Western blotting with specific antibodies. (B) BG-01 cells were transfected with pGL3-Basic or pGL3-miR-302 with either nonspecific control siRNA (NS) or Oct4/Sox2 siRNA (O/S) as described for panel A for 48 h. In addition, all samples were transfected with pRL-CMV as a transfection control. Cell extracts were then harvested and analyzed for relative luciferase activity. A schematic of the cloned promoter region in pGL3-miR-302 is shown. luc/ren, luciferase/ Renilla activities. (C) BG-01 cells were transfected with 100 nM of nonspecific control (NS), Oct4 siRNA (O), Sox2 siRNA (S), or Oct4/Sox2 siRNA (O/S) as described for panel A, and Oct4, Sox2, and β-actin protein expression levels were analyzed by Western blotting with specific antibodies. (D) qRT-PCR was used to analyze the relative expression levels, normalized to 5S rRNA levels, of the mature miR-302a miRNA in the siRNA-depleted cells. (E) HeLa cells were transfected with pGL3-Basic or pGL3-miR-302 with pCAG-Myc-Oct4 and pCAG-HA-Sox2 cotransfection or empty vector cotransfection. Western blots were probed with Oct4, Sox2, and β-actin antibodies. +, present; −, absent. (F) HeLa cell lysates were analyzed for relative luciferase activities; data are expressed in relative units.
Figure Legend Snippet: Oct4 and Sox2 transcription factors are required for expression of miR-302 miRNAs. (A) BG-01 cells were transfected with 200 nM nonspecific control (NS) or 100 nM each of Oct4 and Sox2 (O/S) siRNA oligonucleotides as described in Materials and Methods, and whole-cell extracts were made. Oct4, Sox2, and β-actin protein expression levels were analyzed by Western blotting with specific antibodies. (B) BG-01 cells were transfected with pGL3-Basic or pGL3-miR-302 with either nonspecific control siRNA (NS) or Oct4/Sox2 siRNA (O/S) as described for panel A for 48 h. In addition, all samples were transfected with pRL-CMV as a transfection control. Cell extracts were then harvested and analyzed for relative luciferase activity. A schematic of the cloned promoter region in pGL3-miR-302 is shown. luc/ren, luciferase/ Renilla activities. (C) BG-01 cells were transfected with 100 nM of nonspecific control (NS), Oct4 siRNA (O), Sox2 siRNA (S), or Oct4/Sox2 siRNA (O/S) as described for panel A, and Oct4, Sox2, and β-actin protein expression levels were analyzed by Western blotting with specific antibodies. (D) qRT-PCR was used to analyze the relative expression levels, normalized to 5S rRNA levels, of the mature miR-302a miRNA in the siRNA-depleted cells. (E) HeLa cells were transfected with pGL3-Basic or pGL3-miR-302 with pCAG-Myc-Oct4 and pCAG-HA-Sox2 cotransfection or empty vector cotransfection. Western blots were probed with Oct4, Sox2, and β-actin antibodies. +, present; −, absent. (F) HeLa cell lysates were analyzed for relative luciferase activities; data are expressed in relative units.

Techniques Used: Expressing, Transfection, Western Blot, Luciferase, Activity Assay, Clone Assay, Quantitative RT-PCR, Cotransfection, Plasmid Preparation

Related Articles

Clone Assay:

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Transfection:

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Article Snippet: .. As shown in Figure , transfection of (-1177∼+312)-Luc construct into IPEC-J2 cells resulted in an eightfold induction of luciferase activity compared with the pGL3-Basic vector (p < 0.05). .. Further deletions to bases -777, -345, and -195, respectively, did not cause significant difference of the luciferase activities compared with each other, indicating that there are no important regulatory elements within these regions ( Figure ).

Luciferase:

Article Title: Transcriptional regulation of the potential tumor suppressor ABI3 gene in thyroid carcinomas: interplay between methylation and NKX2-1 availability
Article Snippet: .. The PCR-amplified product was digested with Kpn I and Xho I restriction enzymes (New England Biolabs, Hitchin, UK) and directionally cloned into the same restriction sites upstream to a promoterless firefly luciferase gene of the pGL3-Basic vector and into the pGL3-Control (Promega, Madison, WI), which contains a luciferase gene driven by the SV40 promoter. .. The empty pGL3-Basic and pGL3-Control were used as negative control.

Article Title: TCF/?-catenin plays an important role in HCCR-1 oncogene expression
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Article Title: Cloning and Transcriptional Activity Analysis of the Porcine Abcb1 Gene Promoter: Transcription Factor Sp1 Regulates the Expression of Porcine Abcb1
Article Snippet: .. As shown in Figure , transfection of (-1177∼+312)-Luc construct into IPEC-J2 cells resulted in an eightfold induction of luciferase activity compared with the pGL3-Basic vector (p < 0.05). .. Further deletions to bases -777, -345, and -195, respectively, did not cause significant difference of the luciferase activities compared with each other, indicating that there are no important regulatory elements within these regions ( Figure ).

Reporter Assay:

Article Title: TCF/?-catenin plays an important role in HCCR-1 oncogene expression
Article Snippet: .. Luciferase (LUC) reporter assay K562, HEK/293, and A549 cells were co-transfected with the pGL3-basic vector containing various HCCR-1 promoter regions and pCMV-RL. .. To investigate the effect of the Wnt-signal pathway on HCCR-1 expression, cells were treated with 5 mM LiCl (Sigma-Aldrich Corporation, St. Louis, MO, USA) for 24 h, then harvested.

Mutagenesis:

Article Title: Synergistic impact of mutations in Hepatitis B Virus genome contribute to its occult phenotype in chronic Hepatitis C Virus carriers
Article Snippet: .. In addition, the full-length promoter of GRP78 cloned in pGL3-Basic vector was transfected in Huh7 cells along with pRL-CMV vector and linear monomers wild-type or mutant-HBV (carrying Enh-II mutations as well as the combined PreS2 and X mutations). .. In all cases, after forty eight hours Firefly and Renilla luciferase activities were measured using Dual-Luciferase Reporter Assay kit (Promega) in luminometer.

Construct:

Article Title: Molecular mechanisms governing microRNA-125a expression in human hepatocellular carcinoma cells
Article Snippet: .. The 220 construct was obtained by digestion of the 384 construct with Sma I (restriction site within the genomic fragment) and Hind III (restriction site in the polylinker) and cloning the obtained fragment in the same sites of pGL3-basic vector. .. The 869mut construct with the mutated translation start codons described in Fig. was obtained by site-directed mutagenesis with the PCR-based overlap extension method as described .

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Article Snippet: .. As shown in Figure , transfection of (-1177∼+312)-Luc construct into IPEC-J2 cells resulted in an eightfold induction of luciferase activity compared with the pGL3-Basic vector (p < 0.05). .. Further deletions to bases -777, -345, and -195, respectively, did not cause significant difference of the luciferase activities compared with each other, indicating that there are no important regulatory elements within these regions ( Figure ).

Sequencing:

Article Title: The expression of AURKA is androgen regulated in castration-resistant prostate cancer
Article Snippet: .. ARBS in the AURKA enhancer region were cloned into the pGL3-Basic vector directly upstream of the AURKA promoter sequence. .. PCR was done for normal human leukocyte DNA to amplify the AURKA enhancer ARBS sequence.

Amplification:

Article Title: VEGF Promotes the Transcription of the Human PRL-3 Gene in HUVEC through Transcription Factor MEF2C
Article Snippet: .. Briefly, a DNA fragment containing the PRL-3 promoter region was amplified from HUVEC DNA and cloned into the pGL3 basic vector using primers in . .. Recombination PCR was performed as described previously to introduce mutations into the MEF2 binding sites based on luc-158, using the primers listed in .

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Activity Assay:

Article Title: Cloning and Transcriptional Activity Analysis of the Porcine Abcb1 Gene Promoter: Transcription Factor Sp1 Regulates the Expression of Porcine Abcb1
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Polymerase Chain Reaction:

Article Title: Transcriptional regulation of the potential tumor suppressor ABI3 gene in thyroid carcinomas: interplay between methylation and NKX2-1 availability
Article Snippet: .. The PCR-amplified product was digested with Kpn I and Xho I restriction enzymes (New England Biolabs, Hitchin, UK) and directionally cloned into the same restriction sites upstream to a promoterless firefly luciferase gene of the pGL3-Basic vector and into the pGL3-Control (Promega, Madison, WI), which contains a luciferase gene driven by the SV40 promoter. .. The empty pGL3-Basic and pGL3-Control were used as negative control.

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

Article Title: The expression of AURKA is androgen regulated in castration-resistant prostate cancer
Article Snippet: .. ARBS in the AURKA enhancer region were cloned into the pGL3-Basic vector directly upstream of the AURKA promoter sequence. .. PCR was done for normal human leukocyte DNA to amplify the AURKA enhancer ARBS sequence.

Article Title: Transcriptional regulation of the potential tumor suppressor ABI3 gene in thyroid carcinomas: interplay between methylation and NKX2-1 availability
Article Snippet: .. The PCR-amplified product was digested with Kpn I and Xho I restriction enzymes (New England Biolabs, Hitchin, UK) and directionally cloned into the same restriction sites upstream to a promoterless firefly luciferase gene of the pGL3-Basic vector and into the pGL3-Control (Promega, Madison, WI), which contains a luciferase gene driven by the SV40 promoter. .. The empty pGL3-Basic and pGL3-Control were used as negative control.

Article Title: TCF/?-catenin plays an important role in HCCR-1 oncogene expression
Article Snippet: .. Luciferase (LUC) reporter assay K562, HEK/293, and A549 cells were co-transfected with the pGL3-basic vector containing various HCCR-1 promoter regions and pCMV-RL. .. To investigate the effect of the Wnt-signal pathway on HCCR-1 expression, cells were treated with 5 mM LiCl (Sigma-Aldrich Corporation, St. Louis, MO, USA) for 24 h, then harvested.

Article Title: Molecular mechanisms governing microRNA-125a expression in human hepatocellular carcinoma cells
Article Snippet: .. The 220 construct was obtained by digestion of the 384 construct with Sma I (restriction site within the genomic fragment) and Hind III (restriction site in the polylinker) and cloning the obtained fragment in the same sites of pGL3-basic vector. .. The 869mut construct with the mutated translation start codons described in Fig. was obtained by site-directed mutagenesis with the PCR-based overlap extension method as described .

Article Title: Synergistic impact of mutations in Hepatitis B Virus genome contribute to its occult phenotype in chronic Hepatitis C Virus carriers
Article Snippet: .. In addition, the full-length promoter of GRP78 cloned in pGL3-Basic vector was transfected in Huh7 cells along with pRL-CMV vector and linear monomers wild-type or mutant-HBV (carrying Enh-II mutations as well as the combined PreS2 and X mutations). .. In all cases, after forty eight hours Firefly and Renilla luciferase activities were measured using Dual-Luciferase Reporter Assay kit (Promega) in luminometer.

Article Title: VEGF Promotes the Transcription of the Human PRL-3 Gene in HUVEC through Transcription Factor MEF2C
Article Snippet: .. Briefly, a DNA fragment containing the PRL-3 promoter region was amplified from HUVEC DNA and cloned into the pGL3 basic vector using primers in . .. Recombination PCR was performed as described previously to introduce mutations into the MEF2 binding sites based on luc-158, using the primers listed in .

Article Title: Hepatitis B Virus X Protein Up-Regulates AKR1C1 Expression Through Nuclear Factor-Y in Human Hepatocarcinoma Cells
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 ( , ). .. All the constructs were verified by restriction endonuclease digestion and sequencing.

Article Title: Cloning and Transcriptional Activity Analysis of the Porcine Abcb1 Gene Promoter: Transcription Factor Sp1 Regulates the Expression of Porcine Abcb1
Article Snippet: .. As shown in Figure , transfection of (-1177∼+312)-Luc construct into IPEC-J2 cells resulted in an eightfold induction of luciferase activity compared with the pGL3-Basic vector (p < 0.05). .. Further deletions to bases -777, -345, and -195, respectively, did not cause significant difference of the luciferase activities compared with each other, indicating that there are no important regulatory elements within these regions ( Figure ).

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  • 95
    Promega pgl3 basic vector
    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 <t>pGL3-Basic-AKR1C1-P</t> 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.
    Pgl3 Basic Vector, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 2320 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Promega pgl3 basic reporter vectors
    Luciferase reporter analysis of rs57137919G or A constructs of ABCG1 promoter. (a) Schematic diagram for constructing wild-type or mutant promoter at the ABCG1 rs57137919G > A polymorphism site. Two constructs were subcloned into <t>pGL3-basic</t> plasmid vectors with firefly luciferase reporter gene. pG, -367G (open bar); pA, -367A (grey bar). Luciferase activity assays for two ABCG1 promoter constructs in HEK293T cells (b), THP-1 cells (c), and HepG2 cells (d) are shown. Luciferase activities were measured with the dual-luciferase reporter assay system and normalized to Renilla luciferase activity 24 hours after transfection in the presence or absence of TO901317. The luciferase activity was a corrected relative value. Data are shown as mean ± SD of four independent experiments in triplicate. P
    Pgl3 Basic Reporter Vectors, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    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

    Mutational analysis of the HCCR-1 promoter . The diagrams depict locations of the predicted transcriptional elements in the HCCR-1 promoter. When mutated, the diagrams are indicated by a cross. 4 per construct). " width="100%" height="100%">

    Journal: BMC Molecular Biology

    Article Title: TCF/?-catenin plays an important role in HCCR-1 oncogene expression

    doi: 10.1186/1471-2199-10-42

    Figure Lengend Snippet: Mutational analysis of the HCCR-1 promoter . The diagrams depict locations of the predicted transcriptional elements in the HCCR-1 promoter. When mutated, the diagrams are indicated by a cross. "Tcf" and "My" indicate the consensus sequences for the TCF and C-Myb elements, respectively. Each luciferase reporter construct was co-transfected in K562 cells with pRL-CMV. The horizontal axis shows the ratio of luciferase (LUC) to renilla (REN) activity normalized by LUC/REN (= 100%) displayed by cells co-transfected with pRL-CMV and pGL3-control ( n > 4 per construct).

    Article Snippet: Luciferase (LUC) reporter assay K562, HEK/293, and A549 cells were co-transfected with the pGL3-basic vector containing various HCCR-1 promoter regions and pCMV-RL.

    Techniques: Luciferase, Construct, Transfection, Activity Assay

    Expression of the truncated HCCR-1 promoter in K562, HEK/293, or A549 cells . The DNA constructs containing various lengths of the HCCR-1 promoter region were cloned into upstream of the firefly luciferase (LUC) reporter gene. Each luciferase reporter construct shown in the diagram was co-transfected in K562 ( A and B ; filled bars), HEK293 ( A ; open bars), and A549 ( A ; shadow bars) cells with the pRL-CMV normalizing reporter plasmid encoding with the renilla (REN) gene. The horizontal axis shows the ratio of luciferase to renilla activity normalized by LUC/REN (= 100%) displayed by cells co-transfected with pRL-CMV and pGL3-control. We designated each recombinant vector as pGL3X~Y, where X is the first base and Y the last base of each truncated promoter ( n > 5 per construct).

    Journal: BMC Molecular Biology

    Article Title: TCF/?-catenin plays an important role in HCCR-1 oncogene expression

    doi: 10.1186/1471-2199-10-42

    Figure Lengend Snippet: Expression of the truncated HCCR-1 promoter in K562, HEK/293, or A549 cells . The DNA constructs containing various lengths of the HCCR-1 promoter region were cloned into upstream of the firefly luciferase (LUC) reporter gene. Each luciferase reporter construct shown in the diagram was co-transfected in K562 ( A and B ; filled bars), HEK293 ( A ; open bars), and A549 ( A ; shadow bars) cells with the pRL-CMV normalizing reporter plasmid encoding with the renilla (REN) gene. The horizontal axis shows the ratio of luciferase to renilla activity normalized by LUC/REN (= 100%) displayed by cells co-transfected with pRL-CMV and pGL3-control. We designated each recombinant vector as pGL3X~Y, where X is the first base and Y the last base of each truncated promoter ( n > 5 per construct).

    Article Snippet: Luciferase (LUC) reporter assay K562, HEK/293, and A549 cells were co-transfected with the pGL3-basic vector containing various HCCR-1 promoter regions and pCMV-RL.

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

    MEF2 binding sites are critical for the promoter activity of PRL-3 . ( A ) The region between −158 and −33 is important for the transcriptional activity of the PRL-3 promoter, as measured by luciferase activity. ( B ) The effect of site-directed mutations in the MEF2 binding sites, as measured by luciferase activity. All luciferase activity were normalized to β-galactosidase activity and standardized to the normalized activity of pGL3-Basic. Each column represents the mean ± SD of three independent experiments. ** significantly different from the control at P

    Journal: PLoS ONE

    Article Title: VEGF Promotes the Transcription of the Human PRL-3 Gene in HUVEC through Transcription Factor MEF2C

    doi: 10.1371/journal.pone.0027165

    Figure Lengend Snippet: MEF2 binding sites are critical for the promoter activity of PRL-3 . ( A ) The region between −158 and −33 is important for the transcriptional activity of the PRL-3 promoter, as measured by luciferase activity. ( B ) The effect of site-directed mutations in the MEF2 binding sites, as measured by luciferase activity. All luciferase activity were normalized to β-galactosidase activity and standardized to the normalized activity of pGL3-Basic. Each column represents the mean ± SD of three independent experiments. ** significantly different from the control at P

    Article Snippet: Briefly, a DNA fragment containing the PRL-3 promoter region was amplified from HUVEC DNA and cloned into the pGL3 basic vector using primers in .

    Techniques: Binding Assay, Activity Assay, Luciferase

    Luciferase reporter analysis of rs57137919G or A constructs of ABCG1 promoter. (a) Schematic diagram for constructing wild-type or mutant promoter at the ABCG1 rs57137919G > A polymorphism site. Two constructs were subcloned into pGL3-basic plasmid vectors with firefly luciferase reporter gene. pG, -367G (open bar); pA, -367A (grey bar). Luciferase activity assays for two ABCG1 promoter constructs in HEK293T cells (b), THP-1 cells (c), and HepG2 cells (d) are shown. Luciferase activities were measured with the dual-luciferase reporter assay system and normalized to Renilla luciferase activity 24 hours after transfection in the presence or absence of TO901317. The luciferase activity was a corrected relative value. Data are shown as mean ± SD of four independent experiments in triplicate. P

    Journal: PLoS ONE

    Article Title: ABCG1 rs57137919G > A Polymorphism Is Functionally Associated with Varying Gene Expression and Apoptosis of Macrophages

    doi: 10.1371/journal.pone.0097044

    Figure Lengend Snippet: Luciferase reporter analysis of rs57137919G or A constructs of ABCG1 promoter. (a) Schematic diagram for constructing wild-type or mutant promoter at the ABCG1 rs57137919G > A polymorphism site. Two constructs were subcloned into pGL3-basic plasmid vectors with firefly luciferase reporter gene. pG, -367G (open bar); pA, -367A (grey bar). Luciferase activity assays for two ABCG1 promoter constructs in HEK293T cells (b), THP-1 cells (c), and HepG2 cells (d) are shown. Luciferase activities were measured with the dual-luciferase reporter assay system and normalized to Renilla luciferase activity 24 hours after transfection in the presence or absence of TO901317. The luciferase activity was a corrected relative value. Data are shown as mean ± SD of four independent experiments in triplicate. P

    Article Snippet: A PCR product with wild-type or mutation was then cloned into pGL3-basic reporter vectors with firefly luciferase gene (Promega, Madison, WI, USA); the two constructs were verified by sequencing.

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