Structured Review

Promega luciferase reporter vector pgl4 10
Duplication or deletion of the human SOX9 testicular enhancer eSR-A is associated with DSD. a The 600 kb genomic region upstream of human SOX9 showing the XYSR, RevSex and TESCO candidate regulatory regions. b The XYSR region was defined by deletions in two previously published 46,XY DSD patients (blue) 17 . Two novel duplications in 46,XX DSD patients (grey), allowed us to redefine the minimal overlap to 5.2 kb (green). c Sub-cloning strategy, dark blue lines indicate sub-clones analysed for enhancer activity in the <t>pGL4.10</t> Beta-globin (βg) plasmid using luciferase assays. Predicted transcription factor binding motifs for SF1 (SF1-a and SF1-b) and SOX9 are shown with vertical light blue and magenta lines. Bioinformatic tracks from the UCSC genome browser are shown including the ENCODE track of enhancers present in human mammary epithelial cells (HMEC) (yellow denotes a weak enhancer) 28 and DNaseI hypersensitivity data from human foetal testis and ovary (ROADMap) 23 . This shows a testis-specific peak over the a4 fragment. The 100-vertebrate conservation track shows a spike of conservation beneath the a4 fragment DNaseI peak. d Enhancer activities of sub-cloned fragments a1-a5 as measured by luciferase assays transfected with SF1 and SOX9 ( n = 4). e Mutation of the SF1-a (eSR-A ΔSF1-a), and SOX9 (eSR-A ΔSOX9) binding motifs either separately or together results in a loss of enhancer activity compared to non-mutated eSR-A as assessed by luciferase assays with co-transfection of SF1 and SOX9 ( n = 4). f . Co-transfection of FOXL2 with SF1 + SOX9 shows a repression of eSR-A activity compared to SF1 + SOX9 ( n = 3). All luciferase assays carried out in COS7 cells. Error bars are s.e.m. P -values (two-tailed t tests): * P ≤ 0.05. ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are provided as a Source Data file.
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1) Product Images from "Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9"

Article Title: Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9

Journal: Nature Communications

doi: 10.1038/s41467-018-07784-9

Duplication or deletion of the human SOX9 testicular enhancer eSR-A is associated with DSD. a The 600 kb genomic region upstream of human SOX9 showing the XYSR, RevSex and TESCO candidate regulatory regions. b The XYSR region was defined by deletions in two previously published 46,XY DSD patients (blue) 17 . Two novel duplications in 46,XX DSD patients (grey), allowed us to redefine the minimal overlap to 5.2 kb (green). c Sub-cloning strategy, dark blue lines indicate sub-clones analysed for enhancer activity in the pGL4.10 Beta-globin (βg) plasmid using luciferase assays. Predicted transcription factor binding motifs for SF1 (SF1-a and SF1-b) and SOX9 are shown with vertical light blue and magenta lines. Bioinformatic tracks from the UCSC genome browser are shown including the ENCODE track of enhancers present in human mammary epithelial cells (HMEC) (yellow denotes a weak enhancer) 28 and DNaseI hypersensitivity data from human foetal testis and ovary (ROADMap) 23 . This shows a testis-specific peak over the a4 fragment. The 100-vertebrate conservation track shows a spike of conservation beneath the a4 fragment DNaseI peak. d Enhancer activities of sub-cloned fragments a1-a5 as measured by luciferase assays transfected with SF1 and SOX9 ( n = 4). e Mutation of the SF1-a (eSR-A ΔSF1-a), and SOX9 (eSR-A ΔSOX9) binding motifs either separately or together results in a loss of enhancer activity compared to non-mutated eSR-A as assessed by luciferase assays with co-transfection of SF1 and SOX9 ( n = 4). f . Co-transfection of FOXL2 with SF1 + SOX9 shows a repression of eSR-A activity compared to SF1 + SOX9 ( n = 3). All luciferase assays carried out in COS7 cells. Error bars are s.e.m. P -values (two-tailed t tests): * P ≤ 0.05. ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are provided as a Source Data file.
Figure Legend Snippet: Duplication or deletion of the human SOX9 testicular enhancer eSR-A is associated with DSD. a The 600 kb genomic region upstream of human SOX9 showing the XYSR, RevSex and TESCO candidate regulatory regions. b The XYSR region was defined by deletions in two previously published 46,XY DSD patients (blue) 17 . Two novel duplications in 46,XX DSD patients (grey), allowed us to redefine the minimal overlap to 5.2 kb (green). c Sub-cloning strategy, dark blue lines indicate sub-clones analysed for enhancer activity in the pGL4.10 Beta-globin (βg) plasmid using luciferase assays. Predicted transcription factor binding motifs for SF1 (SF1-a and SF1-b) and SOX9 are shown with vertical light blue and magenta lines. Bioinformatic tracks from the UCSC genome browser are shown including the ENCODE track of enhancers present in human mammary epithelial cells (HMEC) (yellow denotes a weak enhancer) 28 and DNaseI hypersensitivity data from human foetal testis and ovary (ROADMap) 23 . This shows a testis-specific peak over the a4 fragment. The 100-vertebrate conservation track shows a spike of conservation beneath the a4 fragment DNaseI peak. d Enhancer activities of sub-cloned fragments a1-a5 as measured by luciferase assays transfected with SF1 and SOX9 ( n = 4). e Mutation of the SF1-a (eSR-A ΔSF1-a), and SOX9 (eSR-A ΔSOX9) binding motifs either separately or together results in a loss of enhancer activity compared to non-mutated eSR-A as assessed by luciferase assays with co-transfection of SF1 and SOX9 ( n = 4). f . Co-transfection of FOXL2 with SF1 + SOX9 shows a repression of eSR-A activity compared to SF1 + SOX9 ( n = 3). All luciferase assays carried out in COS7 cells. Error bars are s.e.m. P -values (two-tailed t tests): * P ≤ 0.05. ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are provided as a Source Data file.

Techniques Used: Electron Paramagnetic Resonance, Subcloning, Clone Assay, Activity Assay, Plasmid Preparation, Luciferase, Binding Assay, Transfection, Mutagenesis, Cotransfection, Two Tailed Test

2) Product Images from "CHIR99021 enhances Klf4 Expression through β-Catenin Signaling and miR-7a Regulation in J1 Mouse Embryonic Stem Cells"

Article Title: CHIR99021 enhances Klf4 Expression through β-Catenin Signaling and miR-7a Regulation in J1 Mouse Embryonic Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0150936

CHIR regulates Klf4 expression by canonical Wnt pathway activation. (A) : TopFlash and FopFlash assay after CHIR treatment. J1 mESCs were transfected with pSuperTOPFlash reporter plasmids or the pTA-luc control plasmid. At 5 h after transfection, fresh medium was added and 3 μM CHIR or an equal volume of DMSO was added to the transfected cells. 24 h after transfection, luciferase activity was detected using the dual-luciferase reporter assay. (B) : TopFlash and FopFlash assay after overexpression of β -catenin. pCMV-Myc or pCMV-Myc- β -catenin was co-transfected with pSuperTOPFlash reporter plasmids or pTA-luc control plasmid into J1 mESCs, followed by 24 h of incubation. Luciferase activity is expressed relative to that of pTA-luc. Data are presented as the mean ± SD of three independent experiments. (C) : CHIR treatment or β -catenin overexpression promotes nuclear β -catenin expression. J1 mESCs were treated with 3 μM CHIR or equal volume of DMSO (upper panel), or transfected with pCDNA3.1- β -catenin s37a / pCDNA3.1 control plasmid (down panel) for 48 h, The nucleus protein were extracted and the expression of β -catenin was analyzed by western blot. Relative expression levels were compared with PCNA. (D) : β -catenin knockdown. Cells were transfected with siRNA- β -catenin or NC for 48 h, and RT-qPCR (upper panel) or western blot (lower panel) was used to detect the knockdown efficiency of β -catenin. (E) : Knockdown of β -catenin represses Klf4 expression. J1 mESCs were transfected with siRNA- β -catenin or siRNA-NC. At 5 h after transfection, fresh medium was added and 3 μM CHIR or an equal volume of DMSO was added to the transfected cells, followed by 48 h of incubation. Klf4 expression was validated by qPCR. Gapdh was used to normalize template levels. (F) : A novel cis element in the Klf4 gene was activated by β -catenin. The cis-element (WT) and its mutational type (Mut) are shown in the upper panel. A schematic representation of the promoter structure of Klf4 is shown in the lower panel. Control plasmid pGL4.10 was co-transfected with pGL- Klf4 (−1124/+240) or pGL- Klf4 (−1081/+240) promoter reporter plasmid into J1 mESCs. At 5 h after transfection, fresh medium was changed and 3 μM CHIR or an equal volume of DMSO was added to the transfected cells, followed by 24 h of incubation. Luciferase activity is expressed relative to that of pGL4.10. Data are presented as the mean ± SD of three independent experiments. (G) : Chromatin immunoprecipitation assay for the detection of cis-element in Klf4 gene. ChIP was performed using anti-β-catenin antibody and anti-IgG as a control antibody to detect enriched fragments. Data are presented as the mean ± SD of three independent experiments. (*p
Figure Legend Snippet: CHIR regulates Klf4 expression by canonical Wnt pathway activation. (A) : TopFlash and FopFlash assay after CHIR treatment. J1 mESCs were transfected with pSuperTOPFlash reporter plasmids or the pTA-luc control plasmid. At 5 h after transfection, fresh medium was added and 3 μM CHIR or an equal volume of DMSO was added to the transfected cells. 24 h after transfection, luciferase activity was detected using the dual-luciferase reporter assay. (B) : TopFlash and FopFlash assay after overexpression of β -catenin. pCMV-Myc or pCMV-Myc- β -catenin was co-transfected with pSuperTOPFlash reporter plasmids or pTA-luc control plasmid into J1 mESCs, followed by 24 h of incubation. Luciferase activity is expressed relative to that of pTA-luc. Data are presented as the mean ± SD of three independent experiments. (C) : CHIR treatment or β -catenin overexpression promotes nuclear β -catenin expression. J1 mESCs were treated with 3 μM CHIR or equal volume of DMSO (upper panel), or transfected with pCDNA3.1- β -catenin s37a / pCDNA3.1 control plasmid (down panel) for 48 h, The nucleus protein were extracted and the expression of β -catenin was analyzed by western blot. Relative expression levels were compared with PCNA. (D) : β -catenin knockdown. Cells were transfected with siRNA- β -catenin or NC for 48 h, and RT-qPCR (upper panel) or western blot (lower panel) was used to detect the knockdown efficiency of β -catenin. (E) : Knockdown of β -catenin represses Klf4 expression. J1 mESCs were transfected with siRNA- β -catenin or siRNA-NC. At 5 h after transfection, fresh medium was added and 3 μM CHIR or an equal volume of DMSO was added to the transfected cells, followed by 48 h of incubation. Klf4 expression was validated by qPCR. Gapdh was used to normalize template levels. (F) : A novel cis element in the Klf4 gene was activated by β -catenin. The cis-element (WT) and its mutational type (Mut) are shown in the upper panel. A schematic representation of the promoter structure of Klf4 is shown in the lower panel. Control plasmid pGL4.10 was co-transfected with pGL- Klf4 (−1124/+240) or pGL- Klf4 (−1081/+240) promoter reporter plasmid into J1 mESCs. At 5 h after transfection, fresh medium was changed and 3 μM CHIR or an equal volume of DMSO was added to the transfected cells, followed by 24 h of incubation. Luciferase activity is expressed relative to that of pGL4.10. Data are presented as the mean ± SD of three independent experiments. (G) : Chromatin immunoprecipitation assay for the detection of cis-element in Klf4 gene. ChIP was performed using anti-β-catenin antibody and anti-IgG as a control antibody to detect enriched fragments. Data are presented as the mean ± SD of three independent experiments. (*p

Techniques Used: Expressing, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay, Over Expression, Incubation, Western Blot, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

3) Product Images from "Identification of Novel Kaposi's Sarcoma-Associated Herpesvirus Orf50 Transcripts: Discovery of New RTA Isoforms with Variable Transactivation Potential"

Article Title: Identification of Novel Kaposi's Sarcoma-Associated Herpesvirus Orf50 Transcripts: Discovery of New RTA Isoforms with Variable Transactivation Potential

Journal: Journal of Virology

doi: 10.1128/JVI.01434-16

Promoter activity of the proximal, distal, N3/N4, and N5 Orf50 KSHV promoters in various cell types. (A) Vero, 3T12, and RAW 264.7 cells were transfected with pGL4.10[luc] luciferase reporter construct containing 250 bp upstream of the E1 exon. At 48 h posttransfection, luciferase assays were performed. (B) Vero, 3T12, and RAW 264.7 cells were transfected with the pGL4.10[luc] luciferase reporter construct containing 500 bp upstream of the E0A and E0B exon. At 48 h posttransfection, luciferase assays were performed. (C) Vero, 3T12, and RAW 264.7 cells were transfected with the pGL4.10[luc] luciferase reporter construct containing 500 bp upstream of the N3 and N4 exon. At 48 h posttransfection, luciferase assays were performed. (D) Vero, 3T12, and RAW 264.7 cells were transfected with the pGL4.10[luc] luciferase reporter construct containing 500 bp upstream of the N5 exon. At 48 h posttransfection, luciferase assays were performed. Data are presented as the fold difference in firefly luciferase activity versus the pGL4.10 empty vector control. The data represent triplicates of at least three independent transfections.
Figure Legend Snippet: Promoter activity of the proximal, distal, N3/N4, and N5 Orf50 KSHV promoters in various cell types. (A) Vero, 3T12, and RAW 264.7 cells were transfected with pGL4.10[luc] luciferase reporter construct containing 250 bp upstream of the E1 exon. At 48 h posttransfection, luciferase assays were performed. (B) Vero, 3T12, and RAW 264.7 cells were transfected with the pGL4.10[luc] luciferase reporter construct containing 500 bp upstream of the E0A and E0B exon. At 48 h posttransfection, luciferase assays were performed. (C) Vero, 3T12, and RAW 264.7 cells were transfected with the pGL4.10[luc] luciferase reporter construct containing 500 bp upstream of the N3 and N4 exon. At 48 h posttransfection, luciferase assays were performed. (D) Vero, 3T12, and RAW 264.7 cells were transfected with the pGL4.10[luc] luciferase reporter construct containing 500 bp upstream of the N5 exon. At 48 h posttransfection, luciferase assays were performed. Data are presented as the fold difference in firefly luciferase activity versus the pGL4.10 empty vector control. The data represent triplicates of at least three independent transfections.

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

RTA isoforms have the potential to share a RRE binding site. (A and B) Reporter constructs were generated for both the Orf4 and the Orf59 KSHV promoters. We generated 100-bp deletions starting in the context of 507 bp (Orf59) and 500 bp (Orf4) and cloned them into the pGL4.10[luc] luciferase reporter construct. 293T cells were transfected with the reporter constructs in conjunction with the expression constructs pCMV-Flag2B-Iso1 or pCMV-Flag2B-Iso4. At 48 h posttransfection, luciferase assays were performed. Data are plotted as fold changes versus empty vector on a log scale. Experiments were performed in triplicate, and graphs depict the average.
Figure Legend Snippet: RTA isoforms have the potential to share a RRE binding site. (A and B) Reporter constructs were generated for both the Orf4 and the Orf59 KSHV promoters. We generated 100-bp deletions starting in the context of 507 bp (Orf59) and 500 bp (Orf4) and cloned them into the pGL4.10[luc] luciferase reporter construct. 293T cells were transfected with the reporter constructs in conjunction with the expression constructs pCMV-Flag2B-Iso1 or pCMV-Flag2B-Iso4. At 48 h posttransfection, luciferase assays were performed. Data are plotted as fold changes versus empty vector on a log scale. Experiments were performed in triplicate, and graphs depict the average.

Techniques Used: Binding Assay, Construct, Generated, Clone Assay, Luciferase, Transfection, Expressing, Plasmid Preparation

Promoter activity of K-bZIP and PAN when transfected in the presence of RTA isoform 1 or 4, as well as the potential expression of other viral proteins. (A) 293TΔ50BAC cells, containing all other viral proteins but RTA, were transfected with 1.25 μg of K-bZIP pGL4.10[luc] luciferase reporter constructs in conjunction with 1.25 μg of pCMV-Flag2B-Iso1 or pCMV-Flag2B-Iso4. The cells were left either untreated or stimulated with TPA and, at 48 h after transfection, luciferase assays were performed. (B) 293TΔ50BAC cells were transfected with 1.25 μg of PAN pGL4.10[luc] luciferase reporter constructs in conjunction with 1.25 μg of pCMV-Flag2B-Iso1 or pCMV-Flag2B-Iso4. These cells were also left either untreated or stimulated with TPA and, at 48 h after transfection, luciferase assays were performed. Data are presented as the fold change versus the reporter construct plus pCMV-Flag2B-Empty Vector. The data represent triplicates of at least three independent transfections.
Figure Legend Snippet: Promoter activity of K-bZIP and PAN when transfected in the presence of RTA isoform 1 or 4, as well as the potential expression of other viral proteins. (A) 293TΔ50BAC cells, containing all other viral proteins but RTA, were transfected with 1.25 μg of K-bZIP pGL4.10[luc] luciferase reporter constructs in conjunction with 1.25 μg of pCMV-Flag2B-Iso1 or pCMV-Flag2B-Iso4. The cells were left either untreated or stimulated with TPA and, at 48 h after transfection, luciferase assays were performed. (B) 293TΔ50BAC cells were transfected with 1.25 μg of PAN pGL4.10[luc] luciferase reporter constructs in conjunction with 1.25 μg of pCMV-Flag2B-Iso1 or pCMV-Flag2B-Iso4. These cells were also left either untreated or stimulated with TPA and, at 48 h after transfection, luciferase assays were performed. Data are presented as the fold change versus the reporter construct plus pCMV-Flag2B-Empty Vector. The data represent triplicates of at least three independent transfections.

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

RTA isoforms can interact with cellular transcription factors in a synergistic manner. Reporter constructs were generated for the Orf57 KSHV promoter. Four constructs were generated: a full-length Orf57 KSHV promoter, an RTA responsive element (RRE) deletion in the context of the Orf57 KSHV promoter, an RBP-Jk deletion in the context of the Orf57 KSHV promoter, and an RRE plus RBP-Jk deletion in the context of the Orf57 KSHV promoter. All constructs were cloned into the pGL4.10[luc] luciferase reporter and transfected in conjunction with pCMV-Flag2B-Iso1 or pCMV-Flag2B-Iso4 into 293T cells. At 48 h posttransfection, luciferase assays were performed. Data are presented as the fold difference versus the expression construct plus the pCMV-Flag2B-Empty Vector. The data represent triplicates of at least three independent experiments.
Figure Legend Snippet: RTA isoforms can interact with cellular transcription factors in a synergistic manner. Reporter constructs were generated for the Orf57 KSHV promoter. Four constructs were generated: a full-length Orf57 KSHV promoter, an RTA responsive element (RRE) deletion in the context of the Orf57 KSHV promoter, an RBP-Jk deletion in the context of the Orf57 KSHV promoter, and an RRE plus RBP-Jk deletion in the context of the Orf57 KSHV promoter. All constructs were cloned into the pGL4.10[luc] luciferase reporter and transfected in conjunction with pCMV-Flag2B-Iso1 or pCMV-Flag2B-Iso4 into 293T cells. At 48 h posttransfection, luciferase assays were performed. Data are presented as the fold difference versus the expression construct plus the pCMV-Flag2B-Empty Vector. The data represent triplicates of at least three independent experiments.

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

4) Product Images from "The CHR site: definition and genome-wide identification of a cell cycle transcriptional element"

Article Title: The CHR site: definition and genome-wide identification of a cell cycle transcriptional element

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku696

DREAM can bind to non-canonical CHR elements in DNA affinity purification assays. (A) Analysis of DREAM complex binding to the isolated cyclin B2 CDE/CHR element flanked by irrelevant DNA and to a 220-bp cyclin B2 promoter probe. Biotinylated DNA probes based on the mouse Ccnb2 promoter (Ccnb2) and on the CDE/CHR element from the same promoter (CDE/CHR) were cloned in the pGL4.10 vector backbone and subjected to DNA affinity purifications with density-arrested NIH3T3 cells. Binding of DREAM components (p130, E2f4, Lin37, Lin54) to both probes was analyzed with polyclonal antibodies. As a non-DREAM binding negative control, a DNA probe based on the empty pGL4.10 vector was utilized (CTRL). All samples are from the same blot. (B) DREAM complex binding to CHR elements differing from the canonical sequence TTTGAA in one nucleotide analyzed in an in vitro DNA affinity purification assay. Biotinylated probes based on the CDE/CHR probe with all possible 1 bp permutations were prepared and subjected to DNA affinity purifications with nuclear extracts from density-arrested NIH3T3 cells. A DNA probe based on the empty pGL4.10 vector was used as a non-DREAM binding negative control (CTRL). Eluates were probed in western blots for the DREAM components p130, E2f4 and Lin9. All samples were processed in parallel.
Figure Legend Snippet: DREAM can bind to non-canonical CHR elements in DNA affinity purification assays. (A) Analysis of DREAM complex binding to the isolated cyclin B2 CDE/CHR element flanked by irrelevant DNA and to a 220-bp cyclin B2 promoter probe. Biotinylated DNA probes based on the mouse Ccnb2 promoter (Ccnb2) and on the CDE/CHR element from the same promoter (CDE/CHR) were cloned in the pGL4.10 vector backbone and subjected to DNA affinity purifications with density-arrested NIH3T3 cells. Binding of DREAM components (p130, E2f4, Lin37, Lin54) to both probes was analyzed with polyclonal antibodies. As a non-DREAM binding negative control, a DNA probe based on the empty pGL4.10 vector was utilized (CTRL). All samples are from the same blot. (B) DREAM complex binding to CHR elements differing from the canonical sequence TTTGAA in one nucleotide analyzed in an in vitro DNA affinity purification assay. Biotinylated probes based on the CDE/CHR probe with all possible 1 bp permutations were prepared and subjected to DNA affinity purifications with nuclear extracts from density-arrested NIH3T3 cells. A DNA probe based on the empty pGL4.10 vector was used as a non-DREAM binding negative control (CTRL). Eluates were probed in western blots for the DREAM components p130, E2f4 and Lin9. All samples were processed in parallel.

Techniques Used: Affinity Purification, Binding Assay, Isolation, Clone Assay, Plasmid Preparation, Negative Control, Sequencing, In Vitro, Western Blot

Non-canonical CHR elements are essential for cell cycle-dependent gene transcription and DREAM binding. (A) Promoter activity of Bub1 , Chek2 , Melk and Pold1 was analyzed with luciferase reporter assays in NIH3T3 cells synchronized by serum starvation followed by serum re-stimulation. Activities of wild-type promoters were measured in different cell cycle phases and compared to the activity of the corresponding CHR mutants (ΔCHR). pGL4.10 empty vector served as a negative control. FACS analyses for the different time points are shown in Supplementary Figure S1. (B) DREAM binding to promoters with non-canonical CHR elements was analyzed by DNA affinity purification followed by western blot. Proteins from nuclear extracts of density-arrested NIH3T3 cells binding to wild-type promoters (WT) and CHR mutants (ΔCHR) were probed with antibodies targeting p130, E2f4 and Lin37 as representative components of DREAM. A DNA probe of the Gapdhs promoter served as a negative control (CTRL).
Figure Legend Snippet: Non-canonical CHR elements are essential for cell cycle-dependent gene transcription and DREAM binding. (A) Promoter activity of Bub1 , Chek2 , Melk and Pold1 was analyzed with luciferase reporter assays in NIH3T3 cells synchronized by serum starvation followed by serum re-stimulation. Activities of wild-type promoters were measured in different cell cycle phases and compared to the activity of the corresponding CHR mutants (ΔCHR). pGL4.10 empty vector served as a negative control. FACS analyses for the different time points are shown in Supplementary Figure S1. (B) DREAM binding to promoters with non-canonical CHR elements was analyzed by DNA affinity purification followed by western blot. Proteins from nuclear extracts of density-arrested NIH3T3 cells binding to wild-type promoters (WT) and CHR mutants (ΔCHR) were probed with antibodies targeting p130, E2f4 and Lin37 as representative components of DREAM. A DNA probe of the Gapdhs promoter served as a negative control (CTRL).

Techniques Used: Binding Assay, Activity Assay, Luciferase, Plasmid Preparation, Negative Control, FACS, Affinity Purification, Western Blot

CHR elements are enriched in promoters of genes bound by DREAM, MMB and FOXM1. (A) Percentage of promoters with or without CHR elements (+CHR or –CHR) and with or without E2F sites (+E2F or –E2F) in different promoter subsets: all promoters ( All Genes ), DREAM-bound promoters ( D ), promoters bound by the MMB components B-MYB and LIN9 ( MMB ), promoters bound by FOXM1 ( FOXM1 ) and promoters bound by two or all three of the complexes. (B) Fraction of promoters with or without Myb-binding sites (+MBS or –MBS) and with or without CHRs (+CHR, –CHR) in different promoter subsets. (C) Percentage of promoters with or without forkhead-binding sites (+FBS or –FBS) and with or without CHRs (+CHR, –CHR) in different promoter subsets. (D) Binding of MMB to the isolated cyclin B2 CDE/CHR site flanked by irrelevant DNA in comparison to a 220 bp cyclin B2 promoter probe. Biotinylated DNA probes derived from the mouse Ccnb2 promoter (Ccnb2) and from the cyclin B2 CDE/CHR element cloned into the pGL4.10 vector backbone (CDE/CHR) were subjected to DNA affinity purifications with nuclear extracts of proliferating F9 cells which do not form DREAM. MMB components B-Myb, Lin54, Lin37 and Lin9 were detected by western blot. To control the absence of DREAM binding to the probes, E2f4 as part of the DREAM complex was analyzed. As a non-MMB-binding negative control, a DNA probe based on the empty pGL4.10 vector was employed (CTRL).
Figure Legend Snippet: CHR elements are enriched in promoters of genes bound by DREAM, MMB and FOXM1. (A) Percentage of promoters with or without CHR elements (+CHR or –CHR) and with or without E2F sites (+E2F or –E2F) in different promoter subsets: all promoters ( All Genes ), DREAM-bound promoters ( D ), promoters bound by the MMB components B-MYB and LIN9 ( MMB ), promoters bound by FOXM1 ( FOXM1 ) and promoters bound by two or all three of the complexes. (B) Fraction of promoters with or without Myb-binding sites (+MBS or –MBS) and with or without CHRs (+CHR, –CHR) in different promoter subsets. (C) Percentage of promoters with or without forkhead-binding sites (+FBS or –FBS) and with or without CHRs (+CHR, –CHR) in different promoter subsets. (D) Binding of MMB to the isolated cyclin B2 CDE/CHR site flanked by irrelevant DNA in comparison to a 220 bp cyclin B2 promoter probe. Biotinylated DNA probes derived from the mouse Ccnb2 promoter (Ccnb2) and from the cyclin B2 CDE/CHR element cloned into the pGL4.10 vector backbone (CDE/CHR) were subjected to DNA affinity purifications with nuclear extracts of proliferating F9 cells which do not form DREAM. MMB components B-Myb, Lin54, Lin37 and Lin9 were detected by western blot. To control the absence of DREAM binding to the probes, E2f4 as part of the DREAM complex was analyzed. As a non-MMB-binding negative control, a DNA probe based on the empty pGL4.10 vector was employed (CTRL).

Techniques Used: Binding Assay, Isolation, Derivative Assay, Clone Assay, Plasmid Preparation, Western Blot, Negative Control

5) Product Images from "In Vitro and Ex Vivo Analysis of CHRNA3 and CHRNA5 Haplotype Expression"

Article Title: In Vitro and Ex Vivo Analysis of CHRNA3 and CHRNA5 Haplotype Expression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0023373

Relative CHRNA5 promoter activity in BE(2)-C cells. Shown are the averages ± standard deviations of five separate transfections each done in triplicate. The SV40 promoter of the positive control vector pGL4.13[luc2/SV40] is indicated by a yellow box. The “promoterless” negative (background) control vector pGL4.10[luc2] is shown without a promoter region in front of the firefly luciferase (“fLuc”) reporter gene. For experimental CHRNA5 promoter-5′UTR constructs, thin and thick black lines represent the promoter and 5′UTR portion of the construct, respectively. Positions of the Xho I (−1440) and Nco I (+1) sites used for subcloning are indicated, as is an internal Nco I site (−730). The positions and identities of the rs3841324 (−227 to −248), rs503464 (−166W), rs55853698 (−123K) and rs55781567 (−76S) polymorphisms within each construct are indicated. The deletion (“d” in construct name) or normal (“i” in construct name) allele at rs3841324 is indicated by a red triangle or blue square, respectively. The deletion (“d”) constructs exhibited decreased activity (1.6–2.0-fold) relative to the normal (“i”) constructs. *p
Figure Legend Snippet: Relative CHRNA5 promoter activity in BE(2)-C cells. Shown are the averages ± standard deviations of five separate transfections each done in triplicate. The SV40 promoter of the positive control vector pGL4.13[luc2/SV40] is indicated by a yellow box. The “promoterless” negative (background) control vector pGL4.10[luc2] is shown without a promoter region in front of the firefly luciferase (“fLuc”) reporter gene. For experimental CHRNA5 promoter-5′UTR constructs, thin and thick black lines represent the promoter and 5′UTR portion of the construct, respectively. Positions of the Xho I (−1440) and Nco I (+1) sites used for subcloning are indicated, as is an internal Nco I site (−730). The positions and identities of the rs3841324 (−227 to −248), rs503464 (−166W), rs55853698 (−123K) and rs55781567 (−76S) polymorphisms within each construct are indicated. The deletion (“d” in construct name) or normal (“i” in construct name) allele at rs3841324 is indicated by a red triangle or blue square, respectively. The deletion (“d”) constructs exhibited decreased activity (1.6–2.0-fold) relative to the normal (“i”) constructs. *p

Techniques Used: Activity Assay, Transfection, Positive Control, Plasmid Preparation, Luciferase, Construct, Subcloning

Relative CHRNA5 5′UTR variant luciferase activity in BE(2)-C cells. Shown are the averages ± standard deviations of four independent experiments each done in triplicate. The SV40 promoter of the positive control vector pGL4.13[luc2/SV40] is indicated by a yellow box. The “promoterless” negative (background) control vector pGL4.10[luc2] is shown without a promoter region in front of the firefly luciferase (“fLuc”) reporter gene. For experimental CHRNA5 promoter-5′UTR constructs, thin and thick black lines represent the promoter and 5′UTR portion of the construct, respectively. Positions of the Xho I (−1440) and Nco I (+1) sites used for subcloning are indicated, as is an internal Nco I site (−730). The positions and identities of the rs3841324 (−227 to −248, blue box indicates the normal or “i” allele), rs503464 (−166W), rs55853698 (−123K) and rs55781567 (−76S) polymorphisms within each construct are indicated. Note that the “A” allele at rs503464 (−166 W) was not changed to “T” (as defined by haplotypes in Europeans, [26] ) in the pGL(XNNA5iAGG) construct, in order to isolate the 5′UTR SNPs for effects on translation in an equivalent promoter context for each construct. ‡p
Figure Legend Snippet: Relative CHRNA5 5′UTR variant luciferase activity in BE(2)-C cells. Shown are the averages ± standard deviations of four independent experiments each done in triplicate. The SV40 promoter of the positive control vector pGL4.13[luc2/SV40] is indicated by a yellow box. The “promoterless” negative (background) control vector pGL4.10[luc2] is shown without a promoter region in front of the firefly luciferase (“fLuc”) reporter gene. For experimental CHRNA5 promoter-5′UTR constructs, thin and thick black lines represent the promoter and 5′UTR portion of the construct, respectively. Positions of the Xho I (−1440) and Nco I (+1) sites used for subcloning are indicated, as is an internal Nco I site (−730). The positions and identities of the rs3841324 (−227 to −248, blue box indicates the normal or “i” allele), rs503464 (−166W), rs55853698 (−123K) and rs55781567 (−76S) polymorphisms within each construct are indicated. Note that the “A” allele at rs503464 (−166 W) was not changed to “T” (as defined by haplotypes in Europeans, [26] ) in the pGL(XNNA5iAGG) construct, in order to isolate the 5′UTR SNPs for effects on translation in an equivalent promoter context for each construct. ‡p

Techniques Used: Variant Assay, Luciferase, Activity Assay, Positive Control, Plasmid Preparation, Construct, Subcloning

Relative CHRNA3 promoter activity in BE(2)-C cells. Shown are the averages ± standard deviations of at least three independent experiments each done in triplicate. The SV40 promoter of the positive control vector pGL4.13[luc2/SV40] is indicated by a yellow box. The “promoterless” negative (background) control vector pGL4.10[luc2] is shown without a promoter region in front of the firefly luciferase (“fLuc”) reporter gene. For experimental CHRNA3 promoter-5′UTR constructs, thin and thick black lines represent the promoter and 5′UTR portion of the construct, respectively. Positions of the Xho I (−1442) and Nco I (+1) sites used for subcloning are indicated, as is an internal Nco I site (−1064). The positions and identities of the rs13329271 (−1093 K; “initiator”) and rs12911814 (−847 K) polymorphisms, as well as the number of “TTTTG” repeats (−822 to −847) and the number of “GCCC” repeats (−235) within each construct are indicated. There were no statistically significant differences between comparisons of analogous ( i.e. five full length, four 5′ or two 3′ truncated) promoter haplotype constructs by ANOVA with Tukey's HSD post-hoc analysis.
Figure Legend Snippet: Relative CHRNA3 promoter activity in BE(2)-C cells. Shown are the averages ± standard deviations of at least three independent experiments each done in triplicate. The SV40 promoter of the positive control vector pGL4.13[luc2/SV40] is indicated by a yellow box. The “promoterless” negative (background) control vector pGL4.10[luc2] is shown without a promoter region in front of the firefly luciferase (“fLuc”) reporter gene. For experimental CHRNA3 promoter-5′UTR constructs, thin and thick black lines represent the promoter and 5′UTR portion of the construct, respectively. Positions of the Xho I (−1442) and Nco I (+1) sites used for subcloning are indicated, as is an internal Nco I site (−1064). The positions and identities of the rs13329271 (−1093 K; “initiator”) and rs12911814 (−847 K) polymorphisms, as well as the number of “TTTTG” repeats (−822 to −847) and the number of “GCCC” repeats (−235) within each construct are indicated. There were no statistically significant differences between comparisons of analogous ( i.e. five full length, four 5′ or two 3′ truncated) promoter haplotype constructs by ANOVA with Tukey's HSD post-hoc analysis.

Techniques Used: Activity Assay, Positive Control, Plasmid Preparation, Luciferase, Construct, Subcloning

6) Product Images from "Identification of Alternative Transcripts Encoding the Essential Murine Gammaherpesvirus Lytic Transactivator RTA"

Article Title: Identification of Alternative Transcripts Encoding the Essential Murine Gammaherpesvirus Lytic Transactivator RTA

Journal: Journal of Virology

doi: 10.1128/JVI.03110-13

Promoter deletions within the MHV68 E0 250-bp promoter region. Reporter constructs were generated within the context of the 250-bp promoter through overlapping PCR. The 50-bp E0 promoter mutants were cloned into the pGL4.10[luc] luciferase report construct.
Figure Legend Snippet: Promoter deletions within the MHV68 E0 250-bp promoter region. Reporter constructs were generated within the context of the 250-bp promoter through overlapping PCR. The 50-bp E0 promoter mutants were cloned into the pGL4.10[luc] luciferase report construct.

Techniques Used: Construct, Generated, Polymerase Chain Reaction, Clone Assay, Luciferase

Promoter activity in the region immediately 5′ to MHV68 N3, N4, and N5 exons. Vero cells were transfected with pGL4.10[luc] luciferase report constructs containing either 1,000 bp upstream of N3 or 1,000 bp upstream of the N4/N5 exon. Vero cells
Figure Legend Snippet: Promoter activity in the region immediately 5′ to MHV68 N3, N4, and N5 exons. Vero cells were transfected with pGL4.10[luc] luciferase report constructs containing either 1,000 bp upstream of N3 or 1,000 bp upstream of the N4/N5 exon. Vero cells

Techniques Used: Activity Assay, Transfection, Luciferase, Construct

Promoter activity in the region immediately 5′ to the MHV68 proximal promoter and proximal promoter deletion. Vero cells were transfected with pGL4.10[luc] luciferase reporter constructs containing either 410 bp upstream of exon 1 or 340 bp upstream
Figure Legend Snippet: Promoter activity in the region immediately 5′ to the MHV68 proximal promoter and proximal promoter deletion. Vero cells were transfected with pGL4.10[luc] luciferase reporter constructs containing either 410 bp upstream of exon 1 or 340 bp upstream

Techniques Used: Activity Assay, Transfection, Luciferase, Construct

7) Product Images from "Promoter Identification and Transcriptional Regulation of the Goose AMH Gene"

Article Title: Promoter Identification and Transcriptional Regulation of the Goose AMH Gene

Journal: Animals : an Open Access Journal from MDPI

doi: 10.3390/ani9100816

Analysis of AMH gene promoter activity in geese. Luciferase report vectors containing various lengths of the goose AMH promoter region were constructed and transfected into Chinese hamster ovary (CHO) cells. Various lengths of goose AMH region contained pGL4.10-AMH1, deletion of −2344 to −1 bp; pGL4.10-AMH2, deletion of −1896 to −1 bp; pGL4.10-AMH3, deletion of −1452 to −1 bp; pGL4.10-AMH4, deletion of −839 to −1 bp; pGL4.10-AMH5, deletion of −637 to −1 bp; pGL4.10-AMH6, deletion of −331 to −1 bp and pGL4.10-AMH7, deletion of −87 to −1 bp. The left bars indicated truncated AMH promoter sequences linked to luciferase, the bars on the right indicated luciferase activity relative to the pGL4.10-basic negative control vector. Each column represented the mean ± SD ( n = 3). Different lowercase letters represented the evident difference ( p
Figure Legend Snippet: Analysis of AMH gene promoter activity in geese. Luciferase report vectors containing various lengths of the goose AMH promoter region were constructed and transfected into Chinese hamster ovary (CHO) cells. Various lengths of goose AMH region contained pGL4.10-AMH1, deletion of −2344 to −1 bp; pGL4.10-AMH2, deletion of −1896 to −1 bp; pGL4.10-AMH3, deletion of −1452 to −1 bp; pGL4.10-AMH4, deletion of −839 to −1 bp; pGL4.10-AMH5, deletion of −637 to −1 bp; pGL4.10-AMH6, deletion of −331 to −1 bp and pGL4.10-AMH7, deletion of −87 to −1 bp. The left bars indicated truncated AMH promoter sequences linked to luciferase, the bars on the right indicated luciferase activity relative to the pGL4.10-basic negative control vector. Each column represented the mean ± SD ( n = 3). Different lowercase letters represented the evident difference ( p

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

The effect of GATA-4 binding sites on the transcription regulation of AMH gene in geese. ( A ) Overexpression of GATA-4 in the CHO cells. pEGFP-N1 represented an internal control. pEGFP-GATA-4 represented the overexpression plasmid of GATA-4 gene. ( B ) Three binding sites of GATA-4 in the promoter region of goose AMH and mutation constructions were transfected into CHO cells. pGL4.10–AMH2 represented the wild binding sites of GATA-4 in the promoter region of goose AMH . M778, M1399 and M1477 represented the mutation binding sites of GATA-4 in the promoter region of goose AMH , respectively. pEGFP-GATA-4 + pGL4.10-AMH2, pEGFP-GATA-4 + M778, pEGFP-GATA-4 + M1399 and pEGFP-GATA-4 + M1477 represented the co-expression transfection of pEGFP-GATA-4 and three mutation binding sites in the CHO cells, respectively. Each column represented the mean ± SD ( n = 3). Different lowercase letters represented the evident difference ( p
Figure Legend Snippet: The effect of GATA-4 binding sites on the transcription regulation of AMH gene in geese. ( A ) Overexpression of GATA-4 in the CHO cells. pEGFP-N1 represented an internal control. pEGFP-GATA-4 represented the overexpression plasmid of GATA-4 gene. ( B ) Three binding sites of GATA-4 in the promoter region of goose AMH and mutation constructions were transfected into CHO cells. pGL4.10–AMH2 represented the wild binding sites of GATA-4 in the promoter region of goose AMH . M778, M1399 and M1477 represented the mutation binding sites of GATA-4 in the promoter region of goose AMH , respectively. pEGFP-GATA-4 + pGL4.10-AMH2, pEGFP-GATA-4 + M778, pEGFP-GATA-4 + M1399 and pEGFP-GATA-4 + M1477 represented the co-expression transfection of pEGFP-GATA-4 and three mutation binding sites in the CHO cells, respectively. Each column represented the mean ± SD ( n = 3). Different lowercase letters represented the evident difference ( p

Techniques Used: Binding Assay, Over Expression, Plasmid Preparation, Mutagenesis, Transfection, Expressing

8) Product Images from "Pseudoexfoliation syndrome-associated genetic variants affect transcription factor binding and alternative splicing of LOXL1"

Article Title: Pseudoexfoliation syndrome-associated genetic variants affect transcription factor binding and alternative splicing of LOXL1

Journal: Nature Communications

doi: 10.1038/ncomms15466

RXRα influences LOXL1 expression by functioning as a transcriptional repressor. ( a ) ChIP assay for RXRα binding at rs11638944 (SNP12)-containing region of LOXL1 in heterozygous hTCF cell lines ( n =2) using two different antibodies against RXRα (1: D-20, 2: F-1), histone H3 (positive control) and non-immune IgG (negative control); input represents total chromatin applied for immunoprecipitation. DNA isolated from immunoprecipitated complexes was analysed on 2% agarose gel (left) and by qPCR (right) with primers specific for the SNP12 region producing a 148 bp PCR fragment (arrow). Data are expressed as per cent of input (Lane 1: hTCF 1, lane 2: hTCF 2, lane M: DNA marker, lane N: primer control without chromatin). ( b ) Dual luciferase reporter assays using reporter plasmids containing risk (12R) and non-risk (12N) genotypes of SNP 12 alone or co-transfected with RXRα-specific siRNA in human Tenon's capsule fibroblasts (hTCF) compared with the basal LOXL1 promoter activity. Results are expressed as the ratio of Firefly luciferase to Renilla luciferase; the transcriptional activity of the empty pGL4.10 vector was set at 100% (data represent mean values±s.d. of three independent experiments; * P
Figure Legend Snippet: RXRα influences LOXL1 expression by functioning as a transcriptional repressor. ( a ) ChIP assay for RXRα binding at rs11638944 (SNP12)-containing region of LOXL1 in heterozygous hTCF cell lines ( n =2) using two different antibodies against RXRα (1: D-20, 2: F-1), histone H3 (positive control) and non-immune IgG (negative control); input represents total chromatin applied for immunoprecipitation. DNA isolated from immunoprecipitated complexes was analysed on 2% agarose gel (left) and by qPCR (right) with primers specific for the SNP12 region producing a 148 bp PCR fragment (arrow). Data are expressed as per cent of input (Lane 1: hTCF 1, lane 2: hTCF 2, lane M: DNA marker, lane N: primer control without chromatin). ( b ) Dual luciferase reporter assays using reporter plasmids containing risk (12R) and non-risk (12N) genotypes of SNP 12 alone or co-transfected with RXRα-specific siRNA in human Tenon's capsule fibroblasts (hTCF) compared with the basal LOXL1 promoter activity. Results are expressed as the ratio of Firefly luciferase to Renilla luciferase; the transcriptional activity of the empty pGL4.10 vector was set at 100% (data represent mean values±s.d. of three independent experiments; * P

Techniques Used: Expressing, Chromatin Immunoprecipitation, Binding Assay, Positive Control, Negative Control, Immunoprecipitation, Isolation, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Marker, Luciferase, Transfection, Activity Assay, Plasmid Preparation

Effects of risk variants on LOXL1 promoter transcriptional activity in vitro. ( a ) Reporter constructs used in transfection experiments outlining the 14 S/R constructs comprising all 14 SNPs flanked by 51 bp of genomic DNA sequences each (top) and three deletion constructs comprising SNPs 1–5, SNPs 6–10 and SNPs 11–14, respectively (bottom). ( b ) Dual luciferase reporter assays demonstrating regulatory activity of 14 SNP risk (14sR) and non-risk (14sN) haplotypes in human Tenon‘s capsule fibroblasts (hTCF), trabecular meshwork cells (hTMC), nonpigmented ciliary epithelial cells (hNPEC), optic nerve head astrocytes (hONHA), aortic smooth muscle cells (hASMC), limbal epithelial cells (hLEPC), 3T3 fibroblasts and HEK293T cells. Results are expressed as the ratio of Firefly luciferase to Renilla luciferase; the transcriptional activity of the non-risk sequence was set at 100%. ( c ) Regulatory activity of three deletion constructs (SNPs 1–5, 6–10 and 11–14) in hTCF compared with the basal LOXL1 promoter activity; the transcriptional activity of the empty pGL4.10 vector was set at 100%. ( d , e ) Activity assays using reporter plasmids containing each of the four risk or non-risk alleles of individual SNPs 11, 12, 13 and 14 compared with the basal LOXL1 promoter activity in hTCF ( d ) and hASMC ( e ); the transcriptional activity of the empty pGL4.10 vector was set at 100% (data represent mean values±s.d. of five independent experiments; * P
Figure Legend Snippet: Effects of risk variants on LOXL1 promoter transcriptional activity in vitro. ( a ) Reporter constructs used in transfection experiments outlining the 14 S/R constructs comprising all 14 SNPs flanked by 51 bp of genomic DNA sequences each (top) and three deletion constructs comprising SNPs 1–5, SNPs 6–10 and SNPs 11–14, respectively (bottom). ( b ) Dual luciferase reporter assays demonstrating regulatory activity of 14 SNP risk (14sR) and non-risk (14sN) haplotypes in human Tenon‘s capsule fibroblasts (hTCF), trabecular meshwork cells (hTMC), nonpigmented ciliary epithelial cells (hNPEC), optic nerve head astrocytes (hONHA), aortic smooth muscle cells (hASMC), limbal epithelial cells (hLEPC), 3T3 fibroblasts and HEK293T cells. Results are expressed as the ratio of Firefly luciferase to Renilla luciferase; the transcriptional activity of the non-risk sequence was set at 100%. ( c ) Regulatory activity of three deletion constructs (SNPs 1–5, 6–10 and 11–14) in hTCF compared with the basal LOXL1 promoter activity; the transcriptional activity of the empty pGL4.10 vector was set at 100%. ( d , e ) Activity assays using reporter plasmids containing each of the four risk or non-risk alleles of individual SNPs 11, 12, 13 and 14 compared with the basal LOXL1 promoter activity in hTCF ( d ) and hASMC ( e ); the transcriptional activity of the empty pGL4.10 vector was set at 100% (data represent mean values±s.d. of five independent experiments; * P

Techniques Used: Activity Assay, In Vitro, Construct, Transfection, Luciferase, Sequencing, Plasmid Preparation

9) Product Images from "Growth arrest specific gene 2 in tilapia (Oreochromis niloticus): molecular characterization and functional analysis under low-temperature stress"

Article Title: Growth arrest specific gene 2 in tilapia (Oreochromis niloticus): molecular characterization and functional analysis under low-temperature stress

Journal: BMC Molecular Biology

doi: 10.1186/s12867-017-0095-y

Results of 5 fragments analyzed with dual-luciferase reporter system. pGL4-1–5 the activity of 1–5 in pGL4.10 vector, respectively, pGL4.10 negative control, pGL4.51 positive control. Significant differences are indicated with two asterisks at p
Figure Legend Snippet: Results of 5 fragments analyzed with dual-luciferase reporter system. pGL4-1–5 the activity of 1–5 in pGL4.10 vector, respectively, pGL4.10 negative control, pGL4.51 positive control. Significant differences are indicated with two asterisks at p

Techniques Used: Luciferase, Activity Assay, Plasmid Preparation, Negative Control, Positive Control

10) Product Images from "Novel Variant of Thyroglobulin Promoter Triggers Thyroid Autoimmunity through an Epigenetic Interferon ?-modulated Mechanism *"

Article Title: Novel Variant of Thyroglobulin Promoter Triggers Thyroid Autoimmunity through an Epigenetic Interferon ?-modulated Mechanism *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.247510

Functional analysis of IRF-1-dependent TG promoter activity. a , constructs. 2.5-kb sequence, including the rs180195 A allele at the 5′ upstream TG gene, was cloned into pGL4.10 vector to generate pGL4.10-TG(A). The rs180195 G allele as well as
Figure Legend Snippet: Functional analysis of IRF-1-dependent TG promoter activity. a , constructs. 2.5-kb sequence, including the rs180195 A allele at the 5′ upstream TG gene, was cloned into pGL4.10 vector to generate pGL4.10-TG(A). The rs180195 G allele as well as

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

a , reduced IRF-1 mRNA expression in ML-1 cells treated with IRF-1 siRNA. mRNA expression of IRF-1 was assessed by regular RT-PCR and QRT-PCR. b , knockdown of IRF-1 by siRNA reduces the TG promoter activity only in the presence of rs180195 G allele. pGL4.10-TG(A)
Figure Legend Snippet: a , reduced IRF-1 mRNA expression in ML-1 cells treated with IRF-1 siRNA. mRNA expression of IRF-1 was assessed by regular RT-PCR and QRT-PCR. b , knockdown of IRF-1 by siRNA reduces the TG promoter activity only in the presence of rs180195 G allele. pGL4.10-TG(A)

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Activity Assay

INFα stimulates expression of TG in the presence of the disease-associated variant. a , specific effect of IFNα on pGL4.10-TG(G) construct activity. Treatment of ML-1 cells with IFNα increased the TG promoter activity when G allele
Figure Legend Snippet: INFα stimulates expression of TG in the presence of the disease-associated variant. a , specific effect of IFNα on pGL4.10-TG(G) construct activity. Treatment of ML-1 cells with IFNα increased the TG promoter activity when G allele

Techniques Used: Expressing, Variant Assay, Construct, Activity Assay

11) Product Images from "Existence of Two Distinct Infectious Endogenous Retroviruses in Domestic Cats and Their Different Strategies for Adaptation to Transcriptional Regulation"

Article Title: Existence of Two Distinct Infectious Endogenous Retroviruses in Domestic Cats and Their Different Strategies for Adaptation to Transcriptional Regulation

Journal: Journal of Virology

doi: 10.1128/JVI.00716-16

ERV-DC LTR-driven transcription in the presence of CpG methylation. (A) The LTR reporter plasmids (DC7, DC10, DC16, DC19, and empty vector) were methylated in vitro by either M.SssI (Me) or heat-inactivated M.SssI (HI). The DNA was treated with BstUI, separated by gel electrophoresis, and visualized by ethidium bromide staining. M indicates the DNA marker, and Cont indicates the control pGL4.10[luc2] empty vector. (B) The promoter activity levels of methylated or unmethylated (treated with heat-inactivated M.SssI) ERV-DC LTRs were measured by a luciferase reporter assay. The LTR-Luc reporter plasmids were cotransfected with Renilla luciferase-expressing phRL-CMV into HEK293T cells, the cells were harvested at 48 h posttransfection, and luciferase activity was measured. The control was the pGL4.10[luc2] empty vector. The luciferase activity of each LTR was adjusted according to the Renilla luciferase activity level. These results include data from three independent replicates and were statistically analyzed by Student's t test (*, P
Figure Legend Snippet: ERV-DC LTR-driven transcription in the presence of CpG methylation. (A) The LTR reporter plasmids (DC7, DC10, DC16, DC19, and empty vector) were methylated in vitro by either M.SssI (Me) or heat-inactivated M.SssI (HI). The DNA was treated with BstUI, separated by gel electrophoresis, and visualized by ethidium bromide staining. M indicates the DNA marker, and Cont indicates the control pGL4.10[luc2] empty vector. (B) The promoter activity levels of methylated or unmethylated (treated with heat-inactivated M.SssI) ERV-DC LTRs were measured by a luciferase reporter assay. The LTR-Luc reporter plasmids were cotransfected with Renilla luciferase-expressing phRL-CMV into HEK293T cells, the cells were harvested at 48 h posttransfection, and luciferase activity was measured. The control was the pGL4.10[luc2] empty vector. The luciferase activity of each LTR was adjusted according to the Renilla luciferase activity level. These results include data from three independent replicates and were statistically analyzed by Student's t test (*, P

Techniques Used: CpG Methylation Assay, Plasmid Preparation, Methylation, In Vitro, Nucleic Acid Electrophoresis, Staining, Marker, Activity Assay, Luciferase, Reporter Assay, Expressing

Luciferase reporter assay for basal promoter activity of ERV-DC LTRs and identification of LTR cis elements. (A) The basal promoter activity levels of wild-type LTRs were measured by using a luciferase reporter assay. Genotype I (GI) includes DC1 (DC3), DC4, DC8, DC14, DC17, and DC19; genotype II (GII) includes DC7 and DC16; and genotype III (GIII) includes DC10 (DC18). (B) Schematic of chimeric and mutant ERV-DC LTRs that were used to investigate the presence of a cis element in ERV-DC LTRs. The name of each construct is indicated on the left, and the wild-type status or the chimeric or point mutation of the LTRs is indicated on the right. Asterisks indicate a point mutation of the nucleotide. The SmaI restriction enzyme site is located in the LTRs of ERV-DC8 and ERV-DC19 at positions 263 and 267, respectively. (C) The promoter activity levels of chimeric LTRs between the DC19 LTR and DC8 LTR (DC8/19 and DC19/8 LTRs) were measured by a luciferase reporter assay. (D) The promoter activity levels of DC8-mu6 (T280A substitution), DC8-mu7 (A483G substitution), and DC8-mu8 (G508A substitution), which are all DC8 LTRs containing a point mutation, were measured by a luciferase reporter assay. (E) The promoter activity levels of DC10 A281T (A281T substitution) and DC19 A285T (A285T substitution), which are the DC10 and DC19 LTRs with a point mutation, respectively, were measured by a luciferase reporter assay. LTR-Luc reporter plasmids were cotransfected with Renilla luciferase-expressing phRL-CMV into HEK293T cells. The cells were harvested at 48 h posttransfection, and luciferase activity was measured. The control (Cont) was a pGL4.10[luc2] empty vector. The luciferase activity of each LTR was adjusted according to the Renilla luciferase activity. The results from three replicates were collected and statistically analyzed by Student's t test and one-way analysis of variance (*, P
Figure Legend Snippet: Luciferase reporter assay for basal promoter activity of ERV-DC LTRs and identification of LTR cis elements. (A) The basal promoter activity levels of wild-type LTRs were measured by using a luciferase reporter assay. Genotype I (GI) includes DC1 (DC3), DC4, DC8, DC14, DC17, and DC19; genotype II (GII) includes DC7 and DC16; and genotype III (GIII) includes DC10 (DC18). (B) Schematic of chimeric and mutant ERV-DC LTRs that were used to investigate the presence of a cis element in ERV-DC LTRs. The name of each construct is indicated on the left, and the wild-type status or the chimeric or point mutation of the LTRs is indicated on the right. Asterisks indicate a point mutation of the nucleotide. The SmaI restriction enzyme site is located in the LTRs of ERV-DC8 and ERV-DC19 at positions 263 and 267, respectively. (C) The promoter activity levels of chimeric LTRs between the DC19 LTR and DC8 LTR (DC8/19 and DC19/8 LTRs) were measured by a luciferase reporter assay. (D) The promoter activity levels of DC8-mu6 (T280A substitution), DC8-mu7 (A483G substitution), and DC8-mu8 (G508A substitution), which are all DC8 LTRs containing a point mutation, were measured by a luciferase reporter assay. (E) The promoter activity levels of DC10 A281T (A281T substitution) and DC19 A285T (A285T substitution), which are the DC10 and DC19 LTRs with a point mutation, respectively, were measured by a luciferase reporter assay. LTR-Luc reporter plasmids were cotransfected with Renilla luciferase-expressing phRL-CMV into HEK293T cells. The cells were harvested at 48 h posttransfection, and luciferase activity was measured. The control (Cont) was a pGL4.10[luc2] empty vector. The luciferase activity of each LTR was adjusted according to the Renilla luciferase activity. The results from three replicates were collected and statistically analyzed by Student's t test and one-way analysis of variance (*, P

Techniques Used: Luciferase, Reporter Assay, Activity Assay, Mutagenesis, Construct, Expressing, Plasmid Preparation

12) Product Images from "Novel Variant of Thyroglobulin Promoter Triggers Thyroid Autoimmunity through an Epigenetic Interferon ?-modulated Mechanism *"

Article Title: Novel Variant of Thyroglobulin Promoter Triggers Thyroid Autoimmunity through an Epigenetic Interferon ?-modulated Mechanism *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.247510

Functional analysis of IRF-1-dependent TG promoter activity. a , constructs. 2.5-kb sequence, including the rs180195 A allele at the 5′ upstream TG gene, was cloned into pGL4.10 vector to generate pGL4.10-TG(A). The rs180195 G allele as well as
Figure Legend Snippet: Functional analysis of IRF-1-dependent TG promoter activity. a , constructs. 2.5-kb sequence, including the rs180195 A allele at the 5′ upstream TG gene, was cloned into pGL4.10 vector to generate pGL4.10-TG(A). The rs180195 G allele as well as

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

a , reduced IRF-1 mRNA expression in ML-1 cells treated with IRF-1 siRNA. mRNA expression of IRF-1 was assessed by regular RT-PCR and QRT-PCR. b , knockdown of IRF-1 by siRNA reduces the TG promoter activity only in the presence of rs180195 G allele. pGL4.10-TG(A)
Figure Legend Snippet: a , reduced IRF-1 mRNA expression in ML-1 cells treated with IRF-1 siRNA. mRNA expression of IRF-1 was assessed by regular RT-PCR and QRT-PCR. b , knockdown of IRF-1 by siRNA reduces the TG promoter activity only in the presence of rs180195 G allele. pGL4.10-TG(A)

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Activity Assay

INFα stimulates expression of TG in the presence of the disease-associated variant. a , specific effect of IFNα on pGL4.10-TG(G) construct activity. Treatment of ML-1 cells with IFNα increased the TG promoter activity when G allele
Figure Legend Snippet: INFα stimulates expression of TG in the presence of the disease-associated variant. a , specific effect of IFNα on pGL4.10-TG(G) construct activity. Treatment of ML-1 cells with IFNα increased the TG promoter activity when G allele

Techniques Used: Expressing, Variant Assay, Construct, Activity Assay

13) Product Images from "Insulin Gene Expression Is Regulated by DNA Methylation"

Article Title: Insulin Gene Expression Is Regulated by DNA Methylation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0006953

Methylation suppresses the activity of both the mouse and human insulin gene promoters. (A) A diagram of the three luciferase reporter plasmids used to test the effect of methylation on insulin promoter activity. PGL4.10 Ins2 500bp contains bases −480 to +15 (three CpG sites) of the Ins2 gene, pGL4.10 Ins2 MP contains bases −169– +15 bp (no CpG sites), and, pGL4.10 INS 421bp contains bases −385 to +24 of the human INS gene (nine CpG sites). The positions of CpG dinucleotides are represented with “lollipop” markers and the positions of primers for the ChIP experiments by arrows. (B) Methylated (white bar) or mock-methylated (black bar) human INS promoter constructs were transfected into NIT-1 insulinoma cells 48 hours prior to the luciferase assay. The data were normalized with co-transfected control vector and are the average and standard error from three separate experiments of eight replicates each. Data are presented as relative expression compared with the non-methylated, non-stimulated sample. * = p
Figure Legend Snippet: Methylation suppresses the activity of both the mouse and human insulin gene promoters. (A) A diagram of the three luciferase reporter plasmids used to test the effect of methylation on insulin promoter activity. PGL4.10 Ins2 500bp contains bases −480 to +15 (three CpG sites) of the Ins2 gene, pGL4.10 Ins2 MP contains bases −169– +15 bp (no CpG sites), and, pGL4.10 INS 421bp contains bases −385 to +24 of the human INS gene (nine CpG sites). The positions of CpG dinucleotides are represented with “lollipop” markers and the positions of primers for the ChIP experiments by arrows. (B) Methylated (white bar) or mock-methylated (black bar) human INS promoter constructs were transfected into NIT-1 insulinoma cells 48 hours prior to the luciferase assay. The data were normalized with co-transfected control vector and are the average and standard error from three separate experiments of eight replicates each. Data are presented as relative expression compared with the non-methylated, non-stimulated sample. * = p

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

Effect of methylation on transcription factor binding to the Ins2 promoter. (A) Increasing amounts of NIT-1 nuclear extract was bound to methylated or unmethylated mouse Ins2 promoter CRE oligonucleotide in an Electrophoretic Mobility Shift Assay (EMSA). There was a dose-dependent increase in binding to both the methylated and unmethylated CRE with increasing amounts of NIT-1 cell nuclear extract. (B) The binding of NIT-1 nuclear proteins to one nanomolar of either labeled methylated (mCRE) or unmethylated (CRE) was competed with increasing concentrations of unlabeled CRE or mCRE. The figure is one representative of four experiments. (C) Chromatin immunoprecipitation (ChIP) analysis of the endogenous mouse Ins2 gene promoter in NIT-1 cells. Genomic DNA was immunoprecipitated with anti-CREB, anti-ATF2, anti-MECP2, and control IgG. The results indicate that CREB and ATF2, but not MeCP2, are normally associated with the endogenous insulin promoter. (D) Methylated or mock-methylated pGL4.10 Ins2 500bp ( Fig. 2A ) was transfected into NIT-1 cells and then ChIP was carried out with anti-CREB, anti-ATF2, and anti-MeCP2 antibodies. Methylation of the Ins2 promoter decreases the binding of CREB and ATF2, but increases the binding of MeCP2.
Figure Legend Snippet: Effect of methylation on transcription factor binding to the Ins2 promoter. (A) Increasing amounts of NIT-1 nuclear extract was bound to methylated or unmethylated mouse Ins2 promoter CRE oligonucleotide in an Electrophoretic Mobility Shift Assay (EMSA). There was a dose-dependent increase in binding to both the methylated and unmethylated CRE with increasing amounts of NIT-1 cell nuclear extract. (B) The binding of NIT-1 nuclear proteins to one nanomolar of either labeled methylated (mCRE) or unmethylated (CRE) was competed with increasing concentrations of unlabeled CRE or mCRE. The figure is one representative of four experiments. (C) Chromatin immunoprecipitation (ChIP) analysis of the endogenous mouse Ins2 gene promoter in NIT-1 cells. Genomic DNA was immunoprecipitated with anti-CREB, anti-ATF2, anti-MECP2, and control IgG. The results indicate that CREB and ATF2, but not MeCP2, are normally associated with the endogenous insulin promoter. (D) Methylated or mock-methylated pGL4.10 Ins2 500bp ( Fig. 2A ) was transfected into NIT-1 cells and then ChIP was carried out with anti-CREB, anti-ATF2, and anti-MeCP2 antibodies. Methylation of the Ins2 promoter decreases the binding of CREB and ATF2, but increases the binding of MeCP2.

Techniques Used: Methylation, Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Chromatin Immunoprecipitation, Immunoprecipitation, Transfection

14) Product Images from "Insulin Gene Expression Is Regulated by DNA Methylation"

Article Title: Insulin Gene Expression Is Regulated by DNA Methylation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0006953

Methylation suppresses the activity of both the mouse and human insulin gene promoters. (A) A diagram of the three luciferase reporter plasmids used to test the effect of methylation on insulin promoter activity. PGL4.10 Ins2 500bp contains bases −480 to +15 (three CpG sites) of the Ins2 gene, pGL4.10 Ins2 MP contains bases −169– +15 bp (no CpG sites), and, pGL4.10 INS 421bp contains bases −385 to +24 of the human INS gene (nine CpG sites). The positions of CpG dinucleotides are represented with “lollipop” markers and the positions of primers for the ChIP experiments by arrows. (B) Methylated (white bar) or mock-methylated (black bar) human INS promoter constructs were transfected into NIT-1 insulinoma cells 48 hours prior to the luciferase assay. The data were normalized with co-transfected control vector and are the average and standard error from three separate experiments of eight replicates each. Data are presented as relative expression compared with the non-methylated, non-stimulated sample. * = p
Figure Legend Snippet: Methylation suppresses the activity of both the mouse and human insulin gene promoters. (A) A diagram of the three luciferase reporter plasmids used to test the effect of methylation on insulin promoter activity. PGL4.10 Ins2 500bp contains bases −480 to +15 (three CpG sites) of the Ins2 gene, pGL4.10 Ins2 MP contains bases −169– +15 bp (no CpG sites), and, pGL4.10 INS 421bp contains bases −385 to +24 of the human INS gene (nine CpG sites). The positions of CpG dinucleotides are represented with “lollipop” markers and the positions of primers for the ChIP experiments by arrows. (B) Methylated (white bar) or mock-methylated (black bar) human INS promoter constructs were transfected into NIT-1 insulinoma cells 48 hours prior to the luciferase assay. The data were normalized with co-transfected control vector and are the average and standard error from three separate experiments of eight replicates each. Data are presented as relative expression compared with the non-methylated, non-stimulated sample. * = p

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

Effect of methylation on transcription factor binding to the Ins2 promoter. (A) Increasing amounts of NIT-1 nuclear extract was bound to methylated or unmethylated mouse Ins2 promoter CRE oligonucleotide in an Electrophoretic Mobility Shift Assay (EMSA). There was a dose-dependent increase in binding to both the methylated and unmethylated CRE with increasing amounts of NIT-1 cell nuclear extract. (B) The binding of NIT-1 nuclear proteins to one nanomolar of either labeled methylated (mCRE) or unmethylated (CRE) was competed with increasing concentrations of unlabeled CRE or mCRE. The figure is one representative of four experiments. (C) Chromatin immunoprecipitation (ChIP) analysis of the endogenous mouse Ins2 gene promoter in NIT-1 cells. Genomic DNA was immunoprecipitated with anti-CREB, anti-ATF2, anti-MECP2, and control IgG. The results indicate that CREB and ATF2, but not MeCP2, are normally associated with the endogenous insulin promoter. (D) Methylated or mock-methylated pGL4.10 Ins2 500bp ( Fig. 2A ) was transfected into NIT-1 cells and then ChIP was carried out with anti-CREB, anti-ATF2, and anti-MeCP2 antibodies. Methylation of the Ins2 promoter decreases the binding of CREB and ATF2, but increases the binding of MeCP2.
Figure Legend Snippet: Effect of methylation on transcription factor binding to the Ins2 promoter. (A) Increasing amounts of NIT-1 nuclear extract was bound to methylated or unmethylated mouse Ins2 promoter CRE oligonucleotide in an Electrophoretic Mobility Shift Assay (EMSA). There was a dose-dependent increase in binding to both the methylated and unmethylated CRE with increasing amounts of NIT-1 cell nuclear extract. (B) The binding of NIT-1 nuclear proteins to one nanomolar of either labeled methylated (mCRE) or unmethylated (CRE) was competed with increasing concentrations of unlabeled CRE or mCRE. The figure is one representative of four experiments. (C) Chromatin immunoprecipitation (ChIP) analysis of the endogenous mouse Ins2 gene promoter in NIT-1 cells. Genomic DNA was immunoprecipitated with anti-CREB, anti-ATF2, anti-MECP2, and control IgG. The results indicate that CREB and ATF2, but not MeCP2, are normally associated with the endogenous insulin promoter. (D) Methylated or mock-methylated pGL4.10 Ins2 500bp ( Fig. 2A ) was transfected into NIT-1 cells and then ChIP was carried out with anti-CREB, anti-ATF2, and anti-MeCP2 antibodies. Methylation of the Ins2 promoter decreases the binding of CREB and ATF2, but increases the binding of MeCP2.

Techniques Used: Methylation, Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Chromatin Immunoprecipitation, Immunoprecipitation, Transfection

15) Product Images from "Silencing of CHD5 Gene by Promoter Methylation in Leukemia"

Article Title: Silencing of CHD5 Gene by Promoter Methylation in Leukemia

Journal: PLoS ONE

doi: 10.1371/journal.pone.0085172

Identification of the AP2 binding site in the CHD5 promoter regulatory element. ChIP-qPCR assay indicated that AP2 binds to the identified regulatory element of the CHD5 promoter (A, B). Normal IgG was used as a negative control. The pGL4.10-CHD5-2000 to -370 and pGL4.10-CHD5-356 vectors, in which the AP2 binding site was deleted, were measured for luciferase activity in K-562 cells (C). The pGL4.1-CHD5-P-CR vector, harboring the –560 to –240 region, was measured for luciferase activity in K-562 cells (D). ChIP-qPCR analysis showed that DAC treatment enhanced AP2 binding to the CHD5 promoter. All data are presented as mean ± SD. P
Figure Legend Snippet: Identification of the AP2 binding site in the CHD5 promoter regulatory element. ChIP-qPCR assay indicated that AP2 binds to the identified regulatory element of the CHD5 promoter (A, B). Normal IgG was used as a negative control. The pGL4.10-CHD5-2000 to -370 and pGL4.10-CHD5-356 vectors, in which the AP2 binding site was deleted, were measured for luciferase activity in K-562 cells (C). The pGL4.1-CHD5-P-CR vector, harboring the –560 to –240 region, was measured for luciferase activity in K-562 cells (D). ChIP-qPCR analysis showed that DAC treatment enhanced AP2 binding to the CHD5 promoter. All data are presented as mean ± SD. P

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Luciferase, Activity Assay, Plasmid Preparation

Mapping regulatory elements within the CHD5 promoter. The reporter constructs containing the full length (–2000 to –1) CHD5 promoter or partial promoter sequences progressively truncated by 200 bp (A) or 100 bp (B) at the 5' or 3' end were measured for luciferase activity in K-562 cells. The promoterless pGL4.10-Basic vector was used as a negative control. The pGL4.10-CMV vector, which uses the CMV promoter, was used as a positive reporter construct. The relative Luc activity of full length and truncated CHD5 promoters were normalized to Renilla luciferase activity and compared to normalized Luc luciferase activity from pGL4.10-Basic. All data are presented as mean ± SD. P
Figure Legend Snippet: Mapping regulatory elements within the CHD5 promoter. The reporter constructs containing the full length (–2000 to –1) CHD5 promoter or partial promoter sequences progressively truncated by 200 bp (A) or 100 bp (B) at the 5' or 3' end were measured for luciferase activity in K-562 cells. The promoterless pGL4.10-Basic vector was used as a negative control. The pGL4.10-CMV vector, which uses the CMV promoter, was used as a positive reporter construct. The relative Luc activity of full length and truncated CHD5 promoters were normalized to Renilla luciferase activity and compared to normalized Luc luciferase activity from pGL4.10-Basic. All data are presented as mean ± SD. P

Techniques Used: Construct, Luciferase, Activity Assay, Plasmid Preparation, Negative Control

16) Product Images from "Silencing of CHD5 Gene by Promoter Methylation in Leukemia"

Article Title: Silencing of CHD5 Gene by Promoter Methylation in Leukemia

Journal: PLoS ONE

doi: 10.1371/journal.pone.0085172

Identification of the AP2 binding site in the CHD5 promoter regulatory element. ChIP-qPCR assay indicated that AP2 binds to the identified regulatory element of the CHD5 promoter (A, B). Normal IgG was used as a negative control. The pGL4.10-CHD5-2000 to -370 and pGL4.10-CHD5-356 vectors, in which the AP2 binding site was deleted, were measured for luciferase activity in K-562 cells (C). The pGL4.1-CHD5-P-CR vector, harboring the –560 to –240 region, was measured for luciferase activity in K-562 cells (D). ChIP-qPCR analysis showed that DAC treatment enhanced AP2 binding to the CHD5 promoter. All data are presented as mean ± SD. P
Figure Legend Snippet: Identification of the AP2 binding site in the CHD5 promoter regulatory element. ChIP-qPCR assay indicated that AP2 binds to the identified regulatory element of the CHD5 promoter (A, B). Normal IgG was used as a negative control. The pGL4.10-CHD5-2000 to -370 and pGL4.10-CHD5-356 vectors, in which the AP2 binding site was deleted, were measured for luciferase activity in K-562 cells (C). The pGL4.1-CHD5-P-CR vector, harboring the –560 to –240 region, was measured for luciferase activity in K-562 cells (D). ChIP-qPCR analysis showed that DAC treatment enhanced AP2 binding to the CHD5 promoter. All data are presented as mean ± SD. P

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Luciferase, Activity Assay, Plasmid Preparation

Mapping regulatory elements within the CHD5 promoter. The reporter constructs containing the full length (–2000 to –1) CHD5 promoter or partial promoter sequences progressively truncated by 200 bp (A) or 100 bp (B) at the 5' or 3' end were measured for luciferase activity in K-562 cells. The promoterless pGL4.10-Basic vector was used as a negative control. The pGL4.10-CMV vector, which uses the CMV promoter, was used as a positive reporter construct. The relative Luc activity of full length and truncated CHD5 promoters were normalized to Renilla luciferase activity and compared to normalized Luc luciferase activity from pGL4.10-Basic. All data are presented as mean ± SD. P
Figure Legend Snippet: Mapping regulatory elements within the CHD5 promoter. The reporter constructs containing the full length (–2000 to –1) CHD5 promoter or partial promoter sequences progressively truncated by 200 bp (A) or 100 bp (B) at the 5' or 3' end were measured for luciferase activity in K-562 cells. The promoterless pGL4.10-Basic vector was used as a negative control. The pGL4.10-CMV vector, which uses the CMV promoter, was used as a positive reporter construct. The relative Luc activity of full length and truncated CHD5 promoters were normalized to Renilla luciferase activity and compared to normalized Luc luciferase activity from pGL4.10-Basic. All data are presented as mean ± SD. P

Techniques Used: Construct, Luciferase, Activity Assay, Plasmid Preparation, Negative Control

17) Product Images from "Nicotinic Acetylcholine Receptor Subunits ?4 and ?5 Associated with Smoking Behaviour and Lung Cancer Are Regulated by Upstream Open Reading Frames"

Article Title: Nicotinic Acetylcholine Receptor Subunits ?4 and ?5 Associated with Smoking Behaviour and Lung Cancer Are Regulated by Upstream Open Reading Frames

Journal: PLoS ONE

doi: 10.1371/journal.pone.0066157

CHRNA5 5 ′ UTR harbours a functional uORF; CHRNB3 uORFs are not involved in translational control. A: Luciferase assay of CHRNA5 5′UTR resulted in significant increase in protein expression when switching off the uORF. Fold change of firefly activity normalized to pGl4.10 +TK, is illustrated for the five different constructs; 1, pGl4.10+TK; 2, CHRNA5 -1; 3, CHRNA5 -2; 4, CHRNA5 -3; 5, CHRNA5 -4. B: The uORF start codon of CHRNA5 did not initiate translation as efficiently as the firefly start codon. Fold change of firefly activity normalized to pGl4.10 +TK, is illustrated for the five different constructs; 1, pGl4.10+TK; 2, pGL4.10; 3, CHRNA5 -2; 4, CHRNA5 -5; 5, CHRNA5 -6. C: Relative qPCR for CHRNA5 5′UTR showed no significant differences of mRNA amount when comparing intact with deleted uORF. However, significant differences were assessed for the transcription of the two SNP allele variants. Fold change of firefly mRNA normalized to pGl4.10 +TK, is illustrated for the five different constructs; 1, pGl4.10+TK; 2, CHRNA5 -1; 3, CHRNA5 -2; 4, CHRNA5 -3; 5, CHRNA5 -4. D: Luciferase assay of CHRNB3 5′UTR showed no significant results. Fold change of firefly activity normalized to pGl4.10 +TK, is illustrated for the four different constructs; 1, pGl4.10+TK; 2, CHRNB3 -1; 3, CHRNB3 -2; 4, CHRNB3 -3. Error bars represent ± SEM of 3 biological replicates. Asterisks indicate significant differences ( p
Figure Legend Snippet: CHRNA5 5 ′ UTR harbours a functional uORF; CHRNB3 uORFs are not involved in translational control. A: Luciferase assay of CHRNA5 5′UTR resulted in significant increase in protein expression when switching off the uORF. Fold change of firefly activity normalized to pGl4.10 +TK, is illustrated for the five different constructs; 1, pGl4.10+TK; 2, CHRNA5 -1; 3, CHRNA5 -2; 4, CHRNA5 -3; 5, CHRNA5 -4. B: The uORF start codon of CHRNA5 did not initiate translation as efficiently as the firefly start codon. Fold change of firefly activity normalized to pGl4.10 +TK, is illustrated for the five different constructs; 1, pGl4.10+TK; 2, pGL4.10; 3, CHRNA5 -2; 4, CHRNA5 -5; 5, CHRNA5 -6. C: Relative qPCR for CHRNA5 5′UTR showed no significant differences of mRNA amount when comparing intact with deleted uORF. However, significant differences were assessed for the transcription of the two SNP allele variants. Fold change of firefly mRNA normalized to pGl4.10 +TK, is illustrated for the five different constructs; 1, pGl4.10+TK; 2, CHRNA5 -1; 3, CHRNA5 -2; 4, CHRNA5 -3; 5, CHRNA5 -4. D: Luciferase assay of CHRNB3 5′UTR showed no significant results. Fold change of firefly activity normalized to pGl4.10 +TK, is illustrated for the four different constructs; 1, pGl4.10+TK; 2, CHRNB3 -1; 3, CHRNB3 -2; 4, CHRNB3 -3. Error bars represent ± SEM of 3 biological replicates. Asterisks indicate significant differences ( p

Techniques Used: Functional Assay, Luciferase, Expressing, Activity Assay, Construct, Real-time Polymerase Chain Reaction

CHRNA4 isoform 1 contains a post-transcriptionally functional uORF, but not CHRNA4 isoform 2. A: Luciferase assay of CHRNA4 isoform 1 5′UTR resulted in significant increase in protein expression when switching off the uORF. Fold change of firefly activity normalized to pGl4.10 +TK, is illustrated for the three different constructs; 1, pGl4.10+TK; 2, CHRNA4 -iso1-1; 3, CHRNA4 -iso1-2. B: The uORF ATG of CHRNA4 -iso1 is able to initiate translation, resulting in an elongated firefly protein. Fold change of firefly activity normalized to pGl4.10 +TK, is illustrated for the five different constructs; 1, pGl4.10+TK; 2, pGl4.10; 3, CHRNA4 -iso1-2; 4, CHRNA4 -iso1-3; 5, CHRNA4 -iso1-4. C: Relative qPCR for CHRNA4-iso1 5′UTR showed no significant differences of mRNA amount when comparing intact with deleted uORF. Fold change of firefly mRNA normalized to pGl4.10 +TK, is illustrated for the three different constructs; 1, pGl4.10+TK; 2, CHRNA4 -iso1-1; 3, CHRNA4 -iso1-2. D: None of the five uORFs of CHRNA4 -iso2 5′UTR showed significant results. Fold change of firefly activity normalized to pGl4.10 +TK, is illustrated for the seven different constructs; 1, pGl4.10+TK; 2, CHRNA4 -iso2-uORF1-5; 3, CHRNA4 -iso2-uORF1; 4, CHRNA4 -iso2-uORF2; 5, CHRNA4 -iso2-uORF3; 6, CHRNA4 -iso2-uORF4; 7, CHRNA4 -iso2-uORF5. Error bars represent ± SEM of 3 biological replicates. Asterisks indicate significant differences ( p
Figure Legend Snippet: CHRNA4 isoform 1 contains a post-transcriptionally functional uORF, but not CHRNA4 isoform 2. A: Luciferase assay of CHRNA4 isoform 1 5′UTR resulted in significant increase in protein expression when switching off the uORF. Fold change of firefly activity normalized to pGl4.10 +TK, is illustrated for the three different constructs; 1, pGl4.10+TK; 2, CHRNA4 -iso1-1; 3, CHRNA4 -iso1-2. B: The uORF ATG of CHRNA4 -iso1 is able to initiate translation, resulting in an elongated firefly protein. Fold change of firefly activity normalized to pGl4.10 +TK, is illustrated for the five different constructs; 1, pGl4.10+TK; 2, pGl4.10; 3, CHRNA4 -iso1-2; 4, CHRNA4 -iso1-3; 5, CHRNA4 -iso1-4. C: Relative qPCR for CHRNA4-iso1 5′UTR showed no significant differences of mRNA amount when comparing intact with deleted uORF. Fold change of firefly mRNA normalized to pGl4.10 +TK, is illustrated for the three different constructs; 1, pGl4.10+TK; 2, CHRNA4 -iso1-1; 3, CHRNA4 -iso1-2. D: None of the five uORFs of CHRNA4 -iso2 5′UTR showed significant results. Fold change of firefly activity normalized to pGl4.10 +TK, is illustrated for the seven different constructs; 1, pGl4.10+TK; 2, CHRNA4 -iso2-uORF1-5; 3, CHRNA4 -iso2-uORF1; 4, CHRNA4 -iso2-uORF2; 5, CHRNA4 -iso2-uORF3; 6, CHRNA4 -iso2-uORF4; 7, CHRNA4 -iso2-uORF5. Error bars represent ± SEM of 3 biological replicates. Asterisks indicate significant differences ( p

Techniques Used: Functional Assay, Luciferase, Expressing, Activity Assay, Construct, Real-time Polymerase Chain Reaction

18) Product Images from "Integrative analysis of genomic alterations in triple-negative breast cancer in association with homologous recombination deficiency"

Article Title: Integrative analysis of genomic alterations in triple-negative breast cancer in association with homologous recombination deficiency

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1006853

Enhanced expression of TGFA is associated with structural variations (SVs) in the putative regulatory region. ( A ) mRNA expression of TGFA in ER+ breast cancer (BC) (green), HER2+ BC (yellow), and triple-negative BC (TNBC) (red). IDs for samples with SVs within or near the TGFA locus are shown. ( B ) SV break points and copy number (CN) status of samples indicated in ( A ). ( C ) TGFA mRNA expression based on The Cancer Genome Atlas (TCGA) data, depending on whether the SV is within or near the TGFA locus (left) or according to the CN status (right). ( D ) CN status of BCs, based on TCGA data, with presumed SVs within or near the TGFA locus. IDs for TNBC cases are shown in red. ( E ) Acetylation of the lysine residue at position 27 of histone H3 (H3K27ac) detected by ChIP-seq along with the CN status obtained from Cancer Cell Line Encyclopedia (CCLE) data. Data for human mammary epithelial cells (HMECs) were obtained from the Encyclopedia of DNA Elements (ENCODE) project data. ( F ) TGFA mRNA expression in BICR6 cell lines in which H3K27ac-enriched putative enhancers (e1–e7) were deleted using the CRISPR-Cas9 system. P -values were derived from t-tests. ( G ) Luciferase reporter assays measuring the enhancer activity of e6 and e7 in BICR6 cells. The pGL4.10 plasmid without the enhancer region (empty) was used as a negative control. Relative luciferase units were normalized to Renilla luciferase signals. The normalized value for empty vector was set to 1. P -values were derived from t-tests. ( H ) MCF10A cells expressing enhanced green fluorescent protein (EGFP) alone or TGFA together with EGFP, cultured in complete medium or medium without epidermal growth factor.
Figure Legend Snippet: Enhanced expression of TGFA is associated with structural variations (SVs) in the putative regulatory region. ( A ) mRNA expression of TGFA in ER+ breast cancer (BC) (green), HER2+ BC (yellow), and triple-negative BC (TNBC) (red). IDs for samples with SVs within or near the TGFA locus are shown. ( B ) SV break points and copy number (CN) status of samples indicated in ( A ). ( C ) TGFA mRNA expression based on The Cancer Genome Atlas (TCGA) data, depending on whether the SV is within or near the TGFA locus (left) or according to the CN status (right). ( D ) CN status of BCs, based on TCGA data, with presumed SVs within or near the TGFA locus. IDs for TNBC cases are shown in red. ( E ) Acetylation of the lysine residue at position 27 of histone H3 (H3K27ac) detected by ChIP-seq along with the CN status obtained from Cancer Cell Line Encyclopedia (CCLE) data. Data for human mammary epithelial cells (HMECs) were obtained from the Encyclopedia of DNA Elements (ENCODE) project data. ( F ) TGFA mRNA expression in BICR6 cell lines in which H3K27ac-enriched putative enhancers (e1–e7) were deleted using the CRISPR-Cas9 system. P -values were derived from t-tests. ( G ) Luciferase reporter assays measuring the enhancer activity of e6 and e7 in BICR6 cells. The pGL4.10 plasmid without the enhancer region (empty) was used as a negative control. Relative luciferase units were normalized to Renilla luciferase signals. The normalized value for empty vector was set to 1. P -values were derived from t-tests. ( H ) MCF10A cells expressing enhanced green fluorescent protein (EGFP) alone or TGFA together with EGFP, cultured in complete medium or medium without epidermal growth factor.

Techniques Used: Expressing, Chromatin Immunoprecipitation, CRISPR, Derivative Assay, Luciferase, Activity Assay, Plasmid Preparation, Negative Control, Cell Culture

19) Product Images from "Retinoic acid-related orphan receptor ? directly regulates neuronal PAS domain protein 2 transcription in vivo"

Article Title: Retinoic acid-related orphan receptor ? directly regulates neuronal PAS domain protein 2 transcription in vivo

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq1335

Rev-Erbα and the ROR-inverse agonist T0901317 inhibited ROR-induced activation of the Npas2 (–1534/+81) promoter. ( A ) Rev-Erbα expression represses the activation of the Npas2 promoter by RORα and RORγ, Huh-7 cells were transfected with p3xFlag-CMV10-RORγ or p3xFlag-CMV10-RORα, pGL4.10- Npas2 (–1534/+81) and increasing concentrations of p3xFlag-CMV10-Rev-Erbα and 24 h later were assayed for reporter activity. ( B ) The downregulation of basal Npas2 promoter activity by Rev-Erbα was abrogated by mutations in the ROREs. Huh-7 cells were transfected with p3xFlag-CMV10-Rev-Erbα and pGL4.10 driven by the WT Npas2 (–1534/+81) promoter or the promoter with the indicated RORE mutations. About 24 h later cells were assayed for reporter activity. ( C ) The inverse agonist, T0901317, represses the activation of the Npas2 promoter by both RORγ and RORα in Huh-7 cells. Data present mean ± SEM.
Figure Legend Snippet: Rev-Erbα and the ROR-inverse agonist T0901317 inhibited ROR-induced activation of the Npas2 (–1534/+81) promoter. ( A ) Rev-Erbα expression represses the activation of the Npas2 promoter by RORα and RORγ, Huh-7 cells were transfected with p3xFlag-CMV10-RORγ or p3xFlag-CMV10-RORα, pGL4.10- Npas2 (–1534/+81) and increasing concentrations of p3xFlag-CMV10-Rev-Erbα and 24 h later were assayed for reporter activity. ( B ) The downregulation of basal Npas2 promoter activity by Rev-Erbα was abrogated by mutations in the ROREs. Huh-7 cells were transfected with p3xFlag-CMV10-Rev-Erbα and pGL4.10 driven by the WT Npas2 (–1534/+81) promoter or the promoter with the indicated RORE mutations. About 24 h later cells were assayed for reporter activity. ( C ) The inverse agonist, T0901317, represses the activation of the Npas2 promoter by both RORγ and RORα in Huh-7 cells. Data present mean ± SEM.

Techniques Used: Activation Assay, Expressing, Transfection, Activity Assay

Regulation of Npas2 expression by ROR is mediated through ROREs. ( A ) Schematic of the location of the RORE1 and RORE2 in the proximal Npas2 promoter region. The bold and underlined sequences of these ROREs are conserved between the human and mouse genomes. The numbers refer to the distance to the transcription start site. ( B and C ) RORs were able to effectively activate the (–1534/+81) Npas2 proximal promoter in Huh-7 (B) and HEK293T cells (C). Cells were co-transfected with the indicated p3xFlag–CMV10–ROR expression vector, pCMV-β-Gal and a pGL4.10 reporter plasmid driven by either the WT Npas2 (–1534/+81) promoter or the promoter in which RORE1, RORE2 or both were mutated (RORE1m, RORE2m and RORE1m2m, respectively). About 24 h later the relative luciferase reporter activities were determined as described in ‘Materials and Methods’ section. Data present mean ± SEM.
Figure Legend Snippet: Regulation of Npas2 expression by ROR is mediated through ROREs. ( A ) Schematic of the location of the RORE1 and RORE2 in the proximal Npas2 promoter region. The bold and underlined sequences of these ROREs are conserved between the human and mouse genomes. The numbers refer to the distance to the transcription start site. ( B and C ) RORs were able to effectively activate the (–1534/+81) Npas2 proximal promoter in Huh-7 (B) and HEK293T cells (C). Cells were co-transfected with the indicated p3xFlag–CMV10–ROR expression vector, pCMV-β-Gal and a pGL4.10 reporter plasmid driven by either the WT Npas2 (–1534/+81) promoter or the promoter in which RORE1, RORE2 or both were mutated (RORE1m, RORE2m and RORE1m2m, respectively). About 24 h later the relative luciferase reporter activities were determined as described in ‘Materials and Methods’ section. Data present mean ± SEM.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Luciferase

20) Product Images from "Identification of two regulatory binding sites which confer myotube specific expression of the mono-ADP-ribosyltransferase ART1 gene"

Article Title: Identification of two regulatory binding sites which confer myotube specific expression of the mono-ADP-ribosyltransferase ART1 gene

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-9-91

An A/T-rich motif and an E box mediate the induction of the ART1 proximal promoter activity during myogenesis . Candidate motifs for mediating muscle-specific expression of the mouse proximal ART1 promoter were altered by site-directed mutagenesis of the pART1-S (-322/+37) containing pGL4.10 plasmid. Cells at different stages of differentiation were transfected with the plasmids. Promoter activities were determined using the dual luciferase reporter assays normalized to renilla luciferase activity. Following symbols are used: black box – A/T-rich element (A/T); black oval – E box (E); white box – TATA box (TB) and 'luc' boxed – firefly luciferase gene. An arrow indicates the transcription start site numbered with +1. Mutations of symbolised A/T-rich element (A/T) and E box (E) are indicated by an X overlay. The data represent means ± S.D. of three independent experiments in triplicate.
Figure Legend Snippet: An A/T-rich motif and an E box mediate the induction of the ART1 proximal promoter activity during myogenesis . Candidate motifs for mediating muscle-specific expression of the mouse proximal ART1 promoter were altered by site-directed mutagenesis of the pART1-S (-322/+37) containing pGL4.10 plasmid. Cells at different stages of differentiation were transfected with the plasmids. Promoter activities were determined using the dual luciferase reporter assays normalized to renilla luciferase activity. Following symbols are used: black box – A/T-rich element (A/T); black oval – E box (E); white box – TATA box (TB) and 'luc' boxed – firefly luciferase gene. An arrow indicates the transcription start site numbered with +1. Mutations of symbolised A/T-rich element (A/T) and E box (E) are indicated by an X overlay. The data represent means ± S.D. of three independent experiments in triplicate.

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

The activity of a short and a long ART1 promoter fragment is upregulated during myotube formation . (A) Shown is the exon-intron structure of the mouse ART1 gene as described previously [ 58 ]. Exons 1–5 are represented by boxes, introns by thin lines. Coding regions are indicated in black, non-coding regions in white. (B) A long (-1236/+36) and a short (-322/+37) fragment of the putative mouse ART1 promoter region (pART1-L: grey bars and pART1-S: black bars, respectively) was cloned into the pGL4.10 firefly luciferase reporter plasmid. C3H-10T 1/2 (Pg, MyoD-Mb, Mt) and C2C12 (Mb, Mt) cells were transfected with the plasmids and promoter activities were determined using the dual luciferase reporter assays normalized to renilla luciferase activity. Promoter activities are shown as fold activation respective to the pGL4.10 basal activity (pGL4.10 = 1). The data represent means ± S.D. of three independent experiments in triplicate. An arrow indicates the transcription start site numbered with +1 and 'luc' boxed indicates the firefly luciferase gene.
Figure Legend Snippet: The activity of a short and a long ART1 promoter fragment is upregulated during myotube formation . (A) Shown is the exon-intron structure of the mouse ART1 gene as described previously [ 58 ]. Exons 1–5 are represented by boxes, introns by thin lines. Coding regions are indicated in black, non-coding regions in white. (B) A long (-1236/+36) and a short (-322/+37) fragment of the putative mouse ART1 promoter region (pART1-L: grey bars and pART1-S: black bars, respectively) was cloned into the pGL4.10 firefly luciferase reporter plasmid. C3H-10T 1/2 (Pg, MyoD-Mb, Mt) and C2C12 (Mb, Mt) cells were transfected with the plasmids and promoter activities were determined using the dual luciferase reporter assays normalized to renilla luciferase activity. Promoter activities are shown as fold activation respective to the pGL4.10 basal activity (pGL4.10 = 1). The data represent means ± S.D. of three independent experiments in triplicate. An arrow indicates the transcription start site numbered with +1 and 'luc' boxed indicates the firefly luciferase gene.

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

21) Product Images from "Identification of G-quadruplex structures that possess transcriptional regulating functions in the Dele and Cdc6 CpG islands"

Article Title: Identification of G-quadruplex structures that possess transcriptional regulating functions in the Dele and Cdc6 CpG islands

Journal: BMC Molecular Biology

doi: 10.1186/s12867-017-0094-z

Reporter assay for evaluation of the promoter activities of Cdc6 , Dele -F, and Dele -R G4 DNAs. The G4-forming sequences were cloned into the pGL4.10 vector not containing any promoter. Black bars represent the wild-types and white bars represent the mutant-types. Luciferase activities relative to the pGL4.10 vector are shown (mean ± SD, n = 3). Wild- and mutant-types samples t-test differences: ***P
Figure Legend Snippet: Reporter assay for evaluation of the promoter activities of Cdc6 , Dele -F, and Dele -R G4 DNAs. The G4-forming sequences were cloned into the pGL4.10 vector not containing any promoter. Black bars represent the wild-types and white bars represent the mutant-types. Luciferase activities relative to the pGL4.10 vector are shown (mean ± SD, n = 3). Wild- and mutant-types samples t-test differences: ***P

Techniques Used: Reporter Assay, Clone Assay, Plasmid Preparation, Mutagenesis, Luciferase

Reporter assay for evaluation of the transcriptional activity of Cdc6 , Dele -F, and Dele -R G4 DNAs in CGI sequences. Black bars represent the wild-types and white bars represent the mutant-types. Luciferase activity relative to the pGL4.10 is shown (mean ± SD, n = 3). Wild- and mutant-types samples t-test differences: *P
Figure Legend Snippet: Reporter assay for evaluation of the transcriptional activity of Cdc6 , Dele -F, and Dele -R G4 DNAs in CGI sequences. Black bars represent the wild-types and white bars represent the mutant-types. Luciferase activity relative to the pGL4.10 is shown (mean ± SD, n = 3). Wild- and mutant-types samples t-test differences: *P

Techniques Used: Reporter Assay, Activity Assay, Mutagenesis, Luciferase

22) Product Images from "Novel Variant of Thyroglobulin Promoter Triggers Thyroid Autoimmunity through an Epigenetic Interferon ?-modulated Mechanism *"

Article Title: Novel Variant of Thyroglobulin Promoter Triggers Thyroid Autoimmunity through an Epigenetic Interferon ?-modulated Mechanism *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.247510

Functional analysis of IRF-1-dependent TG promoter activity. a , constructs. 2.5-kb sequence, including the rs180195 A allele at the 5′ upstream TG gene, was cloned into pGL4.10 vector to generate pGL4.10-TG(A). The rs180195 G allele as well as
Figure Legend Snippet: Functional analysis of IRF-1-dependent TG promoter activity. a , constructs. 2.5-kb sequence, including the rs180195 A allele at the 5′ upstream TG gene, was cloned into pGL4.10 vector to generate pGL4.10-TG(A). The rs180195 G allele as well as

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

a , reduced IRF-1 mRNA expression in ML-1 cells treated with IRF-1 siRNA. mRNA expression of IRF-1 was assessed by regular RT-PCR and QRT-PCR. b , knockdown of IRF-1 by siRNA reduces the TG promoter activity only in the presence of rs180195 G allele. pGL4.10-TG(A)
Figure Legend Snippet: a , reduced IRF-1 mRNA expression in ML-1 cells treated with IRF-1 siRNA. mRNA expression of IRF-1 was assessed by regular RT-PCR and QRT-PCR. b , knockdown of IRF-1 by siRNA reduces the TG promoter activity only in the presence of rs180195 G allele. pGL4.10-TG(A)

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Activity Assay

INFα stimulates expression of TG in the presence of the disease-associated variant. a , specific effect of IFNα on pGL4.10-TG(G) construct activity. Treatment of ML-1 cells with IFNα increased the TG promoter activity when G allele
Figure Legend Snippet: INFα stimulates expression of TG in the presence of the disease-associated variant. a , specific effect of IFNα on pGL4.10-TG(G) construct activity. Treatment of ML-1 cells with IFNα increased the TG promoter activity when G allele

Techniques Used: Expressing, Variant Assay, Construct, Activity Assay

23) Product Images from "Novel Variant of Thyroglobulin Promoter Triggers Thyroid Autoimmunity through an Epigenetic Interferon ?-modulated Mechanism *"

Article Title: Novel Variant of Thyroglobulin Promoter Triggers Thyroid Autoimmunity through an Epigenetic Interferon ?-modulated Mechanism *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.247510

Functional analysis of IRF-1-dependent TG promoter activity. a , constructs. 2.5-kb sequence, including the rs180195 A allele at the 5′ upstream TG gene, was cloned into pGL4.10 vector to generate pGL4.10-TG(A). The rs180195 G allele as well as
Figure Legend Snippet: Functional analysis of IRF-1-dependent TG promoter activity. a , constructs. 2.5-kb sequence, including the rs180195 A allele at the 5′ upstream TG gene, was cloned into pGL4.10 vector to generate pGL4.10-TG(A). The rs180195 G allele as well as

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

a , reduced IRF-1 mRNA expression in ML-1 cells treated with IRF-1 siRNA. mRNA expression of IRF-1 was assessed by regular RT-PCR and QRT-PCR. b , knockdown of IRF-1 by siRNA reduces the TG promoter activity only in the presence of rs180195 G allele. pGL4.10-TG(A)
Figure Legend Snippet: a , reduced IRF-1 mRNA expression in ML-1 cells treated with IRF-1 siRNA. mRNA expression of IRF-1 was assessed by regular RT-PCR and QRT-PCR. b , knockdown of IRF-1 by siRNA reduces the TG promoter activity only in the presence of rs180195 G allele. pGL4.10-TG(A)

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Activity Assay

INFα stimulates expression of TG in the presence of the disease-associated variant. a , specific effect of IFNα on pGL4.10-TG(G) construct activity. Treatment of ML-1 cells with IFNα increased the TG promoter activity when G allele
Figure Legend Snippet: INFα stimulates expression of TG in the presence of the disease-associated variant. a , specific effect of IFNα on pGL4.10-TG(G) construct activity. Treatment of ML-1 cells with IFNα increased the TG promoter activity when G allele

Techniques Used: Expressing, Variant Assay, Construct, Activity Assay

24) Product Images from "Novel Variant of Thyroglobulin Promoter Triggers Thyroid Autoimmunity through an Epigenetic Interferon ?-modulated Mechanism *"

Article Title: Novel Variant of Thyroglobulin Promoter Triggers Thyroid Autoimmunity through an Epigenetic Interferon ?-modulated Mechanism *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.247510

Functional analysis of IRF-1-dependent TG promoter activity. a , constructs. 2.5-kb sequence, including the rs180195 A allele at the 5′ upstream TG gene, was cloned into pGL4.10 vector to generate pGL4.10-TG(A). The rs180195 G allele as well as
Figure Legend Snippet: Functional analysis of IRF-1-dependent TG promoter activity. a , constructs. 2.5-kb sequence, including the rs180195 A allele at the 5′ upstream TG gene, was cloned into pGL4.10 vector to generate pGL4.10-TG(A). The rs180195 G allele as well as

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

a , reduced IRF-1 mRNA expression in ML-1 cells treated with IRF-1 siRNA. mRNA expression of IRF-1 was assessed by regular RT-PCR and QRT-PCR. b , knockdown of IRF-1 by siRNA reduces the TG promoter activity only in the presence of rs180195 G allele. pGL4.10-TG(A)
Figure Legend Snippet: a , reduced IRF-1 mRNA expression in ML-1 cells treated with IRF-1 siRNA. mRNA expression of IRF-1 was assessed by regular RT-PCR and QRT-PCR. b , knockdown of IRF-1 by siRNA reduces the TG promoter activity only in the presence of rs180195 G allele. pGL4.10-TG(A)

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Activity Assay

INFα stimulates expression of TG in the presence of the disease-associated variant. a , specific effect of IFNα on pGL4.10-TG(G) construct activity. Treatment of ML-1 cells with IFNα increased the TG promoter activity when G allele
Figure Legend Snippet: INFα stimulates expression of TG in the presence of the disease-associated variant. a , specific effect of IFNα on pGL4.10-TG(G) construct activity. Treatment of ML-1 cells with IFNα increased the TG promoter activity when G allele

Techniques Used: Expressing, Variant Assay, Construct, Activity Assay

25) Product Images from "Identification of two regulatory binding sites which confer myotube specific expression of the mono-ADP-ribosyltransferase ART1 gene"

Article Title: Identification of two regulatory binding sites which confer myotube specific expression of the mono-ADP-ribosyltransferase ART1 gene

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-9-91

An A/T-rich motif and an E box mediate the induction of the ART1 proximal promoter activity during myogenesis . Candidate motifs for mediating muscle-specific expression of the mouse proximal ART1 promoter were altered by site-directed mutagenesis of the pART1-S (-322/+37) containing pGL4.10 plasmid. Cells at different stages of differentiation were transfected with the plasmids. Promoter activities were determined using the dual luciferase reporter assays normalized to renilla luciferase activity. Following symbols are used: black box – A/T-rich element (A/T); black oval – E box (E); white box – TATA box (TB) and 'luc' boxed – firefly luciferase gene. An arrow indicates the transcription start site numbered with +1. Mutations of symbolised A/T-rich element (A/T) and E box (E) are indicated by an X overlay. The data represent means ± S.D. of three independent experiments in triplicate.
Figure Legend Snippet: An A/T-rich motif and an E box mediate the induction of the ART1 proximal promoter activity during myogenesis . Candidate motifs for mediating muscle-specific expression of the mouse proximal ART1 promoter were altered by site-directed mutagenesis of the pART1-S (-322/+37) containing pGL4.10 plasmid. Cells at different stages of differentiation were transfected with the plasmids. Promoter activities were determined using the dual luciferase reporter assays normalized to renilla luciferase activity. Following symbols are used: black box – A/T-rich element (A/T); black oval – E box (E); white box – TATA box (TB) and 'luc' boxed – firefly luciferase gene. An arrow indicates the transcription start site numbered with +1. Mutations of symbolised A/T-rich element (A/T) and E box (E) are indicated by an X overlay. The data represent means ± S.D. of three independent experiments in triplicate.

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

The activity of a short and a long ART1 promoter fragment is upregulated during myotube formation . (A) Shown is the exon-intron structure of the mouse ART1 gene as described previously [ 58 ]. Exons 1–5 are represented by boxes, introns by thin lines. Coding regions are indicated in black, non-coding regions in white. (B) A long (-1236/+36) and a short (-322/+37) fragment of the putative mouse ART1 promoter region (pART1-L: grey bars and pART1-S: black bars, respectively) was cloned into the pGL4.10 firefly luciferase reporter plasmid. C3H-10T 1/2 (Pg, MyoD-Mb, Mt) and C2C12 (Mb, Mt) cells were transfected with the plasmids and promoter activities were determined using the dual luciferase reporter assays normalized to renilla luciferase activity. Promoter activities are shown as fold activation respective to the pGL4.10 basal activity (pGL4.10 = 1). The data represent means ± S.D. of three independent experiments in triplicate. An arrow indicates the transcription start site numbered with +1 and 'luc' boxed indicates the firefly luciferase gene.
Figure Legend Snippet: The activity of a short and a long ART1 promoter fragment is upregulated during myotube formation . (A) Shown is the exon-intron structure of the mouse ART1 gene as described previously [ 58 ]. Exons 1–5 are represented by boxes, introns by thin lines. Coding regions are indicated in black, non-coding regions in white. (B) A long (-1236/+36) and a short (-322/+37) fragment of the putative mouse ART1 promoter region (pART1-L: grey bars and pART1-S: black bars, respectively) was cloned into the pGL4.10 firefly luciferase reporter plasmid. C3H-10T 1/2 (Pg, MyoD-Mb, Mt) and C2C12 (Mb, Mt) cells were transfected with the plasmids and promoter activities were determined using the dual luciferase reporter assays normalized to renilla luciferase activity. Promoter activities are shown as fold activation respective to the pGL4.10 basal activity (pGL4.10 = 1). The data represent means ± S.D. of three independent experiments in triplicate. An arrow indicates the transcription start site numbered with +1 and 'luc' boxed indicates the firefly luciferase gene.

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

26) Product Images from "REGULATION OF EXPRESSION OF STROMAL-DERIVED FACTOR-1 (SDF-1) RECEPTORS: CXCR4 AND CXCR7 IN HUMAN RHADOMYOSARCOMAS"

Article Title: REGULATION OF EXPRESSION OF STROMAL-DERIVED FACTOR-1 (SDF-1) RECEPTORS: CXCR4 AND CXCR7 IN HUMAN RHADOMYOSARCOMAS

Journal: Molecular cancer research : MCR

doi: 10.1158/1541-7786.MCR-09-0259

CXCR4 promoter deletion studies and transcription binding sites analysis Panel A. Constructed CXCR4 promoter inserts. The promoter region of the CXCR4 gene from −2,237 to +62 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 was sequentially shortened according to the positions of PAX3 binding sites, and NRF-1 binding sites as depicted in the theoretical model of the cloned sequences of the promoter. The position of PAX3-, NRF-1- HRE-, NF-κb and YY1 binding sites as well as P1-3 primer pairs used in the ChIP experiment are shown. Panel B. CXCR4 promoter activity studies. RD and RD/PAX3-FKHR cells were transfected with the appropriate plasmids. Cultured cells were harvested after 24 hours and assayed for the amount of luciferase activity. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag1–7 p
Figure Legend Snippet: CXCR4 promoter deletion studies and transcription binding sites analysis Panel A. Constructed CXCR4 promoter inserts. The promoter region of the CXCR4 gene from −2,237 to +62 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 was sequentially shortened according to the positions of PAX3 binding sites, and NRF-1 binding sites as depicted in the theoretical model of the cloned sequences of the promoter. The position of PAX3-, NRF-1- HRE-, NF-κb and YY1 binding sites as well as P1-3 primer pairs used in the ChIP experiment are shown. Panel B. CXCR4 promoter activity studies. RD and RD/PAX3-FKHR cells were transfected with the appropriate plasmids. Cultured cells were harvested after 24 hours and assayed for the amount of luciferase activity. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag1–7 p

Techniques Used: Binding Assay, Construct, Clone Assay, Chromatin Immunoprecipitation, Activity Assay, Transfection, Cell Culture, Luciferase, Plasmid Preparation

CXCR7 promoter deletion studies and transcription binding sites analysis Panel A. CXCR7 promoter inserts. The promoter region of the CXCR7 gene from −2,409 to +89 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 shorter derivatives were obtained according to the positions of NRF-1, HRE binding sites, and NF-κB binding sites. The individual constructs were cloned to pGL4.10 vector, transfected into either RD or RD/PAX3-FKHR cells, and used in Dual Luciferase assays. (X) – mutated NF-κB and HRE binding sites. Primer pairs used in the ChIP experiment (N1–N5) flanked 5 different potential binding sites for NF-κB transcription factor. Panel B. NF-κB as a crucial transcription factor that drives CXCR7 promoter activity. Activity of particular CXCR7 promoter constructs was assayed by Dual Luciferase showing an important role of NF-κB transcription factors. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag 1–6 p
Figure Legend Snippet: CXCR7 promoter deletion studies and transcription binding sites analysis Panel A. CXCR7 promoter inserts. The promoter region of the CXCR7 gene from −2,409 to +89 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 shorter derivatives were obtained according to the positions of NRF-1, HRE binding sites, and NF-κB binding sites. The individual constructs were cloned to pGL4.10 vector, transfected into either RD or RD/PAX3-FKHR cells, and used in Dual Luciferase assays. (X) – mutated NF-κB and HRE binding sites. Primer pairs used in the ChIP experiment (N1–N5) flanked 5 different potential binding sites for NF-κB transcription factor. Panel B. NF-κB as a crucial transcription factor that drives CXCR7 promoter activity. Activity of particular CXCR7 promoter constructs was assayed by Dual Luciferase showing an important role of NF-κB transcription factors. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag 1–6 p

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

27) Product Images from "Silencing of CHD5 Gene by Promoter Methylation in Leukemia"

Article Title: Silencing of CHD5 Gene by Promoter Methylation in Leukemia

Journal: PLoS ONE

doi: 10.1371/journal.pone.0085172

Identification of the AP2 binding site in the CHD5 promoter regulatory element. ChIP-qPCR assay indicated that AP2 binds to the identified regulatory element of the CHD5 promoter (A, B). Normal IgG was used as a negative control. The pGL4.10-CHD5-2000 to -370 and pGL4.10-CHD5-356 vectors, in which the AP2 binding site was deleted, were measured for luciferase activity in K-562 cells (C). The pGL4.1-CHD5-P-CR vector, harboring the –560 to –240 region, was measured for luciferase activity in K-562 cells (D). ChIP-qPCR analysis showed that DAC treatment enhanced AP2 binding to the CHD5 promoter. All data are presented as mean ± SD. P
Figure Legend Snippet: Identification of the AP2 binding site in the CHD5 promoter regulatory element. ChIP-qPCR assay indicated that AP2 binds to the identified regulatory element of the CHD5 promoter (A, B). Normal IgG was used as a negative control. The pGL4.10-CHD5-2000 to -370 and pGL4.10-CHD5-356 vectors, in which the AP2 binding site was deleted, were measured for luciferase activity in K-562 cells (C). The pGL4.1-CHD5-P-CR vector, harboring the –560 to –240 region, was measured for luciferase activity in K-562 cells (D). ChIP-qPCR analysis showed that DAC treatment enhanced AP2 binding to the CHD5 promoter. All data are presented as mean ± SD. P

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Luciferase, Activity Assay, Plasmid Preparation

Mapping regulatory elements within the CHD5 promoter. The reporter constructs containing the full length (–2000 to –1) CHD5 promoter or partial promoter sequences progressively truncated by 200 bp (A) or 100 bp (B) at the 5' or 3' end were measured for luciferase activity in K-562 cells. The promoterless pGL4.10-Basic vector was used as a negative control. The pGL4.10-CMV vector, which uses the CMV promoter, was used as a positive reporter construct. The relative Luc activity of full length and truncated CHD5 promoters were normalized to Renilla luciferase activity and compared to normalized Luc luciferase activity from pGL4.10-Basic. All data are presented as mean ± SD. P
Figure Legend Snippet: Mapping regulatory elements within the CHD5 promoter. The reporter constructs containing the full length (–2000 to –1) CHD5 promoter or partial promoter sequences progressively truncated by 200 bp (A) or 100 bp (B) at the 5' or 3' end were measured for luciferase activity in K-562 cells. The promoterless pGL4.10-Basic vector was used as a negative control. The pGL4.10-CMV vector, which uses the CMV promoter, was used as a positive reporter construct. The relative Luc activity of full length and truncated CHD5 promoters were normalized to Renilla luciferase activity and compared to normalized Luc luciferase activity from pGL4.10-Basic. All data are presented as mean ± SD. P

Techniques Used: Construct, Luciferase, Activity Assay, Plasmid Preparation, Negative Control

28) Product Images from "Retinoic acid-related orphan receptor ? directly regulates neuronal PAS domain protein 2 transcription in vivo"

Article Title: Retinoic acid-related orphan receptor ? directly regulates neuronal PAS domain protein 2 transcription in vivo

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq1335

Rev-Erbα and the ROR-inverse agonist T0901317 inhibited ROR-induced activation of the Npas2 (–1534/+81) promoter. ( A ) Rev-Erbα expression represses the activation of the Npas2 promoter by RORα and RORγ, Huh-7 cells were transfected with p3xFlag-CMV10-RORγ or p3xFlag-CMV10-RORα, pGL4.10- Npas2 (–1534/+81) and increasing concentrations of p3xFlag-CMV10-Rev-Erbα and 24 h later were assayed for reporter activity. ( B ) The downregulation of basal Npas2 promoter activity by Rev-Erbα was abrogated by mutations in the ROREs. Huh-7 cells were transfected with p3xFlag-CMV10-Rev-Erbα and pGL4.10 driven by the WT Npas2 (–1534/+81) promoter or the promoter with the indicated RORE mutations. About 24 h later cells were assayed for reporter activity. ( C ) The inverse agonist, T0901317, represses the activation of the Npas2 promoter by both RORγ and RORα in Huh-7 cells. Data present mean ± SEM.
Figure Legend Snippet: Rev-Erbα and the ROR-inverse agonist T0901317 inhibited ROR-induced activation of the Npas2 (–1534/+81) promoter. ( A ) Rev-Erbα expression represses the activation of the Npas2 promoter by RORα and RORγ, Huh-7 cells were transfected with p3xFlag-CMV10-RORγ or p3xFlag-CMV10-RORα, pGL4.10- Npas2 (–1534/+81) and increasing concentrations of p3xFlag-CMV10-Rev-Erbα and 24 h later were assayed for reporter activity. ( B ) The downregulation of basal Npas2 promoter activity by Rev-Erbα was abrogated by mutations in the ROREs. Huh-7 cells were transfected with p3xFlag-CMV10-Rev-Erbα and pGL4.10 driven by the WT Npas2 (–1534/+81) promoter or the promoter with the indicated RORE mutations. About 24 h later cells were assayed for reporter activity. ( C ) The inverse agonist, T0901317, represses the activation of the Npas2 promoter by both RORγ and RORα in Huh-7 cells. Data present mean ± SEM.

Techniques Used: Activation Assay, Expressing, Transfection, Activity Assay

Regulation of Npas2 expression by ROR is mediated through ROREs. ( A ) Schematic of the location of the RORE1 and RORE2 in the proximal Npas2 promoter region. The bold and underlined sequences of these ROREs are conserved between the human and mouse genomes. The numbers refer to the distance to the transcription start site. ( B and C ) RORs were able to effectively activate the (–1534/+81) Npas2 proximal promoter in Huh-7 (B) and HEK293T cells (C). Cells were co-transfected with the indicated p3xFlag–CMV10–ROR expression vector, pCMV-β-Gal and a pGL4.10 reporter plasmid driven by either the WT Npas2 (–1534/+81) promoter or the promoter in which RORE1, RORE2 or both were mutated (RORE1m, RORE2m and RORE1m2m, respectively). About 24 h later the relative luciferase reporter activities were determined as described in ‘Materials and Methods’ section. Data present mean ± SEM.
Figure Legend Snippet: Regulation of Npas2 expression by ROR is mediated through ROREs. ( A ) Schematic of the location of the RORE1 and RORE2 in the proximal Npas2 promoter region. The bold and underlined sequences of these ROREs are conserved between the human and mouse genomes. The numbers refer to the distance to the transcription start site. ( B and C ) RORs were able to effectively activate the (–1534/+81) Npas2 proximal promoter in Huh-7 (B) and HEK293T cells (C). Cells were co-transfected with the indicated p3xFlag–CMV10–ROR expression vector, pCMV-β-Gal and a pGL4.10 reporter plasmid driven by either the WT Npas2 (–1534/+81) promoter or the promoter in which RORE1, RORE2 or both were mutated (RORE1m, RORE2m and RORE1m2m, respectively). About 24 h later the relative luciferase reporter activities were determined as described in ‘Materials and Methods’ section. Data present mean ± SEM.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Luciferase

29) Product Images from "Existence of Two Distinct Infectious Endogenous Retroviruses in Domestic Cats and Their Different Strategies for Adaptation to Transcriptional Regulation"

Article Title: Existence of Two Distinct Infectious Endogenous Retroviruses in Domestic Cats and Their Different Strategies for Adaptation to Transcriptional Regulation

Journal: Journal of Virology

doi: 10.1128/JVI.00716-16

ERV-DC LTR-driven transcription in the presence of CpG methylation. (A) The LTR reporter plasmids (DC7, DC10, DC16, DC19, and empty vector) were methylated in vitro by either M.SssI (Me) or heat-inactivated M.SssI (HI). The DNA was treated with BstUI, separated by gel electrophoresis, and visualized by ethidium bromide staining. M indicates the DNA marker, and Cont indicates the control pGL4.10[luc2] empty vector. (B) The promoter activity levels of methylated or unmethylated (treated with heat-inactivated M.SssI) ERV-DC LTRs were measured by a luciferase reporter assay. The LTR-Luc reporter plasmids were cotransfected with Renilla luciferase-expressing phRL-CMV into HEK293T cells, the cells were harvested at 48 h posttransfection, and luciferase activity was measured. The control was the pGL4.10[luc2] empty vector. The luciferase activity of each LTR was adjusted according to the Renilla luciferase activity level. These results include data from three independent replicates and were statistically analyzed by Student's t test (*, P
Figure Legend Snippet: ERV-DC LTR-driven transcription in the presence of CpG methylation. (A) The LTR reporter plasmids (DC7, DC10, DC16, DC19, and empty vector) were methylated in vitro by either M.SssI (Me) or heat-inactivated M.SssI (HI). The DNA was treated with BstUI, separated by gel electrophoresis, and visualized by ethidium bromide staining. M indicates the DNA marker, and Cont indicates the control pGL4.10[luc2] empty vector. (B) The promoter activity levels of methylated or unmethylated (treated with heat-inactivated M.SssI) ERV-DC LTRs were measured by a luciferase reporter assay. The LTR-Luc reporter plasmids were cotransfected with Renilla luciferase-expressing phRL-CMV into HEK293T cells, the cells were harvested at 48 h posttransfection, and luciferase activity was measured. The control was the pGL4.10[luc2] empty vector. The luciferase activity of each LTR was adjusted according to the Renilla luciferase activity level. These results include data from three independent replicates and were statistically analyzed by Student's t test (*, P

Techniques Used: CpG Methylation Assay, Plasmid Preparation, Methylation, In Vitro, Nucleic Acid Electrophoresis, Staining, Marker, Activity Assay, Luciferase, Reporter Assay, Expressing

Luciferase reporter assay for basal promoter activity of ERV-DC LTRs and identification of LTR cis elements. (A) The basal promoter activity levels of wild-type LTRs were measured by using a luciferase reporter assay. Genotype I (GI) includes DC1 (DC3), DC4, DC8, DC14, DC17, and DC19; genotype II (GII) includes DC7 and DC16; and genotype III (GIII) includes DC10 (DC18). (B) Schematic of chimeric and mutant ERV-DC LTRs that were used to investigate the presence of a cis element in ERV-DC LTRs. The name of each construct is indicated on the left, and the wild-type status or the chimeric or point mutation of the LTRs is indicated on the right. Asterisks indicate a point mutation of the nucleotide. The SmaI restriction enzyme site is located in the LTRs of ERV-DC8 and ERV-DC19 at positions 263 and 267, respectively. (C) The promoter activity levels of chimeric LTRs between the DC19 LTR and DC8 LTR (DC8/19 and DC19/8 LTRs) were measured by a luciferase reporter assay. (D) The promoter activity levels of DC8-mu6 (T280A substitution), DC8-mu7 (A483G substitution), and DC8-mu8 (G508A substitution), which are all DC8 LTRs containing a point mutation, were measured by a luciferase reporter assay. (E) The promoter activity levels of DC10 A281T (A281T substitution) and DC19 A285T (A285T substitution), which are the DC10 and DC19 LTRs with a point mutation, respectively, were measured by a luciferase reporter assay. LTR-Luc reporter plasmids were cotransfected with Renilla luciferase-expressing phRL-CMV into HEK293T cells. The cells were harvested at 48 h posttransfection, and luciferase activity was measured. The control (Cont) was a pGL4.10[luc2] empty vector. The luciferase activity of each LTR was adjusted according to the Renilla luciferase activity. The results from three replicates were collected and statistically analyzed by Student's t test and one-way analysis of variance (*, P
Figure Legend Snippet: Luciferase reporter assay for basal promoter activity of ERV-DC LTRs and identification of LTR cis elements. (A) The basal promoter activity levels of wild-type LTRs were measured by using a luciferase reporter assay. Genotype I (GI) includes DC1 (DC3), DC4, DC8, DC14, DC17, and DC19; genotype II (GII) includes DC7 and DC16; and genotype III (GIII) includes DC10 (DC18). (B) Schematic of chimeric and mutant ERV-DC LTRs that were used to investigate the presence of a cis element in ERV-DC LTRs. The name of each construct is indicated on the left, and the wild-type status or the chimeric or point mutation of the LTRs is indicated on the right. Asterisks indicate a point mutation of the nucleotide. The SmaI restriction enzyme site is located in the LTRs of ERV-DC8 and ERV-DC19 at positions 263 and 267, respectively. (C) The promoter activity levels of chimeric LTRs between the DC19 LTR and DC8 LTR (DC8/19 and DC19/8 LTRs) were measured by a luciferase reporter assay. (D) The promoter activity levels of DC8-mu6 (T280A substitution), DC8-mu7 (A483G substitution), and DC8-mu8 (G508A substitution), which are all DC8 LTRs containing a point mutation, were measured by a luciferase reporter assay. (E) The promoter activity levels of DC10 A281T (A281T substitution) and DC19 A285T (A285T substitution), which are the DC10 and DC19 LTRs with a point mutation, respectively, were measured by a luciferase reporter assay. LTR-Luc reporter plasmids were cotransfected with Renilla luciferase-expressing phRL-CMV into HEK293T cells. The cells were harvested at 48 h posttransfection, and luciferase activity was measured. The control (Cont) was a pGL4.10[luc2] empty vector. The luciferase activity of each LTR was adjusted according to the Renilla luciferase activity. The results from three replicates were collected and statistically analyzed by Student's t test and one-way analysis of variance (*, P

Techniques Used: Luciferase, Reporter Assay, Activity Assay, Mutagenesis, Construct, Expressing, Plasmid Preparation

30) Product Images from "Existence of Two Distinct Infectious Endogenous Retroviruses in Domestic Cats and Their Different Strategies for Adaptation to Transcriptional Regulation"

Article Title: Existence of Two Distinct Infectious Endogenous Retroviruses in Domestic Cats and Their Different Strategies for Adaptation to Transcriptional Regulation

Journal: Journal of Virology

doi: 10.1128/JVI.00716-16

ERV-DC LTR-driven transcription in the presence of CpG methylation. (A) The LTR reporter plasmids (DC7, DC10, DC16, DC19, and empty vector) were methylated in vitro by either M.SssI (Me) or heat-inactivated M.SssI (HI). The DNA was treated with BstUI, separated by gel electrophoresis, and visualized by ethidium bromide staining. M indicates the DNA marker, and Cont indicates the control pGL4.10[luc2] empty vector. (B) The promoter activity levels of methylated or unmethylated (treated with heat-inactivated M.SssI) ERV-DC LTRs were measured by a luciferase reporter assay. The LTR-Luc reporter plasmids were cotransfected with Renilla luciferase-expressing phRL-CMV into HEK293T cells, the cells were harvested at 48 h posttransfection, and luciferase activity was measured. The control was the pGL4.10[luc2] empty vector. The luciferase activity of each LTR was adjusted according to the Renilla luciferase activity level. These results include data from three independent replicates and were statistically analyzed by Student's t test (*, P
Figure Legend Snippet: ERV-DC LTR-driven transcription in the presence of CpG methylation. (A) The LTR reporter plasmids (DC7, DC10, DC16, DC19, and empty vector) were methylated in vitro by either M.SssI (Me) or heat-inactivated M.SssI (HI). The DNA was treated with BstUI, separated by gel electrophoresis, and visualized by ethidium bromide staining. M indicates the DNA marker, and Cont indicates the control pGL4.10[luc2] empty vector. (B) The promoter activity levels of methylated or unmethylated (treated with heat-inactivated M.SssI) ERV-DC LTRs were measured by a luciferase reporter assay. The LTR-Luc reporter plasmids were cotransfected with Renilla luciferase-expressing phRL-CMV into HEK293T cells, the cells were harvested at 48 h posttransfection, and luciferase activity was measured. The control was the pGL4.10[luc2] empty vector. The luciferase activity of each LTR was adjusted according to the Renilla luciferase activity level. These results include data from three independent replicates and were statistically analyzed by Student's t test (*, P

Techniques Used: CpG Methylation Assay, Plasmid Preparation, Methylation, In Vitro, Nucleic Acid Electrophoresis, Staining, Marker, Activity Assay, Luciferase, Reporter Assay, Expressing

Luciferase reporter assay for basal promoter activity of ERV-DC LTRs and identification of LTR cis elements. (A) The basal promoter activity levels of wild-type LTRs were measured by using a luciferase reporter assay. Genotype I (GI) includes DC1 (DC3), DC4, DC8, DC14, DC17, and DC19; genotype II (GII) includes DC7 and DC16; and genotype III (GIII) includes DC10 (DC18). (B) Schematic of chimeric and mutant ERV-DC LTRs that were used to investigate the presence of a cis element in ERV-DC LTRs. The name of each construct is indicated on the left, and the wild-type status or the chimeric or point mutation of the LTRs is indicated on the right. Asterisks indicate a point mutation of the nucleotide. The SmaI restriction enzyme site is located in the LTRs of ERV-DC8 and ERV-DC19 at positions 263 and 267, respectively. (C) The promoter activity levels of chimeric LTRs between the DC19 LTR and DC8 LTR (DC8/19 and DC19/8 LTRs) were measured by a luciferase reporter assay. (D) The promoter activity levels of DC8-mu6 (T280A substitution), DC8-mu7 (A483G substitution), and DC8-mu8 (G508A substitution), which are all DC8 LTRs containing a point mutation, were measured by a luciferase reporter assay. (E) The promoter activity levels of DC10 A281T (A281T substitution) and DC19 A285T (A285T substitution), which are the DC10 and DC19 LTRs with a point mutation, respectively, were measured by a luciferase reporter assay. LTR-Luc reporter plasmids were cotransfected with Renilla luciferase-expressing phRL-CMV into HEK293T cells. The cells were harvested at 48 h posttransfection, and luciferase activity was measured. The control (Cont) was a pGL4.10[luc2] empty vector. The luciferase activity of each LTR was adjusted according to the Renilla luciferase activity. The results from three replicates were collected and statistically analyzed by Student's t test and one-way analysis of variance (*, P
Figure Legend Snippet: Luciferase reporter assay for basal promoter activity of ERV-DC LTRs and identification of LTR cis elements. (A) The basal promoter activity levels of wild-type LTRs were measured by using a luciferase reporter assay. Genotype I (GI) includes DC1 (DC3), DC4, DC8, DC14, DC17, and DC19; genotype II (GII) includes DC7 and DC16; and genotype III (GIII) includes DC10 (DC18). (B) Schematic of chimeric and mutant ERV-DC LTRs that were used to investigate the presence of a cis element in ERV-DC LTRs. The name of each construct is indicated on the left, and the wild-type status or the chimeric or point mutation of the LTRs is indicated on the right. Asterisks indicate a point mutation of the nucleotide. The SmaI restriction enzyme site is located in the LTRs of ERV-DC8 and ERV-DC19 at positions 263 and 267, respectively. (C) The promoter activity levels of chimeric LTRs between the DC19 LTR and DC8 LTR (DC8/19 and DC19/8 LTRs) were measured by a luciferase reporter assay. (D) The promoter activity levels of DC8-mu6 (T280A substitution), DC8-mu7 (A483G substitution), and DC8-mu8 (G508A substitution), which are all DC8 LTRs containing a point mutation, were measured by a luciferase reporter assay. (E) The promoter activity levels of DC10 A281T (A281T substitution) and DC19 A285T (A285T substitution), which are the DC10 and DC19 LTRs with a point mutation, respectively, were measured by a luciferase reporter assay. LTR-Luc reporter plasmids were cotransfected with Renilla luciferase-expressing phRL-CMV into HEK293T cells. The cells were harvested at 48 h posttransfection, and luciferase activity was measured. The control (Cont) was a pGL4.10[luc2] empty vector. The luciferase activity of each LTR was adjusted according to the Renilla luciferase activity. The results from three replicates were collected and statistically analyzed by Student's t test and one-way analysis of variance (*, P

Techniques Used: Luciferase, Reporter Assay, Activity Assay, Mutagenesis, Construct, Expressing, Plasmid Preparation

31) Product Images from "An Intronic Enhancer Element Regulates Angiotensin II Type 2 Receptor Expression during Satellite Cell Differentiation, and Its Activity Is Suppressed in Congestive Heart Failure *"

Article Title: An Intronic Enhancer Element Regulates Angiotensin II Type 2 Receptor Expression during Satellite Cell Differentiation, and Its Activity Is Suppressed in Congestive Heart Failure *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.752501

AT2R promoter activity during myoblast differentiation. Mouse AT2R genomic DNA sequence upstream of TSS (+1) together with exon 1, intron 1, exon 2, intron 2, and the 5′-UTR of exon 3 were subcloned into the pGL4.10[luc2] vector. Four vectors were generated with different sized 5′ sequence (3000, 2000, 1000, and 500 bp). These vectors were transfected into SCs and C2C12 myoblasts. After 2 days of transfection, cells were induced to differentiate and harvested on days 0–3, and luciferase activity was measured using Renilla luciferase as an internal control. Mean ± S.E., n = 4; *, p
Figure Legend Snippet: AT2R promoter activity during myoblast differentiation. Mouse AT2R genomic DNA sequence upstream of TSS (+1) together with exon 1, intron 1, exon 2, intron 2, and the 5′-UTR of exon 3 were subcloned into the pGL4.10[luc2] vector. Four vectors were generated with different sized 5′ sequence (3000, 2000, 1000, and 500 bp). These vectors were transfected into SCs and C2C12 myoblasts. After 2 days of transfection, cells were induced to differentiate and harvested on days 0–3, and luciferase activity was measured using Renilla luciferase as an internal control. Mean ± S.E., n = 4; *, p

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

32) Product Images from "Autosomal recessive retinitis pigmentosa with homozygous rhodopsin mutation E150K and non-coding cis-regulatory variants in CRX-binding regions of SAMD7"

Article Title: Autosomal recessive retinitis pigmentosa with homozygous rhodopsin mutation E150K and non-coding cis-regulatory variants in CRX-binding regions of SAMD7

Journal: Scientific Reports

doi: 10.1038/srep21307

Retinal immunostaining of SAMD7 and transcriptional activity of SAMD7 variants. ( a ) Human retinal SAMD7 localization. Representative fluorescent images of horizontal cross-sections of human retina stained with anti-SAMD7 antibody (red, 1:250). Retinal counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI) (blue). SAMD7 immunoreactivity is predominantly detected in the photoreceptor nuclei, located in the ONL. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. ( b ) Luciferase experiments. Luciferase assays were performed in HEK cells, using different SAMD7 reporter constructs. The first construct consisted of CBR1 cloned in the pGL4.10 reporter vector containing a luciferase expressing gene without promoter. For the second construct CBR2 was cloned in the pGL3 promoter vector, upstream of a luciferase reporter gene and a minimal basal promoter. Finally, the third construct was obtained by cloning CBR2 upstream of CBR1 in the pGL4.10 vector. Two types of each construct were created, one containing the wt sequence of a healthy control, the other one consisting of the patient (pat) sequences containing the SAMD7 CBR variants. Cis -regulatory activity could be demonstrated for the CBR1 constructs, while for CBR2 only very little luciferase expression could be measured. In both cases, no significant difference in luciferase expression has been observed between patient and control. However, when combining CBR2 and CBR1, luciferase expression increases for the control construct, while for the patient construct there is a significant decrease in expression. *corresponds with p
Figure Legend Snippet: Retinal immunostaining of SAMD7 and transcriptional activity of SAMD7 variants. ( a ) Human retinal SAMD7 localization. Representative fluorescent images of horizontal cross-sections of human retina stained with anti-SAMD7 antibody (red, 1:250). Retinal counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI) (blue). SAMD7 immunoreactivity is predominantly detected in the photoreceptor nuclei, located in the ONL. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. ( b ) Luciferase experiments. Luciferase assays were performed in HEK cells, using different SAMD7 reporter constructs. The first construct consisted of CBR1 cloned in the pGL4.10 reporter vector containing a luciferase expressing gene without promoter. For the second construct CBR2 was cloned in the pGL3 promoter vector, upstream of a luciferase reporter gene and a minimal basal promoter. Finally, the third construct was obtained by cloning CBR2 upstream of CBR1 in the pGL4.10 vector. Two types of each construct were created, one containing the wt sequence of a healthy control, the other one consisting of the patient (pat) sequences containing the SAMD7 CBR variants. Cis -regulatory activity could be demonstrated for the CBR1 constructs, while for CBR2 only very little luciferase expression could be measured. In both cases, no significant difference in luciferase expression has been observed between patient and control. However, when combining CBR2 and CBR1, luciferase expression increases for the control construct, while for the patient construct there is a significant decrease in expression. *corresponds with p

Techniques Used: Immunostaining, Activity Assay, Staining, Luciferase, Construct, Clone Assay, Plasmid Preparation, Expressing, Sequencing

33) Product Images from "MMP13 inhibition rescues cognitive decline in Alzheimer transgenic mice via BACE1 regulation"

Article Title: MMP13 inhibition rescues cognitive decline in Alzheimer transgenic mice via BACE1 regulation

Journal: Brain

doi: 10.1093/brain/awy305

CL82198 inhibits BACE1 protein expression through MMP13. ( A ) HEK293 cells were transfected with the luciferase reporter plasmids pGL4.21-BACE1 and pGL4.10 (negative control) in the absence or presence of 5 μM CL82198 or 10 nM WAY170523 for 48 h. Luciferase assays were performed with a GloMax 96 microplate luminometer. Firefly luciferase activity was normalized to that of the plasmid pGL4.10. ( B ) BACE1 was knocked down with BACE1 siRNA (siBACE-1 and -6) or control siRNA (NC) in HEK293 cells for 48 h. A dramatic decrease in the amount of BACE1 protein is shown at ∼70 kD. M = protein marker. ( C ) HEK293 cells were treated with WAY170523 (WAY, 10 nM) for 48 h, while control cells were treated with DMSO (CTRL). ( D ) SH-SY5Y cells were treated with 5 μM CL82198 for 6, 12, 24 and 48 h. ( E ) SH-SY5Y cells were treated with 1, 2.5, 5 and 10 μM of CL82198 for 48 h. ( F ) SH-SY5Y cells were treated with an MMP13 neutralizing antibody (anti-MMP13, 1:500) or control rabbit IgG antibody (CTRL) for 48 h. ( G – I ) Mmp13 was knocked down with shRNA-1 (shMMP13-1, G ) or shRNA-2 (shMMP13-2, H ) in HT22 cells for 72 h or was overexpressed with an MMP13 vector in HEK293 cells for 48 h ( I ). CTRL = control shRNA; MOCK = control vector. ( J ) HEK293 cells were transfected with either the control vector (MOCK) or MMP13 vector for 48 h in the absence or presence of 5 μM CL82198 (CL). Cell lysates were subjected to western blotting analysis. Representative western blots for BACE1 are shown on the top , and quantifications are shown below. ( K ) Primary cultured cortical neurons were treated with 0.1, 0.5, 1, 2, 5 and 10 μM SC205756 for 48 h. ( L ) HEK293 cells were treated with 1 μM SC205756 for 48 h. ( M ) HEK-APP cells were treated with 5 µM CL82198 (CL) for 48 h. Cell lysates were prepared and subjected to western blotting analysis for APP and ADAM10. sAPPβ was analysed in conditioned media using an sAPPβ antibody. All values were normalized to CTRL or MOCK (1.0) within each experiment. The error bars are the SEM. n.s. = no significant difference; * P
Figure Legend Snippet: CL82198 inhibits BACE1 protein expression through MMP13. ( A ) HEK293 cells were transfected with the luciferase reporter plasmids pGL4.21-BACE1 and pGL4.10 (negative control) in the absence or presence of 5 μM CL82198 or 10 nM WAY170523 for 48 h. Luciferase assays were performed with a GloMax 96 microplate luminometer. Firefly luciferase activity was normalized to that of the plasmid pGL4.10. ( B ) BACE1 was knocked down with BACE1 siRNA (siBACE-1 and -6) or control siRNA (NC) in HEK293 cells for 48 h. A dramatic decrease in the amount of BACE1 protein is shown at ∼70 kD. M = protein marker. ( C ) HEK293 cells were treated with WAY170523 (WAY, 10 nM) for 48 h, while control cells were treated with DMSO (CTRL). ( D ) SH-SY5Y cells were treated with 5 μM CL82198 for 6, 12, 24 and 48 h. ( E ) SH-SY5Y cells were treated with 1, 2.5, 5 and 10 μM of CL82198 for 48 h. ( F ) SH-SY5Y cells were treated with an MMP13 neutralizing antibody (anti-MMP13, 1:500) or control rabbit IgG antibody (CTRL) for 48 h. ( G – I ) Mmp13 was knocked down with shRNA-1 (shMMP13-1, G ) or shRNA-2 (shMMP13-2, H ) in HT22 cells for 72 h or was overexpressed with an MMP13 vector in HEK293 cells for 48 h ( I ). CTRL = control shRNA; MOCK = control vector. ( J ) HEK293 cells were transfected with either the control vector (MOCK) or MMP13 vector for 48 h in the absence or presence of 5 μM CL82198 (CL). Cell lysates were subjected to western blotting analysis. Representative western blots for BACE1 are shown on the top , and quantifications are shown below. ( K ) Primary cultured cortical neurons were treated with 0.1, 0.5, 1, 2, 5 and 10 μM SC205756 for 48 h. ( L ) HEK293 cells were treated with 1 μM SC205756 for 48 h. ( M ) HEK-APP cells were treated with 5 µM CL82198 (CL) for 48 h. Cell lysates were prepared and subjected to western blotting analysis for APP and ADAM10. sAPPβ was analysed in conditioned media using an sAPPβ antibody. All values were normalized to CTRL or MOCK (1.0) within each experiment. The error bars are the SEM. n.s. = no significant difference; * P

Techniques Used: Expressing, Transfection, Luciferase, Negative Control, Activity Assay, Plasmid Preparation, Marker, shRNA, Western Blot, Cell Culture

34) Product Images from "Targeting Cancer Cells with a Bisphosphonate Prodrug"

Article Title: Targeting Cancer Cells with a Bisphosphonate Prodrug

Journal: ChemMedChem

doi: 10.1002/cmdc.201600465

Compound 7 inhibits EJ-1 bladder carcinoma growth in immunodeficient NOG mice. (A) Whole-body bioluminescence imaging of tumor cells. Immunodeficient NOG mice were intraperitoneally (i.p.) inoculated with 1 × 10 6 EJ-1 cells transfected with pGL4.10[luc2] on day 0 and treated with 2 μg/0.1 mL compound 7 (97.1 μg/kg) or 0.1 mL of PBS i.p. on days 3 and 6. On day 7, 0.1 mL of 15 mg/mL VivoGlo™ luciferin (Xenogen, Alameda, CA, USA) was injected i.p. and the mice were placed in the specimen chamber and photon emissions transmitted from the mice were then measured. This treatment regimen was repeated 7 times and the images from day 28 are shown. Luciferase units are photons/second/mouse. (B) Inhibition of tumor growth by compound 7 in vivo. The growth of EJ-1 was monitored for weeks 2 through 7 as described in (A) and the average photon flux/sec for four to five mice is plotted ± SEM. * p
Figure Legend Snippet: Compound 7 inhibits EJ-1 bladder carcinoma growth in immunodeficient NOG mice. (A) Whole-body bioluminescence imaging of tumor cells. Immunodeficient NOG mice were intraperitoneally (i.p.) inoculated with 1 × 10 6 EJ-1 cells transfected with pGL4.10[luc2] on day 0 and treated with 2 μg/0.1 mL compound 7 (97.1 μg/kg) or 0.1 mL of PBS i.p. on days 3 and 6. On day 7, 0.1 mL of 15 mg/mL VivoGlo™ luciferin (Xenogen, Alameda, CA, USA) was injected i.p. and the mice were placed in the specimen chamber and photon emissions transmitted from the mice were then measured. This treatment regimen was repeated 7 times and the images from day 28 are shown. Luciferase units are photons/second/mouse. (B) Inhibition of tumor growth by compound 7 in vivo. The growth of EJ-1 was monitored for weeks 2 through 7 as described in (A) and the average photon flux/sec for four to five mice is plotted ± SEM. * p

Techniques Used: Mouse Assay, Imaging, Transfection, Injection, Luciferase, Inhibition, In Vivo, Size-exclusion Chromatography

35) Product Images from "MMP13 inhibition rescues cognitive decline in Alzheimer transgenic mice via BACE1 regulation"

Article Title: MMP13 inhibition rescues cognitive decline in Alzheimer transgenic mice via BACE1 regulation

Journal: Brain

doi: 10.1093/brain/awy305

CL82198 inhibits BACE1 protein expression through MMP13. ( A ) HEK293 cells were transfected with the luciferase reporter plasmids pGL4.21-BACE1 and pGL4.10 (negative control) in the absence or presence of 5 μM CL82198 or 10 nM WAY170523 for 48 h. Luciferase assays were performed with a GloMax 96 microplate luminometer. Firefly luciferase activity was normalized to that of the plasmid pGL4.10. ( B ) BACE1 was knocked down with BACE1 siRNA (siBACE-1 and -6) or control siRNA (NC) in HEK293 cells for 48 h. A dramatic decrease in the amount of BACE1 protein is shown at ∼70 kD. M = protein marker. ( C ) HEK293 cells were treated with WAY170523 (WAY, 10 nM) for 48 h, while control cells were treated with DMSO (CTRL). ( D ) SH-SY5Y cells were treated with 5 μM CL82198 for 6, 12, 24 and 48 h. ( E ) SH-SY5Y cells were treated with 1, 2.5, 5 and 10 μM of CL82198 for 48 h. ( F ) SH-SY5Y cells were treated with an MMP13 neutralizing antibody (anti-MMP13, 1:500) or control rabbit IgG antibody (CTRL) for 48 h. ( G – I ) Mmp13 was knocked down with shRNA-1 (shMMP13-1, G ) or shRNA-2 (shMMP13-2, H ) in HT22 cells for 72 h or was overexpressed with an MMP13 vector in HEK293 cells for 48 h ( I ). CTRL = control shRNA; MOCK = control vector. ( J ) HEK293 cells were transfected with either the control vector (MOCK) or MMP13 vector for 48 h in the absence or presence of 5 μM CL82198 (CL). Cell lysates were subjected to western blotting analysis. Representative western blots for BACE1 are shown on the top , and quantifications are shown below. ( K ) Primary cultured cortical neurons were treated with 0.1, 0.5, 1, 2, 5 and 10 μM SC205756 for 48 h. ( L ) HEK293 cells were treated with 1 μM SC205756 for 48 h. ( M ) HEK-APP cells were treated with 5 µM CL82198 (CL) for 48 h. Cell lysates were prepared and subjected to western blotting analysis for APP and ADAM10. sAPPβ was analysed in conditioned media using an sAPPβ antibody. All values were normalized to CTRL or MOCK (1.0) within each experiment. The error bars are the SEM. n.s. = no significant difference; * P
Figure Legend Snippet: CL82198 inhibits BACE1 protein expression through MMP13. ( A ) HEK293 cells were transfected with the luciferase reporter plasmids pGL4.21-BACE1 and pGL4.10 (negative control) in the absence or presence of 5 μM CL82198 or 10 nM WAY170523 for 48 h. Luciferase assays were performed with a GloMax 96 microplate luminometer. Firefly luciferase activity was normalized to that of the plasmid pGL4.10. ( B ) BACE1 was knocked down with BACE1 siRNA (siBACE-1 and -6) or control siRNA (NC) in HEK293 cells for 48 h. A dramatic decrease in the amount of BACE1 protein is shown at ∼70 kD. M = protein marker. ( C ) HEK293 cells were treated with WAY170523 (WAY, 10 nM) for 48 h, while control cells were treated with DMSO (CTRL). ( D ) SH-SY5Y cells were treated with 5 μM CL82198 for 6, 12, 24 and 48 h. ( E ) SH-SY5Y cells were treated with 1, 2.5, 5 and 10 μM of CL82198 for 48 h. ( F ) SH-SY5Y cells were treated with an MMP13 neutralizing antibody (anti-MMP13, 1:500) or control rabbit IgG antibody (CTRL) for 48 h. ( G – I ) Mmp13 was knocked down with shRNA-1 (shMMP13-1, G ) or shRNA-2 (shMMP13-2, H ) in HT22 cells for 72 h or was overexpressed with an MMP13 vector in HEK293 cells for 48 h ( I ). CTRL = control shRNA; MOCK = control vector. ( J ) HEK293 cells were transfected with either the control vector (MOCK) or MMP13 vector for 48 h in the absence or presence of 5 μM CL82198 (CL). Cell lysates were subjected to western blotting analysis. Representative western blots for BACE1 are shown on the top , and quantifications are shown below. ( K ) Primary cultured cortical neurons were treated with 0.1, 0.5, 1, 2, 5 and 10 μM SC205756 for 48 h. ( L ) HEK293 cells were treated with 1 μM SC205756 for 48 h. ( M ) HEK-APP cells were treated with 5 µM CL82198 (CL) for 48 h. Cell lysates were prepared and subjected to western blotting analysis for APP and ADAM10. sAPPβ was analysed in conditioned media using an sAPPβ antibody. All values were normalized to CTRL or MOCK (1.0) within each experiment. The error bars are the SEM. n.s. = no significant difference; * P

Techniques Used: Expressing, Transfection, Luciferase, Negative Control, Activity Assay, Plasmid Preparation, Marker, shRNA, Western Blot, Cell Culture

36) Product Images from "Transcriptional control of the MUC16 promoter facilitates follicle-stimulating hormone peptide-conjugated shRNA nanoparticle-mediated inhibition of ovarian carcinoma in vivo"

Article Title: Transcriptional control of the MUC16 promoter facilitates follicle-stimulating hormone peptide-conjugated shRNA nanoparticle-mediated inhibition of ovarian carcinoma in vivo

Journal: Drug Delivery

doi: 10.1080/10717544.2018.1451934

MUC16 expression and transcriptional activity of MUC16 promoters. (A) MUC16 expression in ovarian cancer cells by immunocytochemistry. 1, HEY; 2, Caov3; 3, SKOV-3; 4, RMUG-L. (B) Transcriptional activity of the MUC16 promoters in ovarian cancer cells per dual-luciferase reporter assays. Cells were transfected with pGL4.10 vectors containing different MUC16 promoters (pGL-MUC16.1, pGL-MUC16.2, pGL-MUC16.3 and pGL-MUC16.4). The pGL4.10 vector with the CMV promoter (pGL-CMV) was used as the positive control. pGL4.10 without a promoter (pGL-Basic) was used as the negative control. * P
Figure Legend Snippet: MUC16 expression and transcriptional activity of MUC16 promoters. (A) MUC16 expression in ovarian cancer cells by immunocytochemistry. 1, HEY; 2, Caov3; 3, SKOV-3; 4, RMUG-L. (B) Transcriptional activity of the MUC16 promoters in ovarian cancer cells per dual-luciferase reporter assays. Cells were transfected with pGL4.10 vectors containing different MUC16 promoters (pGL-MUC16.1, pGL-MUC16.2, pGL-MUC16.3 and pGL-MUC16.4). The pGL4.10 vector with the CMV promoter (pGL-CMV) was used as the positive control. pGL4.10 without a promoter (pGL-Basic) was used as the negative control. * P

Techniques Used: Expressing, Activity Assay, Immunocytochemistry, Luciferase, Transfection, Plasmid Preparation, Positive Control, Negative Control

37) Product Images from "Androgen receptor and chemokine receptors 4 and 7 form a signaling axis to regulate CXCL12-dependent cellular motility"

Article Title: Androgen receptor and chemokine receptors 4 and 7 form a signaling axis to regulate CXCL12-dependent cellular motility

Journal: BMC Cancer

doi: 10.1186/s12885-015-1201-5

CXCR7 modulates AR transcriptional activity. (A) Luciferase assay testing the effects of CXCR7 overexpression on the AR-target promoter probasin in LNCaP cells. LNCaP cells were co-transfected with the pGL4.10-Luc2- probasin and pRLSV40 Renilla vectors along with increasing amounts (30 ng experimental + 270 ng pcDNA3, 100 ng experimental + 200 ng pcDNA3, 300 ng experimental + 0 ng pcDNA3) of CXCR7 or ACTN4 cDNA mammalian expression vectors. The maximal amount (300 ng) of the pcDNA3 mammalian expression vector served as the positive control. Cells were subsequently treated with androgen (1 nM R1881) or vehicle (ethanol) and tested for dual luciferase activity. Student’s t -test was used to calculate significant differences (* p ≤ 0.05, n = 3) between control and experimental cells within the androgen-treatment group. (B) Luciferase assay testing the effects of CXCR7 siRNA knockdown and treatment with CXCR7 ligand on the AR-target promoter probasin . LNCaP cells were co-transfected with the pGL4.10-Luc2- probasin and pRLSV40- renilla vectors, along with control or experimental siRNAs (50 nM). Next, cells were pre-treated with indicated ligands (BSA, CXCL11, or CXCL12) for 30 min. Cells were then subsequently treated with androgen (1 nM R1881) or vehicle (ethanol) for 18 hrs and tested for dual luciferase activity. Student’s t -test was used to calculate significant differences (* p ≤ 0.05, n = 3) between control cells and experimental cells within the androgen-treatment group. (C) RNA isolated from LNCaP cells treated with vehicle (0.1% BSA), CXCL11 (10 nM), or CXCL12 (10 nM) for 30 min and subsequently treated with vehicle or androgen (1 nM R1881) for 18 hrs were subjected to qPCR analysis for AR , FASN , NKX3.1 , PSA , and TMPRSS2 gene expressions. Student’s t -test was used to calculate significant differences (* p ≤ 0.05, n = 3) between control and chemokine ligand-treated cells.
Figure Legend Snippet: CXCR7 modulates AR transcriptional activity. (A) Luciferase assay testing the effects of CXCR7 overexpression on the AR-target promoter probasin in LNCaP cells. LNCaP cells were co-transfected with the pGL4.10-Luc2- probasin and pRLSV40 Renilla vectors along with increasing amounts (30 ng experimental + 270 ng pcDNA3, 100 ng experimental + 200 ng pcDNA3, 300 ng experimental + 0 ng pcDNA3) of CXCR7 or ACTN4 cDNA mammalian expression vectors. The maximal amount (300 ng) of the pcDNA3 mammalian expression vector served as the positive control. Cells were subsequently treated with androgen (1 nM R1881) or vehicle (ethanol) and tested for dual luciferase activity. Student’s t -test was used to calculate significant differences (* p ≤ 0.05, n = 3) between control and experimental cells within the androgen-treatment group. (B) Luciferase assay testing the effects of CXCR7 siRNA knockdown and treatment with CXCR7 ligand on the AR-target promoter probasin . LNCaP cells were co-transfected with the pGL4.10-Luc2- probasin and pRLSV40- renilla vectors, along with control or experimental siRNAs (50 nM). Next, cells were pre-treated with indicated ligands (BSA, CXCL11, or CXCL12) for 30 min. Cells were then subsequently treated with androgen (1 nM R1881) or vehicle (ethanol) for 18 hrs and tested for dual luciferase activity. Student’s t -test was used to calculate significant differences (* p ≤ 0.05, n = 3) between control cells and experimental cells within the androgen-treatment group. (C) RNA isolated from LNCaP cells treated with vehicle (0.1% BSA), CXCL11 (10 nM), or CXCL12 (10 nM) for 30 min and subsequently treated with vehicle or androgen (1 nM R1881) for 18 hrs were subjected to qPCR analysis for AR , FASN , NKX3.1 , PSA , and TMPRSS2 gene expressions. Student’s t -test was used to calculate significant differences (* p ≤ 0.05, n = 3) between control and chemokine ligand-treated cells.

Techniques Used: Activity Assay, Luciferase, Over Expression, Transfection, Expressing, Plasmid Preparation, Positive Control, Isolation, Real-time Polymerase Chain Reaction

38) Product Images from "KChIP2 is a core transcriptional regulator of cardiac excitability"

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

Journal: eLife

doi: 10.7554/eLife.17304

KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p
Figure Legend Snippet: KChIP2 represses miR-34b/c expression by direct interaction with a putative DRE motif in promoter. ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p

Techniques Used: Expressing, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Plasmid Preparation, Activation Assay

39) Product Images from "Polycomb- and Methylation-Independent Roles of EZH2 as a Transcription Activator"

Article Title: Polycomb- and Methylation-Independent Roles of EZH2 as a Transcription Activator

Journal: Cell reports

doi: 10.1016/j.celrep.2018.11.035

EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the pRetroX-Tight-Pur-Luc vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.
Figure Legend Snippet: EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the pRetroX-Tight-Pur-Luc vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.

Techniques Used: Chromatin Immunoprecipitation, Over Expression, Real-time Polymerase Chain Reaction, Binding Assay, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Transfection, Sequencing, Luciferase, Labeling, Activation Assay, Infection, CRISPR, Reverse Transcription Polymerase Chain Reaction

40) Product Images from "CEBPA-dependent HK3 and KLF5 expression in primary AML and during AML differentiation"

Article Title: CEBPA-dependent HK3 and KLF5 expression in primary AML and during AML differentiation

Journal: Scientific Reports

doi: 10.1038/srep04261

CEBPA binds to and activates the HK3 and the KLF5 promoters. Schematic representation of a 6 kb human HK3 (a) and a 5 kb human KLF5 (c) genomic regions retrieved from an online database. MatInspector software predicted two putative CEBPA binding sites (squares) in the DNA sequences analyzed. In vivo binding of CEBPA to these CEBPA consensus sites in the HK3 (b) or KLF5 (d) genomic regions was shown by chromatin immunoprecipitation (ChIP) in NB4 APL cells. As a negative control for the different pull downs, absence of GAPDH amplification is shown. *unspecific band, primer dimer. Two HK3 (e–f) and one KLF5 genomic region (g) containing the CEBPA binding sites were PCR amplified from genomic DNA of NB4 cells using proof reading Pfu DNA polymerase and cloned into the pGL4.10-basic vector. H1299 cells were transiently transfected with 40 ng of either HK3 promoter reporter construct A (e), construct A with mutated CEBPA binding site (f, wild-type GAAAGAC, mutated GGTCGAC) or the KLF5 promoter reporter construct (g), together with pcDNA3.1 empty vector or increasing concentrations (40–80–120 ng) (e,g) or 80 ng of CEBPA expression vector (f). The promoter activity is shown as relative light units (RLU) relative to pcDNA3.1 control transfected cells. Results are the means ± s.d. of at least triplicate transfections. MWU: **p
Figure Legend Snippet: CEBPA binds to and activates the HK3 and the KLF5 promoters. Schematic representation of a 6 kb human HK3 (a) and a 5 kb human KLF5 (c) genomic regions retrieved from an online database. MatInspector software predicted two putative CEBPA binding sites (squares) in the DNA sequences analyzed. In vivo binding of CEBPA to these CEBPA consensus sites in the HK3 (b) or KLF5 (d) genomic regions was shown by chromatin immunoprecipitation (ChIP) in NB4 APL cells. As a negative control for the different pull downs, absence of GAPDH amplification is shown. *unspecific band, primer dimer. Two HK3 (e–f) and one KLF5 genomic region (g) containing the CEBPA binding sites were PCR amplified from genomic DNA of NB4 cells using proof reading Pfu DNA polymerase and cloned into the pGL4.10-basic vector. H1299 cells were transiently transfected with 40 ng of either HK3 promoter reporter construct A (e), construct A with mutated CEBPA binding site (f, wild-type GAAAGAC, mutated GGTCGAC) or the KLF5 promoter reporter construct (g), together with pcDNA3.1 empty vector or increasing concentrations (40–80–120 ng) (e,g) or 80 ng of CEBPA expression vector (f). The promoter activity is shown as relative light units (RLU) relative to pcDNA3.1 control transfected cells. Results are the means ± s.d. of at least triplicate transfections. MWU: **p

Techniques Used: Software, Binding Assay, In Vivo, Chromatin Immunoprecipitation, Negative Control, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Transfection, Construct, Expressing, Activity Assay

41) Product Images from "Cortisol overproduction results from DNA methylation of CYP11B1 in hypercortisolemia"

Article Title: Cortisol overproduction results from DNA methylation of CYP11B1 in hypercortisolemia

Journal: Scientific Reports

doi: 10.1038/s41598-017-11435-2

DNA methylation suppresses CYP11B1 promoter activity. ( a ) Confirmation of CYP11B1 promoter activity. H295R cells were transiently transfected with pGL4.10[luc2] (control) or pGL4-cyp11b1[−302/+7](CYP11B1(−302/+7)). Two days after transfection, cells were lysed and luciferase activity was measured. The luciferase activity of the control sample was set to 1.0, and data are shown as the mean ± SEM (n = 6), and analyzed with the Mann-Whitney U test. ** P
Figure Legend Snippet: DNA methylation suppresses CYP11B1 promoter activity. ( a ) Confirmation of CYP11B1 promoter activity. H295R cells were transiently transfected with pGL4.10[luc2] (control) or pGL4-cyp11b1[−302/+7](CYP11B1(−302/+7)). Two days after transfection, cells were lysed and luciferase activity was measured. The luciferase activity of the control sample was set to 1.0, and data are shown as the mean ± SEM (n = 6), and analyzed with the Mann-Whitney U test. ** P

Techniques Used: DNA Methylation Assay, Activity Assay, Transfection, Luciferase, MANN-WHITNEY

42) Product Images from "Identification and Characterization of the Human Xylosyltransferase I Gene Promoter Region *"

Article Title: Identification and Characterization of the Human Xylosyltransferase I Gene Promoter Region *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.016592

Functional analysis of human XYLT1 promoter constructs. A , functional analysis of human XYLT1 promoter activity using 5′ deletion constructs in SW1353 cells. Bars show fold increase in luciferase activity for the XYLT1 promoter constructs cloned into pGL4.10 vector compared with promoterless pGL4.10 vector (negative control). The numbers on the left indicate the 5′ ends of the constructs relative to the translation initiation start site. The values are the mean ± S.E. of triplicates from at least three independent experiments. B , effect of 218-bp 3′ deletions on XYLT1 promoter activity for selected promoter constructs. C , comparison of the activities of the wild-type ( wt ) XYLT1 promoter fragment ranging from −797 to the translation start site (− 797 + 1 wt ) and the equivalent 218-bp 3′ truncated form (− 797 − 218 wt ) to constructs in which the AP-1 binding site is either mutated ( mut ) or deleted ( del ). The mutated bases are underlined and printed in bold letters ; the deletion is indicated by a gap .
Figure Legend Snippet: Functional analysis of human XYLT1 promoter constructs. A , functional analysis of human XYLT1 promoter activity using 5′ deletion constructs in SW1353 cells. Bars show fold increase in luciferase activity for the XYLT1 promoter constructs cloned into pGL4.10 vector compared with promoterless pGL4.10 vector (negative control). The numbers on the left indicate the 5′ ends of the constructs relative to the translation initiation start site. The values are the mean ± S.E. of triplicates from at least three independent experiments. B , effect of 218-bp 3′ deletions on XYLT1 promoter activity for selected promoter constructs. C , comparison of the activities of the wild-type ( wt ) XYLT1 promoter fragment ranging from −797 to the translation start site (− 797 + 1 wt ) and the equivalent 218-bp 3′ truncated form (− 797 − 218 wt ) to constructs in which the AP-1 binding site is either mutated ( mut ) or deleted ( del ). The mutated bases are underlined and printed in bold letters ; the deletion is indicated by a gap .

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

43) Product Images from "Overexpression of CLC-3 is regulated by XRCC5 and is a poor prognostic biomarker for gastric cancer"

Article Title: Overexpression of CLC-3 is regulated by XRCC5 and is a poor prognostic biomarker for gastric cancer

Journal: Journal of Hematology & Oncology

doi: 10.1186/s13045-018-0660-y

The expression of CLC-3 was regulated at the transcriptional level by XRCC5 interacting with PARP1. a The binding of XRCC5 to the CLC-3 DNA in SGC-7901 cells was suppressed after XRCC5 knockdown. b Knockdown of XRCC5 impaired the promoter activities of the pGL4.10-CLC-3 − 248 and pGL4.10-CLC-3 − 538 reporter plasmids in SGC-7901 cells ( n = 3). c The RNA level of CLC-3 was reduced after XRCC5 knockdown and increased after XRCC5 overexpression ( n = 3). d Knockdown of XRCC5 decreased the levels of key targets in the PI3K/Akt signaling pathway by downregulating CLC-3 in SGC-7901 cells. e – g PARP1 was identified as an interaction partner of XRCC5 by IP and MS in nuclear protein extracts of SGC-7901 cells. h The interaction between XRCC5 and PARP1 was confirmed by Co-IP in stable SGC-7901 cells with XRCC5 overexpression. i Binding between PARP1 and the CLC-3 promoter was detected by WB in the nuclear protein/DNA complex using synthesized probe or NSP. j The co-localization of XRCC5 and PARP1 was observed in the nucleus by confocal microscopy analysis. k Overexpression of XRCC5 increased the expression of CLC-3 in SGC-7901 cells, and the increase effect could be reversed by the PARP1 knockdown. l , m The proliferation and clonogenicity of SGC-7901 cells were promoted by XRCC5 overexpression, and the promotion effects were reversed by PARP1 knockdown ( n = 3). * P
Figure Legend Snippet: The expression of CLC-3 was regulated at the transcriptional level by XRCC5 interacting with PARP1. a The binding of XRCC5 to the CLC-3 DNA in SGC-7901 cells was suppressed after XRCC5 knockdown. b Knockdown of XRCC5 impaired the promoter activities of the pGL4.10-CLC-3 − 248 and pGL4.10-CLC-3 − 538 reporter plasmids in SGC-7901 cells ( n = 3). c The RNA level of CLC-3 was reduced after XRCC5 knockdown and increased after XRCC5 overexpression ( n = 3). d Knockdown of XRCC5 decreased the levels of key targets in the PI3K/Akt signaling pathway by downregulating CLC-3 in SGC-7901 cells. e – g PARP1 was identified as an interaction partner of XRCC5 by IP and MS in nuclear protein extracts of SGC-7901 cells. h The interaction between XRCC5 and PARP1 was confirmed by Co-IP in stable SGC-7901 cells with XRCC5 overexpression. i Binding between PARP1 and the CLC-3 promoter was detected by WB in the nuclear protein/DNA complex using synthesized probe or NSP. j The co-localization of XRCC5 and PARP1 was observed in the nucleus by confocal microscopy analysis. k Overexpression of XRCC5 increased the expression of CLC-3 in SGC-7901 cells, and the increase effect could be reversed by the PARP1 knockdown. l , m The proliferation and clonogenicity of SGC-7901 cells were promoted by XRCC5 overexpression, and the promotion effects were reversed by PARP1 knockdown ( n = 3). * P

Techniques Used: Expressing, Binding Assay, Over Expression, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot, Synthesized, Confocal Microscopy

44) Product Images from "Tumor Necrosis Factor ?-Mediated Induction of Interleukin 17C in Human Keratinocytes Is Controlled by Nuclear Factor ?B"

Article Title: Tumor Necrosis Factor ?-Mediated Induction of Interleukin 17C in Human Keratinocytes Is Controlled by Nuclear Factor ?B

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.240671

IL-17C promoter activation by TNFα is mediated by a NF-κB-dependent mechanism. Cultured normal human keratinocytes were transfected with wild-type (IL-17C-2-3204-luc2) IL-17C-promoter-luciferase plasmids together with an internal control
Figure Legend Snippet: IL-17C promoter activation by TNFα is mediated by a NF-κB-dependent mechanism. Cultured normal human keratinocytes were transfected with wild-type (IL-17C-2-3204-luc2) IL-17C-promoter-luciferase plasmids together with an internal control

Techniques Used: Activation Assay, Cell Culture, Transfection, Luciferase

45) Product Images from "Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells"

Article Title: Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells

Journal: Scientific Reports

doi: 10.1038/s41598-019-38809-y

Luciferase assay of Kaiso regulatory region of the Ctse gene. ( A ) Nucleotide sequences of CGCG motif in Kaiso regulatory region expanding chromosome 1: 131641487–131642069, and AGGAG motif in PU.1 promoter region expanding chromosome 1: 131661764–131662059 in prepared pGL4.10 [ luc2 ] Vector constructs. ( B ) pGL4.10 [ luc2 ] Vector constructs (1 μg) were transfected into EL4 cells with pRL-TK plasmid and Firefly/Renilla ratio of pGL4.10 (empty vector) was set as 1.0. MRL/MRL construct showed significantly higher luciferase activity compared with pGL4.10-B6/B6 construct (***p
Figure Legend Snippet: Luciferase assay of Kaiso regulatory region of the Ctse gene. ( A ) Nucleotide sequences of CGCG motif in Kaiso regulatory region expanding chromosome 1: 131641487–131642069, and AGGAG motif in PU.1 promoter region expanding chromosome 1: 131661764–131662059 in prepared pGL4.10 [ luc2 ] Vector constructs. ( B ) pGL4.10 [ luc2 ] Vector constructs (1 μg) were transfected into EL4 cells with pRL-TK plasmid and Firefly/Renilla ratio of pGL4.10 (empty vector) was set as 1.0. MRL/MRL construct showed significantly higher luciferase activity compared with pGL4.10-B6/B6 construct (***p

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

46) Product Images from "REGULATION OF EXPRESSION OF STROMAL-DERIVED FACTOR-1 (SDF-1) RECEPTORS: CXCR4 AND CXCR7 IN HUMAN RHADOMYOSARCOMAS"

Article Title: REGULATION OF EXPRESSION OF STROMAL-DERIVED FACTOR-1 (SDF-1) RECEPTORS: CXCR4 AND CXCR7 IN HUMAN RHADOMYOSARCOMAS

Journal: Molecular cancer research : MCR

doi: 10.1158/1541-7786.MCR-09-0259

CXCR4 promoter deletion studies and transcription binding sites analysis Panel A. Constructed CXCR4 promoter inserts. The promoter region of the CXCR4 gene from −2,237 to +62 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 was sequentially shortened according to the positions of PAX3 binding sites, and NRF-1 binding sites as depicted in the theoretical model of the cloned sequences of the promoter. The position of PAX3-, NRF-1- HRE-, NF-κb and YY1 binding sites as well as P1-3 primer pairs used in the ChIP experiment are shown. Panel B. CXCR4 promoter activity studies. RD and RD/PAX3-FKHR cells were transfected with the appropriate plasmids. Cultured cells were harvested after 24 hours and assayed for the amount of luciferase activity. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag1–7 p
Figure Legend Snippet: CXCR4 promoter deletion studies and transcription binding sites analysis Panel A. Constructed CXCR4 promoter inserts. The promoter region of the CXCR4 gene from −2,237 to +62 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 was sequentially shortened according to the positions of PAX3 binding sites, and NRF-1 binding sites as depicted in the theoretical model of the cloned sequences of the promoter. The position of PAX3-, NRF-1- HRE-, NF-κb and YY1 binding sites as well as P1-3 primer pairs used in the ChIP experiment are shown. Panel B. CXCR4 promoter activity studies. RD and RD/PAX3-FKHR cells were transfected with the appropriate plasmids. Cultured cells were harvested after 24 hours and assayed for the amount of luciferase activity. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag1–7 p

Techniques Used: Binding Assay, Construct, Clone Assay, Chromatin Immunoprecipitation, Activity Assay, Transfection, Cell Culture, Luciferase, Plasmid Preparation

CXCR7 promoter deletion studies and transcription binding sites analysis Panel A. CXCR7 promoter inserts. The promoter region of the CXCR7 gene from −2,409 to +89 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 shorter derivatives were obtained according to the positions of NRF-1, HRE binding sites, and NF-κB binding sites. The individual constructs were cloned to pGL4.10 vector, transfected into either RD or RD/PAX3-FKHR cells, and used in Dual Luciferase assays. (X) – mutated NF-κB and HRE binding sites. Primer pairs used in the ChIP experiment (N1–N5) flanked 5 different potential binding sites for NF-κB transcription factor. Panel B. NF-κB as a crucial transcription factor that drives CXCR7 promoter activity. Activity of particular CXCR7 promoter constructs was assayed by Dual Luciferase showing an important role of NF-κB transcription factors. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag 1–6 p
Figure Legend Snippet: CXCR7 promoter deletion studies and transcription binding sites analysis Panel A. CXCR7 promoter inserts. The promoter region of the CXCR7 gene from −2,409 to +89 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 shorter derivatives were obtained according to the positions of NRF-1, HRE binding sites, and NF-κB binding sites. The individual constructs were cloned to pGL4.10 vector, transfected into either RD or RD/PAX3-FKHR cells, and used in Dual Luciferase assays. (X) – mutated NF-κB and HRE binding sites. Primer pairs used in the ChIP experiment (N1–N5) flanked 5 different potential binding sites for NF-κB transcription factor. Panel B. NF-κB as a crucial transcription factor that drives CXCR7 promoter activity. Activity of particular CXCR7 promoter constructs was assayed by Dual Luciferase showing an important role of NF-κB transcription factors. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag 1–6 p

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

47) Product Images from "Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells"

Article Title: Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.221

Expression and stability of Fra-2 phospho-mutants in myogenic cells. ( a ) Western blot analysis showing the expression of Fra-2 (wild-type), Fra-2 DEF and mutated proteins: Fra-2 S120A, S230A, S320A, Fra-2 S120A, S200A, S230A, Fra-2 S120A, S200A, S230A, S320A and T322A. ( b ) Western blot analysis showing expression of Fra-2 (wild-type), Fra-2 DEF, Fra-2 S320A, Fra-2 T322A, Fra-2 S320A/T322A, Fra-2 S320D and Fra-2 T322D in myogenic cells. ( c ) C2C12 cells treated with MG132 (5 μ M) for 5 h. Cells were harvested and expression of wild-type and mutated Fra-2 were analyzed by western blotting. Actin was used as a loading control and dsRed was used as a marking of transfection efficiency. ( d ) Limited proteolytic digestion of GST-Fra-2. An in vitro kinase assay using 10 μ g of GST-Fra-2 and 20 ng of activated ERK 2 was performed. The reaction was divided into seven tubes and trypsin was added at a ratio of 100 : 1 and incubated for the indicated times. Samples were run on a 10% SDS-PAGE and Fra-2 was detected by western blotting. ( e ) C2C12 cells were transfected with wild-type Fra-2, Fra-2 S320D or T322D, and a myogenin promoter reporter gene (pGL4-10- myogenin -luc). Cells were maintained in DM conditions for 24 h before harvesting. Luciferase values were normalized to renilla
Figure Legend Snippet: Expression and stability of Fra-2 phospho-mutants in myogenic cells. ( a ) Western blot analysis showing the expression of Fra-2 (wild-type), Fra-2 DEF and mutated proteins: Fra-2 S120A, S230A, S320A, Fra-2 S120A, S200A, S230A, Fra-2 S120A, S200A, S230A, S320A and T322A. ( b ) Western blot analysis showing expression of Fra-2 (wild-type), Fra-2 DEF, Fra-2 S320A, Fra-2 T322A, Fra-2 S320A/T322A, Fra-2 S320D and Fra-2 T322D in myogenic cells. ( c ) C2C12 cells treated with MG132 (5 μ M) for 5 h. Cells were harvested and expression of wild-type and mutated Fra-2 were analyzed by western blotting. Actin was used as a loading control and dsRed was used as a marking of transfection efficiency. ( d ) Limited proteolytic digestion of GST-Fra-2. An in vitro kinase assay using 10 μ g of GST-Fra-2 and 20 ng of activated ERK 2 was performed. The reaction was divided into seven tubes and trypsin was added at a ratio of 100 : 1 and incubated for the indicated times. Samples were run on a 10% SDS-PAGE and Fra-2 was detected by western blotting. ( e ) C2C12 cells were transfected with wild-type Fra-2, Fra-2 S320D or T322D, and a myogenin promoter reporter gene (pGL4-10- myogenin -luc). Cells were maintained in DM conditions for 24 h before harvesting. Luciferase values were normalized to renilla

Techniques Used: Expressing, Western Blot, Transfection, In Vitro, Kinase Assay, Incubation, SDS Page, Luciferase

48) Product Images from "REGULATION OF EXPRESSION OF STROMAL-DERIVED FACTOR-1 (SDF-1) RECEPTORS: CXCR4 AND CXCR7 IN HUMAN RHADOMYOSARCOMAS"

Article Title: REGULATION OF EXPRESSION OF STROMAL-DERIVED FACTOR-1 (SDF-1) RECEPTORS: CXCR4 AND CXCR7 IN HUMAN RHADOMYOSARCOMAS

Journal: Molecular cancer research : MCR

doi: 10.1158/1541-7786.MCR-09-0259

CXCR4 promoter deletion studies and transcription binding sites analysis Panel A. Constructed CXCR4 promoter inserts. The promoter region of the CXCR4 gene from −2,237 to +62 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 was sequentially shortened according to the positions of PAX3 binding sites, and NRF-1 binding sites as depicted in the theoretical model of the cloned sequences of the promoter. The position of PAX3-, NRF-1- HRE-, NF-κb and YY1 binding sites as well as P1-3 primer pairs used in the ChIP experiment are shown. Panel B. CXCR4 promoter activity studies. RD and RD/PAX3-FKHR cells were transfected with the appropriate plasmids. Cultured cells were harvested after 24 hours and assayed for the amount of luciferase activity. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag1–7 p
Figure Legend Snippet: CXCR4 promoter deletion studies and transcription binding sites analysis Panel A. Constructed CXCR4 promoter inserts. The promoter region of the CXCR4 gene from −2,237 to +62 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 was sequentially shortened according to the positions of PAX3 binding sites, and NRF-1 binding sites as depicted in the theoretical model of the cloned sequences of the promoter. The position of PAX3-, NRF-1- HRE-, NF-κb and YY1 binding sites as well as P1-3 primer pairs used in the ChIP experiment are shown. Panel B. CXCR4 promoter activity studies. RD and RD/PAX3-FKHR cells were transfected with the appropriate plasmids. Cultured cells were harvested after 24 hours and assayed for the amount of luciferase activity. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag1–7 p

Techniques Used: Binding Assay, Construct, Clone Assay, Chromatin Immunoprecipitation, Activity Assay, Transfection, Cell Culture, Luciferase, Plasmid Preparation

CXCR7 promoter deletion studies and transcription binding sites analysis Panel A. CXCR7 promoter inserts. The promoter region of the CXCR7 gene from −2,409 to +89 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 shorter derivatives were obtained according to the positions of NRF-1, HRE binding sites, and NF-κB binding sites. The individual constructs were cloned to pGL4.10 vector, transfected into either RD or RD/PAX3-FKHR cells, and used in Dual Luciferase assays. (X) – mutated NF-κB and HRE binding sites. Primer pairs used in the ChIP experiment (N1–N5) flanked 5 different potential binding sites for NF-κB transcription factor. Panel B. NF-κB as a crucial transcription factor that drives CXCR7 promoter activity. Activity of particular CXCR7 promoter constructs was assayed by Dual Luciferase showing an important role of NF-κB transcription factors. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag 1–6 p
Figure Legend Snippet: CXCR7 promoter deletion studies and transcription binding sites analysis Panel A. CXCR7 promoter inserts. The promoter region of the CXCR7 gene from −2,409 to +89 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 shorter derivatives were obtained according to the positions of NRF-1, HRE binding sites, and NF-κB binding sites. The individual constructs were cloned to pGL4.10 vector, transfected into either RD or RD/PAX3-FKHR cells, and used in Dual Luciferase assays. (X) – mutated NF-κB and HRE binding sites. Primer pairs used in the ChIP experiment (N1–N5) flanked 5 different potential binding sites for NF-κB transcription factor. Panel B. NF-κB as a crucial transcription factor that drives CXCR7 promoter activity. Activity of particular CXCR7 promoter constructs was assayed by Dual Luciferase showing an important role of NF-κB transcription factors. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag 1–6 p

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

49) Product Images from "Characterization of the 5’-flanking region of the human TP53 gene and its response to the natural compound, Resveratrol"

Article Title: Characterization of the 5’-flanking region of the human TP53 gene and its response to the natural compound, Resveratrol

Journal: Journal of Biochemistry

doi: 10.1093/jb/mvv126

Effect of Rsv on human TP53 promoter activity. ( A–D ) (Left panel) The 5’-flanking region of the human TP53 gene, which has been ligated upstream of the Luciferase gene of the pGL4.10[ luc 2], is shown. The 5’-end of the cDNA is designated
Figure Legend Snippet: Effect of Rsv on human TP53 promoter activity. ( A–D ) (Left panel) The 5’-flanking region of the human TP53 gene, which has been ligated upstream of the Luciferase gene of the pGL4.10[ luc 2], is shown. The 5’-end of the cDNA is designated

Techniques Used: Activity Assay, Luciferase

50) Product Images from "REGULATION OF EXPRESSION OF STROMAL-DERIVED FACTOR-1 (SDF-1) RECEPTORS: CXCR4 AND CXCR7 IN HUMAN RHADOMYOSARCOMAS"

Article Title: REGULATION OF EXPRESSION OF STROMAL-DERIVED FACTOR-1 (SDF-1) RECEPTORS: CXCR4 AND CXCR7 IN HUMAN RHADOMYOSARCOMAS

Journal: Molecular cancer research : MCR

doi: 10.1158/1541-7786.MCR-09-0259

CXCR4 promoter deletion studies and transcription binding sites analysis Panel A. Constructed CXCR4 promoter inserts. The promoter region of the CXCR4 gene from −2,237 to +62 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 was sequentially shortened according to the positions of PAX3 binding sites, and NRF-1 binding sites as depicted in the theoretical model of the cloned sequences of the promoter. The position of PAX3-, NRF-1- HRE-, NF-κb and YY1 binding sites as well as P1-3 primer pairs used in the ChIP experiment are shown. Panel B. CXCR4 promoter activity studies. RD and RD/PAX3-FKHR cells were transfected with the appropriate plasmids. Cultured cells were harvested after 24 hours and assayed for the amount of luciferase activity. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag1–7 p
Figure Legend Snippet: CXCR4 promoter deletion studies and transcription binding sites analysis Panel A. Constructed CXCR4 promoter inserts. The promoter region of the CXCR4 gene from −2,237 to +62 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 was sequentially shortened according to the positions of PAX3 binding sites, and NRF-1 binding sites as depicted in the theoretical model of the cloned sequences of the promoter. The position of PAX3-, NRF-1- HRE-, NF-κb and YY1 binding sites as well as P1-3 primer pairs used in the ChIP experiment are shown. Panel B. CXCR4 promoter activity studies. RD and RD/PAX3-FKHR cells were transfected with the appropriate plasmids. Cultured cells were harvested after 24 hours and assayed for the amount of luciferase activity. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag1–7 p

Techniques Used: Binding Assay, Construct, Clone Assay, Chromatin Immunoprecipitation, Activity Assay, Transfection, Cell Culture, Luciferase, Plasmid Preparation

CXCR7 promoter deletion studies and transcription binding sites analysis Panel A. CXCR7 promoter inserts. The promoter region of the CXCR7 gene from −2,409 to +89 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 shorter derivatives were obtained according to the positions of NRF-1, HRE binding sites, and NF-κB binding sites. The individual constructs were cloned to pGL4.10 vector, transfected into either RD or RD/PAX3-FKHR cells, and used in Dual Luciferase assays. (X) – mutated NF-κB and HRE binding sites. Primer pairs used in the ChIP experiment (N1–N5) flanked 5 different potential binding sites for NF-κB transcription factor. Panel B. NF-κB as a crucial transcription factor that drives CXCR7 promoter activity. Activity of particular CXCR7 promoter constructs was assayed by Dual Luciferase showing an important role of NF-κB transcription factors. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag 1–6 p
Figure Legend Snippet: CXCR7 promoter deletion studies and transcription binding sites analysis Panel A. CXCR7 promoter inserts. The promoter region of the CXCR7 gene from −2,409 to +89 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 shorter derivatives were obtained according to the positions of NRF-1, HRE binding sites, and NF-κB binding sites. The individual constructs were cloned to pGL4.10 vector, transfected into either RD or RD/PAX3-FKHR cells, and used in Dual Luciferase assays. (X) – mutated NF-κB and HRE binding sites. Primer pairs used in the ChIP experiment (N1–N5) flanked 5 different potential binding sites for NF-κB transcription factor. Panel B. NF-κB as a crucial transcription factor that drives CXCR7 promoter activity. Activity of particular CXCR7 promoter constructs was assayed by Dual Luciferase showing an important role of NF-κB transcription factors. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag 1–6 p

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

51) Product Images from "A novel promoter controls Cyp19a1 gene expression in mouse adipose tissue"

Article Title: A novel promoter controls Cyp19a1 gene expression in mouse adipose tissue

Journal: Reproductive Biology and Endocrinology : RB & E

doi: 10.1186/1477-7827-7-37

Regulation of the adipose-specific promoter of the Cyp19a1 gene by dexamethasone (A) and Bt2cAMP plus PDA (B) . Luciferase plasmids containing the 5'-flanking promoter region of the mouse Cyp19a1 gene in gonadal fat (-343/-1 bp and -1637/-1 bp) were transfected into murine 3T3 L1 cells for 24 hours. pGL4.74 [hRluc] Renilla was used as an internal control for transfection efficiency. Promoter activity was normalized to pGL4.74 [hRluc] Renilla and was represented as the average of data from triplicate replicates; the empty luciferase vector PGL4.10 [ luc2 ] without treatment was arbitrarily assigned a unit of 1, and results are expressed as multiples of the PGL4.10 [ luc2 ] vector. All results are given as mean ± SE from at least 3 independent experiments performed in triplicate. *p
Figure Legend Snippet: Regulation of the adipose-specific promoter of the Cyp19a1 gene by dexamethasone (A) and Bt2cAMP plus PDA (B) . Luciferase plasmids containing the 5'-flanking promoter region of the mouse Cyp19a1 gene in gonadal fat (-343/-1 bp and -1637/-1 bp) were transfected into murine 3T3 L1 cells for 24 hours. pGL4.74 [hRluc] Renilla was used as an internal control for transfection efficiency. Promoter activity was normalized to pGL4.74 [hRluc] Renilla and was represented as the average of data from triplicate replicates; the empty luciferase vector PGL4.10 [ luc2 ] without treatment was arbitrarily assigned a unit of 1, and results are expressed as multiples of the PGL4.10 [ luc2 ] vector. All results are given as mean ± SE from at least 3 independent experiments performed in triplicate. *p

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

52) Product Images from "CaMKII Signaling Stimulates Mef2c Activity In Vitro but Only Minimally Affects Murine Long Bone Development in vivo"

Article Title: CaMKII Signaling Stimulates Mef2c Activity In Vitro but Only Minimally Affects Murine Long Bone Development in vivo

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2017.00020

CaMKII activity translocates β-catenin into the nucleus and regulates Runx2 and Mef2c activity in a dose dependent manner. (A) qPCR showing the expression levels of the four Camk2 isoforms, α, β, γ, δ, in chondrocytes isolated from E12.5 skeletal elements. Expression levels were normalized to Gapdh and Actb and ploted relative to the expression of CaMKIIγ. n = 3. Error bars indicate ±SEM. (B) Epiphyseal chondrocytes from P6 wild-type limbs were transfected with an expression vector encoding DaCaMKII::eGFP or eGFP (control). Nuclear localization of β-catenin (white arrows) was observed in DaCaMKII transfected cells. (C) Primary chondrocytes from E13.5 appendicular skeletal elements were co-transfected with 6X Ose2 luciferase (OSE2-luc) reporter and TK-renilla reporter plus increasing amounts of the expression vector encoding DaCaMKII or rKIIN. Ratios of luciferase activity with respect to control (Ctrl, black bar) are plotted as bar charts: DaCaMKII (green bars) and rKIIN (red bars). (D) Relative Runx2 expression levels, determined by qPCR, in primary chondrocytes from E13.5 appendicular skeletal elements co-transfected with increasing amounts of an expression vector encoding DaCaMKII or rKIIN used for the OSE2-Luc assay. (E) Primary chondrocytes from E13.5 wild-type appendicular skeletal elements were co-transfected with 3X Mef2 luciferase reporter (MEF2-Luc), TK-renilla reporter, and increasing amounts of the expression vector encoding DaCaMKII or rKIIN. Bar graph showing the ratio of luciferase activity with respect to control (Ctrl, black bar), DaCaMKII (green bars), and rKIIN (red bars) from four independent transfection experiments. The immunoblot below shows the corresponding increase in CaMKII levels. (F) Relative Mef2c expression levels, determined by qPCR, in primary chondrocytes from MEF2-luc assays co-transfected with increasing amounts of expression vector encoding DaCaMKII or rKIIN. * p
Figure Legend Snippet: CaMKII activity translocates β-catenin into the nucleus and regulates Runx2 and Mef2c activity in a dose dependent manner. (A) qPCR showing the expression levels of the four Camk2 isoforms, α, β, γ, δ, in chondrocytes isolated from E12.5 skeletal elements. Expression levels were normalized to Gapdh and Actb and ploted relative to the expression of CaMKIIγ. n = 3. Error bars indicate ±SEM. (B) Epiphyseal chondrocytes from P6 wild-type limbs were transfected with an expression vector encoding DaCaMKII::eGFP or eGFP (control). Nuclear localization of β-catenin (white arrows) was observed in DaCaMKII transfected cells. (C) Primary chondrocytes from E13.5 appendicular skeletal elements were co-transfected with 6X Ose2 luciferase (OSE2-luc) reporter and TK-renilla reporter plus increasing amounts of the expression vector encoding DaCaMKII or rKIIN. Ratios of luciferase activity with respect to control (Ctrl, black bar) are plotted as bar charts: DaCaMKII (green bars) and rKIIN (red bars). (D) Relative Runx2 expression levels, determined by qPCR, in primary chondrocytes from E13.5 appendicular skeletal elements co-transfected with increasing amounts of an expression vector encoding DaCaMKII or rKIIN used for the OSE2-Luc assay. (E) Primary chondrocytes from E13.5 wild-type appendicular skeletal elements were co-transfected with 3X Mef2 luciferase reporter (MEF2-Luc), TK-renilla reporter, and increasing amounts of the expression vector encoding DaCaMKII or rKIIN. Bar graph showing the ratio of luciferase activity with respect to control (Ctrl, black bar), DaCaMKII (green bars), and rKIIN (red bars) from four independent transfection experiments. The immunoblot below shows the corresponding increase in CaMKII levels. (F) Relative Mef2c expression levels, determined by qPCR, in primary chondrocytes from MEF2-luc assays co-transfected with increasing amounts of expression vector encoding DaCaMKII or rKIIN. * p

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

53) Product Images from "Identification and characterization of a FOXA2-regulated transcriptional enhancer at a type 2 diabetes intronic locus that controls GCKR expression in liver cells"

Article Title: Identification and characterization of a FOXA2-regulated transcriptional enhancer at a type 2 diabetes intronic locus that controls GCKR expression in liver cells

Journal: Genome Medicine

doi: 10.1186/s13073-017-0453-x

Haplotype-specific transcriptional activity. Luciferase reporter assays. Firefly luciferase activity was normalized to the Renilla luciferase signal and the data are shown as proportions of control vector pGL4.10-TK. a HepG2 cells with 638-bp constructs. Right panel : FOXA2 induced a haplotype-specific transcriptional activity (CGG > TAC) compared to the control pGL4.10-TK. Error bars represent the standard deviation of five experiments ( n = 5) with three, three, eight, four, and four technical replicates (*** p ≤ 0.005; two-tailed t -test). b HepG2 cells with 3-kb constructs. Right panel : FOXA2 induced the transcriptional activity of the inserts in a haplotype-specific way (CGG > TAC) compared to the control pGL4.10-TK. Error bars represent the standard deviation of six experiments ( n = 6) with four, four, eight, eight, four, and four technical replicates (*** p ≤ 0.005; two-tailed t -test). c MPHs with 3-kb constructs. Right panel : insulin reduced FOXA2-induced transcriptional activity in MPHs. Error bars represent standard deviation of four biological replicates corresponding to four livers ( n = 4) with two technical replicates each (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.005; t -test for comparison between haplotypes within treatment; one-way ANOVA for comparisons between the treatments). a – c Left panels : in co-transfection with the pCMV6-XL5 vector without a TF insert, none of the haplotypes show transcriptional activity with respect to the control plasmid pGL4.10-TK. d HepG2 cells with 3-kb constructs. Left panel : in co-transfection with MAFK and pCMV6-XL5, none of the haplotypes show transcriptional activity. Right panel : co-transfection of FOXA2 and MAFK induced transcriptional activity from both 3-kb regions but inverted the haplotype bias compared to FOXA2 alone (TAC > CGG). Error bars represent standard deviation of six experiments ( n = 6) with six, six, six, six, four, and four technical replicates (*** p ≤ 0.005; two-tailed t -test)
Figure Legend Snippet: Haplotype-specific transcriptional activity. Luciferase reporter assays. Firefly luciferase activity was normalized to the Renilla luciferase signal and the data are shown as proportions of control vector pGL4.10-TK. a HepG2 cells with 638-bp constructs. Right panel : FOXA2 induced a haplotype-specific transcriptional activity (CGG > TAC) compared to the control pGL4.10-TK. Error bars represent the standard deviation of five experiments ( n = 5) with three, three, eight, four, and four technical replicates (*** p ≤ 0.005; two-tailed t -test). b HepG2 cells with 3-kb constructs. Right panel : FOXA2 induced the transcriptional activity of the inserts in a haplotype-specific way (CGG > TAC) compared to the control pGL4.10-TK. Error bars represent the standard deviation of six experiments ( n = 6) with four, four, eight, eight, four, and four technical replicates (*** p ≤ 0.005; two-tailed t -test). c MPHs with 3-kb constructs. Right panel : insulin reduced FOXA2-induced transcriptional activity in MPHs. Error bars represent standard deviation of four biological replicates corresponding to four livers ( n = 4) with two technical replicates each (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.005; t -test for comparison between haplotypes within treatment; one-way ANOVA for comparisons between the treatments). a – c Left panels : in co-transfection with the pCMV6-XL5 vector without a TF insert, none of the haplotypes show transcriptional activity with respect to the control plasmid pGL4.10-TK. d HepG2 cells with 3-kb constructs. Left panel : in co-transfection with MAFK and pCMV6-XL5, none of the haplotypes show transcriptional activity. Right panel : co-transfection of FOXA2 and MAFK induced transcriptional activity from both 3-kb regions but inverted the haplotype bias compared to FOXA2 alone (TAC > CGG). Error bars represent standard deviation of six experiments ( n = 6) with six, six, six, six, four, and four technical replicates (*** p ≤ 0.005; two-tailed t -test)

Techniques Used: Activity Assay, Luciferase, Plasmid Preparation, Construct, Standard Deviation, Two Tailed Test, Cotransfection

54) Product Images from "Dynamic Regulation of the Adenosine Kinase Gene during Early Postnatal Brain Development and Maturation"

Article Title: Dynamic Regulation of the Adenosine Kinase Gene during Early Postnatal Brain Development and Maturation

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2016.00099

Identification of transcription factor binding. (A) Upper panel: SP1 binding sites (indicated in red) at the amplified region of Adk 1d primers (indicated in blue) around the TSS (pos. +1, bold). Lower panel: Binding of SP1 to the promoter region of the long Adk isoform are shown relative to a positive control primer in P4 and P14 rat hippocampal neurons. The analyzed promoter region spanned over 1 kb between position −735 and +470 relative to the TSS. There is significant enrichment of SP1 binding around the TSS of the Adk-L promoter in P14 rat hippocampal neurons, which may be implicated in the suppression of the long Adk isoform. Inset displays enrichment of positive control for Sp1 antibody presented as percent of the total input chromatin (% input). (B) Luciferase assay was performed to analyze the binding of SP1 to the Adk promoter and its impact on gene expression. Therefore, the Adk-L promoter including 1.000 bp upstream the TSS was subcloned in front of luciferase 2 (luc2) gene in the pGL4.10 vector and transfected into HEK293 cells. Transfected cells were incubated with increasing concentrations of recombinant SP1 for 3 h and then used for luciferase activity measurements. (C) We identified a concentration dependent negative regulation of the Adk-L promoter and luc2 gene through transcription factor SP1. Adk, Adenosine kinase; Adk-L promoter, promoter regulating expression of the long Adk isoform; Ap3m1, adaptor-related protein complex 3, mu 1 subunit; ChIP, chromatin immunoprecipitation; CpG, cytosine guanine dinucleotide; DHFR, Dihydrofolate reductase; kb, kilobase; SP1, specificity protein 1;TSS, transcriptional start site. * indicates significance.
Figure Legend Snippet: Identification of transcription factor binding. (A) Upper panel: SP1 binding sites (indicated in red) at the amplified region of Adk 1d primers (indicated in blue) around the TSS (pos. +1, bold). Lower panel: Binding of SP1 to the promoter region of the long Adk isoform are shown relative to a positive control primer in P4 and P14 rat hippocampal neurons. The analyzed promoter region spanned over 1 kb between position −735 and +470 relative to the TSS. There is significant enrichment of SP1 binding around the TSS of the Adk-L promoter in P14 rat hippocampal neurons, which may be implicated in the suppression of the long Adk isoform. Inset displays enrichment of positive control for Sp1 antibody presented as percent of the total input chromatin (% input). (B) Luciferase assay was performed to analyze the binding of SP1 to the Adk promoter and its impact on gene expression. Therefore, the Adk-L promoter including 1.000 bp upstream the TSS was subcloned in front of luciferase 2 (luc2) gene in the pGL4.10 vector and transfected into HEK293 cells. Transfected cells were incubated with increasing concentrations of recombinant SP1 for 3 h and then used for luciferase activity measurements. (C) We identified a concentration dependent negative regulation of the Adk-L promoter and luc2 gene through transcription factor SP1. Adk, Adenosine kinase; Adk-L promoter, promoter regulating expression of the long Adk isoform; Ap3m1, adaptor-related protein complex 3, mu 1 subunit; ChIP, chromatin immunoprecipitation; CpG, cytosine guanine dinucleotide; DHFR, Dihydrofolate reductase; kb, kilobase; SP1, specificity protein 1;TSS, transcriptional start site. * indicates significance.

Techniques Used: Binding Assay, Amplification, Positive Control, Luciferase, Expressing, Plasmid Preparation, Transfection, Incubation, Recombinant, Activity Assay, Concentration Assay, Chromatin Immunoprecipitation

55) Product Images from "Wip1 promotes RUNX2-dependent apoptosis in p53-negative tumors and protects normal tissues during treatment with anticancer agents"

Article Title: Wip1 promotes RUNX2-dependent apoptosis in p53-negative tumors and protects normal tissues during treatment with anticancer agents

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

doi: 10.1073/pnas.1107017108

Role of RUNX2 transcriptional factor in Wip1-dependent cisplatin-induced apoptosis. ( A ) RUNX2 silencing abrogates Wip1-mediated cell death after cisplatin (CDDP) treatment in Saos-2-Wip1-on cells. Saos-2-Wip1-on cells were transfected with control scrambled siRNA or RUNX2 siRNA. Forty-eight hours after transfection, cells were treated with CDDP for 48 h, harvested, and subjected to Guava ViaCount cell death assay. ( B ) RUNX2 silencing abrogates Bax mRNA induction in Saos2-Wip1-on cells after CDDP treatment. Saos2-Wip1-on cells were transfected with control scrambled siRNA or RUNX2 siRNA. Thirty hours after transfection, Wip1 was induced by doxycycline. Forty-eight hours after transfection, cells were treated with cisplatin for 6 h and harvested, and mRNA was purified and subjected to quantitative PCR analysis. ( C ) Wip1 interacts with endogenous RUNX2. Saos-2-FLAG-Wip1-on cells were cultivated with or without doxycycline. Twenty-four hours later, cells were lysed and immunoprecipitated with FLAG M2 or RUNX2 antibody, and immunoblotted with FLAG, Wip1, or RUNX2 antibody as indicated. Whole-cell lysates were also immunoblotted. ( D ) Transactivation of the human Bax gene promoter by Runx2 mutants. Firefly luciferase reporter vectors contained a 1.2-kb fragment of the human bax gene promoter. Saos-2 cells were cotransfected with the reporter plasmid, a WT Runx2 or mutant Runx2 expression plasmid, and a renilla luciferase plasmid as a reference. The values shown are the means ± SE from three experiments. ( E ) Phosphatase activity of Wip1 on Runx2 phosphopeptides. Phosphate release by Wip1 with 0 to 400 μM 432pS (○), and 465pS (◆) phosphopeptides was measured at 30 °C. Wip1 activity was measured in the same conditions with 0 to 100 μM ATM-1981pS (▵) phosphopeptide as a positive control. The values shown represent the means ± SEM for A , B , and D , or means ± SD for E of three to four independent experiments (* P
Figure Legend Snippet: Role of RUNX2 transcriptional factor in Wip1-dependent cisplatin-induced apoptosis. ( A ) RUNX2 silencing abrogates Wip1-mediated cell death after cisplatin (CDDP) treatment in Saos-2-Wip1-on cells. Saos-2-Wip1-on cells were transfected with control scrambled siRNA or RUNX2 siRNA. Forty-eight hours after transfection, cells were treated with CDDP for 48 h, harvested, and subjected to Guava ViaCount cell death assay. ( B ) RUNX2 silencing abrogates Bax mRNA induction in Saos2-Wip1-on cells after CDDP treatment. Saos2-Wip1-on cells were transfected with control scrambled siRNA or RUNX2 siRNA. Thirty hours after transfection, Wip1 was induced by doxycycline. Forty-eight hours after transfection, cells were treated with cisplatin for 6 h and harvested, and mRNA was purified and subjected to quantitative PCR analysis. ( C ) Wip1 interacts with endogenous RUNX2. Saos-2-FLAG-Wip1-on cells were cultivated with or without doxycycline. Twenty-four hours later, cells were lysed and immunoprecipitated with FLAG M2 or RUNX2 antibody, and immunoblotted with FLAG, Wip1, or RUNX2 antibody as indicated. Whole-cell lysates were also immunoblotted. ( D ) Transactivation of the human Bax gene promoter by Runx2 mutants. Firefly luciferase reporter vectors contained a 1.2-kb fragment of the human bax gene promoter. Saos-2 cells were cotransfected with the reporter plasmid, a WT Runx2 or mutant Runx2 expression plasmid, and a renilla luciferase plasmid as a reference. The values shown are the means ± SE from three experiments. ( E ) Phosphatase activity of Wip1 on Runx2 phosphopeptides. Phosphate release by Wip1 with 0 to 400 μM 432pS (○), and 465pS (◆) phosphopeptides was measured at 30 °C. Wip1 activity was measured in the same conditions with 0 to 100 μM ATM-1981pS (▵) phosphopeptide as a positive control. The values shown represent the means ± SEM for A , B , and D , or means ± SD for E of three to four independent experiments (* P

Techniques Used: Transfection, Purification, Real-time Polymerase Chain Reaction, Immunoprecipitation, Luciferase, Plasmid Preparation, Mutagenesis, Expressing, Activity Assay, Positive Control

56) Product Images from "Epigenetic regulation of Runx2 transcription and osteoblast differentiation by nicotinamide phosphoribosyltransferase"

Article Title: Epigenetic regulation of Runx2 transcription and osteoblast differentiation by nicotinamide phosphoribosyltransferase

Journal: Cell & Bioscience

doi: 10.1186/s13578-017-0154-6

Regulative effects of Nampt on Runx2 transcription in MC3T3-E1 cell differentiation. MC3T3 cells were differentiated for 48 h and then co-transfected with pGL4.10-mRunX2pro firefly luciferase reporter (100 ng), pGL4.75 Renilla reporter (4 ng) and either 100 µM of scrambled RNA or Nampt shRNA using Lipofectamine 3000. Transfected MC3T3 cells were incubated for 24 h and luciferase activity was determined using Promega’s Dual-Glo Luciferase Assay Kit. Background corrected firefly luminescence values were normalized with Renilla luminescence values. Relative luciferase activities were normalized against differentiated MC3T3 cells transfected with pGL4.10 empty vector. n = 4; Bars are mean ± SD. **p
Figure Legend Snippet: Regulative effects of Nampt on Runx2 transcription in MC3T3-E1 cell differentiation. MC3T3 cells were differentiated for 48 h and then co-transfected with pGL4.10-mRunX2pro firefly luciferase reporter (100 ng), pGL4.75 Renilla reporter (4 ng) and either 100 µM of scrambled RNA or Nampt shRNA using Lipofectamine 3000. Transfected MC3T3 cells were incubated for 24 h and luciferase activity was determined using Promega’s Dual-Glo Luciferase Assay Kit. Background corrected firefly luminescence values were normalized with Renilla luminescence values. Relative luciferase activities were normalized against differentiated MC3T3 cells transfected with pGL4.10 empty vector. n = 4; Bars are mean ± SD. **p

Techniques Used: Cell Differentiation, Transfection, Luciferase, shRNA, Incubation, Activity Assay, Plasmid Preparation

57) Product Images from "c-Maf Transcription Factor Regulates ADAMTS-12 Expression in Human Chondrogenic Cells"

Article Title: c-Maf Transcription Factor Regulates ADAMTS-12 Expression in Human Chondrogenic Cells

Journal: Cartilage

doi: 10.1177/1947603512472697

The activity of ADAMTS-12 promoter is up-regulated by c-Maf. ( A ) Schematic diagram of the sequential 5′ deletions of ADAMTS-12 promoter region that were used to map c-Maf-response element. Ovals represent predicted putative Maf binding sites. Not drawn to scale. ( B ) Basal transcriptional activity of this 3-kb promoter was measured in three different cell lines using luciferase assays. Bars indicate fold change from pGL4.10 control vector. ( C ) The basal activities of each truncated ADAMTS-12 promoter were measured in the SW1353 chondrosarcoma cell line. ( D ) To measure the activation of the ADAMTS-12 promoter by c-Maf above basal levels, c-Maf or empty expression vector (v) were cotransfected with the various ADAMTS-12 promoter truncation constructs. Assays were run in triplicate and the average from three independent experiments is shown with standard deviation. (*) Indicates statistically significant difference in luciferase activity relative to the respective controls, with p
Figure Legend Snippet: The activity of ADAMTS-12 promoter is up-regulated by c-Maf. ( A ) Schematic diagram of the sequential 5′ deletions of ADAMTS-12 promoter region that were used to map c-Maf-response element. Ovals represent predicted putative Maf binding sites. Not drawn to scale. ( B ) Basal transcriptional activity of this 3-kb promoter was measured in three different cell lines using luciferase assays. Bars indicate fold change from pGL4.10 control vector. ( C ) The basal activities of each truncated ADAMTS-12 promoter were measured in the SW1353 chondrosarcoma cell line. ( D ) To measure the activation of the ADAMTS-12 promoter by c-Maf above basal levels, c-Maf or empty expression vector (v) were cotransfected with the various ADAMTS-12 promoter truncation constructs. Assays were run in triplicate and the average from three independent experiments is shown with standard deviation. (*) Indicates statistically significant difference in luciferase activity relative to the respective controls, with p

Techniques Used: Activity Assay, Binding Assay, Luciferase, Plasmid Preparation, Activation Assay, Expressing, Construct, Standard Deviation

58) Product Images from "Polo-like kinase 4 transcription is activated via CRE and NRF1 elements, repressed by DREAM through CDE/CHR sites and deregulated by HPV E7 protein"

Article Title: Polo-like kinase 4 transcription is activated via CRE and NRF1 elements, repressed by DREAM through CDE/CHR sites and deregulated by HPV E7 protein

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt849

The DREAM complex components bind through the CDE and CHR and not through NRF1 or CRE sites to the Plk4 promoter. ( A ) Nuclear extracts from density-arrested NIH3T3 cells were analyzed by DNA affinity purification with wt, CRE/NRF1, CDE or CHR mutant Plk4 promoter probes followed by western blot. An irrelevant segment from the pGL4.10 vector served as negative control. Band intensities were quantified by densitometric analyses. The intensities relative to the control are given below the bands. ( B ) In vivo protein binding to the Plk4 promoter in serum-starved and restimulated T98G cells was tested by ChIP.
Figure Legend Snippet: The DREAM complex components bind through the CDE and CHR and not through NRF1 or CRE sites to the Plk4 promoter. ( A ) Nuclear extracts from density-arrested NIH3T3 cells were analyzed by DNA affinity purification with wt, CRE/NRF1, CDE or CHR mutant Plk4 promoter probes followed by western blot. An irrelevant segment from the pGL4.10 vector served as negative control. Band intensities were quantified by densitometric analyses. The intensities relative to the control are given below the bands. ( B ) In vivo protein binding to the Plk4 promoter in serum-starved and restimulated T98G cells was tested by ChIP.

Techniques Used: Affinity Purification, Mutagenesis, Western Blot, Plasmid Preparation, Negative Control, In Vivo, Protein Binding, Chromatin Immunoprecipitation

59) Product Images from "Identification of G-quadruplex structures that possess transcriptional regulating functions in the Dele and Cdc6 CpG islands"

Article Title: Identification of G-quadruplex structures that possess transcriptional regulating functions in the Dele and Cdc6 CpG islands

Journal: BMC Molecular Biology

doi: 10.1186/s12867-017-0094-z

Reporter assay for evaluation of the promoter activities of Cdc6 , Dele -F, and Dele -R G4 DNAs. The G4-forming sequences were cloned into the pGL4.10 vector not containing any promoter. Black bars represent the wild-types and white bars represent the mutant-types. Luciferase activities relative to the pGL4.10 vector are shown (mean ± SD, n = 3). Wild- and mutant-types samples t-test differences: ***P
Figure Legend Snippet: Reporter assay for evaluation of the promoter activities of Cdc6 , Dele -F, and Dele -R G4 DNAs. The G4-forming sequences were cloned into the pGL4.10 vector not containing any promoter. Black bars represent the wild-types and white bars represent the mutant-types. Luciferase activities relative to the pGL4.10 vector are shown (mean ± SD, n = 3). Wild- and mutant-types samples t-test differences: ***P

Techniques Used: Reporter Assay, Clone Assay, Plasmid Preparation, Mutagenesis, Luciferase

Reporter assay for evaluation of the transcriptional activity of Cdc6 , Dele -F, and Dele -R G4 DNAs in CGI sequences. Black bars represent the wild-types and white bars represent the mutant-types. Luciferase activity relative to the pGL4.10 is shown (mean ± SD, n = 3). Wild- and mutant-types samples t-test differences: *P
Figure Legend Snippet: Reporter assay for evaluation of the transcriptional activity of Cdc6 , Dele -F, and Dele -R G4 DNAs in CGI sequences. Black bars represent the wild-types and white bars represent the mutant-types. Luciferase activity relative to the pGL4.10 is shown (mean ± SD, n = 3). Wild- and mutant-types samples t-test differences: *P

Techniques Used: Reporter Assay, Activity Assay, Mutagenesis, Luciferase

60) Product Images from "Unbiased Mutagenesis of MHV68 LANA Reveals a DNA-Binding Domain Required for LANA Function In Vitro and In Vivo"

Article Title: Unbiased Mutagenesis of MHV68 LANA Reveals a DNA-Binding Domain Required for LANA Function In Vitro and In Vivo

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002906

Deletion analysis of TR reveals DNA sequence required for mLANA-mediated repression. (A) Schematic of pGL-TR, noting the numbering scheme with regard to the NotI site. Coordinate 1 is corresponds to MHV68 WUMS [31] bp 119,105, proceeding in order towards the right end of the unique sequence. Coordinate 894 is the end of one TR unit fused to the beginning of the next. Indicated on the diagram are two known sites of transcription initiation: (i) (73p1) starts transcription between coordinates 423–466 (118,683–118,640) depending on cell type [29] , [32] and the partial exon splices to the full 73E1 exon (118,695–118,605) in the next repeat unit; (ii) initiates transcription of one species of ORF75a beginning between coordinates 885–891 (118221–118216) [32] (promoter elements are likely shared between ORF75a and ORF73 transcripts that initiate at 73E2 [29] , and in distal copies may splice into 73E1). (B) Serial deletions of the TR were made in the pGL-TR vector by PCR and are named accordingly. Each TR deletion, including the full-length TR (FL) was co-transfected into 293T cells, along with either an mLANA-GFP or empty GFP expression vector, as in Fig. 1 , in triplicate. Here, data are normalized by taking the ratio of each deletion construct over its respective pGL4.10 empty reporter control, setting pGL4.10 in each case to 1. Deleting the 3′ end gives a large uptick in activity, which is still repressed by mLANA, until the second deletion removes mLANA sensitivity. 5′ deletions had no effect on mLANA sensitivity.
Figure Legend Snippet: Deletion analysis of TR reveals DNA sequence required for mLANA-mediated repression. (A) Schematic of pGL-TR, noting the numbering scheme with regard to the NotI site. Coordinate 1 is corresponds to MHV68 WUMS [31] bp 119,105, proceeding in order towards the right end of the unique sequence. Coordinate 894 is the end of one TR unit fused to the beginning of the next. Indicated on the diagram are two known sites of transcription initiation: (i) (73p1) starts transcription between coordinates 423–466 (118,683–118,640) depending on cell type [29] , [32] and the partial exon splices to the full 73E1 exon (118,695–118,605) in the next repeat unit; (ii) initiates transcription of one species of ORF75a beginning between coordinates 885–891 (118221–118216) [32] (promoter elements are likely shared between ORF75a and ORF73 transcripts that initiate at 73E2 [29] , and in distal copies may splice into 73E1). (B) Serial deletions of the TR were made in the pGL-TR vector by PCR and are named accordingly. Each TR deletion, including the full-length TR (FL) was co-transfected into 293T cells, along with either an mLANA-GFP or empty GFP expression vector, as in Fig. 1 , in triplicate. Here, data are normalized by taking the ratio of each deletion construct over its respective pGL4.10 empty reporter control, setting pGL4.10 in each case to 1. Deleting the 3′ end gives a large uptick in activity, which is still repressed by mLANA, until the second deletion removes mLANA sensitivity. 5′ deletions had no effect on mLANA sensitivity.

Techniques Used: Sequencing, Plasmid Preparation, Polymerase Chain Reaction, Transfection, Expressing, Construct, Activity Assay

61) Product Images from "Negative feedback loop between ZBTB7A and TGF-β in breast cancer"

Article Title: Negative feedback loop between ZBTB7A and TGF-β in breast cancer

Journal: Oncology Letters

doi: 10.3892/ol.2017.6291

ZBTB7A suppresses the promoter activity of TGF-β1 indirectly. (A) ZBTB7A inhibited the promoter activity of TGF-β1. MCF-7 and 293T cells were co-transfected with plasmids of ZBTB7A, PGL4.10-TGF-β1 PP1 or PGL4.10-TGF-β1 PP2, and Renilla . Subsequently, the cells were harvested and subjected to a dual-luciferase reporter assay. (B) Direct binding analysis between ZBTB7A and promoter sequences of TGF-β1 in MCF-7 cells. An electrophoretic mobility shift assay was performed. IgG was used as a negative control. Data are presented as the mean ± standard deviation following three independent experiments. ***P
Figure Legend Snippet: ZBTB7A suppresses the promoter activity of TGF-β1 indirectly. (A) ZBTB7A inhibited the promoter activity of TGF-β1. MCF-7 and 293T cells were co-transfected with plasmids of ZBTB7A, PGL4.10-TGF-β1 PP1 or PGL4.10-TGF-β1 PP2, and Renilla . Subsequently, the cells were harvested and subjected to a dual-luciferase reporter assay. (B) Direct binding analysis between ZBTB7A and promoter sequences of TGF-β1 in MCF-7 cells. An electrophoretic mobility shift assay was performed. IgG was used as a negative control. Data are presented as the mean ± standard deviation following three independent experiments. ***P

Techniques Used: Activity Assay, Transfection, Luciferase, Reporter Assay, Binding Assay, Electrophoretic Mobility Shift Assay, Negative Control, Standard Deviation

62) Product Images from "Toll-like receptor 9 (TLR9) polymorphism associated with symptomatic malaria: a cohort study"

Article Title: Toll-like receptor 9 (TLR9) polymorphism associated with symptomatic malaria: a cohort study

Journal: Malaria Journal

doi: 10.1186/1475-2875-11-168

Luciferase reporter activity of TLR9 (promoter-intron) plasmid constructs. a ), schematic of four reporter gene constructs that contains TLR9 promoter- intron region with TTA, CCG, CTG and TCG at position −1486, -1237, and +1174 polymorphic sites, respectively. b ), luciferase expression of the four constructs in THP-1 cells and pGL4.10 as negative control. The luciferase activity levels are mean values of three (3) independent experiments that were all carried out in triplicate. Anova test and t-test were used to test for statistical significance. P value of
Figure Legend Snippet: Luciferase reporter activity of TLR9 (promoter-intron) plasmid constructs. a ), schematic of four reporter gene constructs that contains TLR9 promoter- intron region with TTA, CCG, CTG and TCG at position −1486, -1237, and +1174 polymorphic sites, respectively. b ), luciferase expression of the four constructs in THP-1 cells and pGL4.10 as negative control. The luciferase activity levels are mean values of three (3) independent experiments that were all carried out in triplicate. Anova test and t-test were used to test for statistical significance. P value of

Techniques Used: Luciferase, Activity Assay, Plasmid Preparation, Construct, CTG Assay, Expressing, Negative Control

63) Product Images from "Transcriptional Regulation of the Tumor Suppressor FHL2 by p53 in Human Kidney and Liver Cells"

Article Title: Transcriptional Regulation of the Tumor Suppressor FHL2 by p53 in Human Kidney and Liver Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0099359

Schematic diagram of truncated 5′- flanking sequences of FHL2 exon 1a. These truncations were fused to the luciferase reporter gene in the pGL4.10-basic vector. Numbers indicate positions relative to the transcription start site (TSS, +1).
Figure Legend Snippet: Schematic diagram of truncated 5′- flanking sequences of FHL2 exon 1a. These truncations were fused to the luciferase reporter gene in the pGL4.10-basic vector. Numbers indicate positions relative to the transcription start site (TSS, +1).

Techniques Used: Luciferase, Plasmid Preparation

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Clone Assay:

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Luciferase:

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Reporter Assay:

Article Title: Long non‐coding RNA NNT‐ AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer, et al. Long non‐coding RNA NNT‐AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer
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Article Snippet: Transfection and luciferase assays The Ascl2 5′-flanking sequence (-2588/+620 bp region), amplified using PCR from intestinal tissue DNA, was inserted into the luciferase reporter vector pGL3-Basic (Promega, Madison, WI, USA). .. Luciferase activity was measured 48 h post-transfection using the Dual-Luciferase Reporter Assay System (Promega) as described in our previous report [ ].

Article Title: Linc-POU3F3 is overexpressed in in-stent restenosis patients and induces VSMC phenotypic transformation via POU3F3/miR-449a/KLF4 signaling pathway
Article Snippet: Paragraph title: Luciferase reporter assay ... A fragment of KLF-4 3’-UTR and the mutated variants of the predicted miR-449a binding site on KLF-4 3’-UTR were PCR-amplified and fused to the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA).

Article Title: Long non‐coding RNA NNT‐ AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer, et al. Long non‐coding RNA NNT‐AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer
Article Snippet: Paragraph title: 2.7. Luciferase reporter assay ... For Luciferase assays, NNT‐AS1 wild type with potential miR‐424 binding sites or mutant sites was generated by Sangon Biotech (Shanghai, China), and then fused with the luciferase reporter vector psi‐CHECK‐2 (Promega, Madison, WI, USA).

Article Title: CtBP2 is an independent prognostic marker that promotes GLI1 induced epithelial-mesenchymal transition in hepatocellular carcinoma
Article Snippet: .. Luciferase reporter assay Four CtBP2 promoter fragments (−2000/−1 bp, −1307/−1 bp, −675/−1 bp and −1350/−652 bp from the transcription start site) were isolated by PCR amplification and ligated into the luciferase reporter vector pGL3-Basic from Promega (Fitchburg, WI, USA). .. Huh7 cells were plated on 24-well plates one day prior to the transfection.

Article Title: LncRNA ADAMTS9-AS2 inhibits cell proliferation and decreases chemoresistance in clear cell renal cell carcinoma via the miR-27a-3p/FOXO1 axis
Article Snippet: .. Dual-luciferase reporter assay A fragment of WT ADAMTS9-AS2 with potential miR-27a-3p binding sites or MUT ADAMTS9-AS2 with nonfunctional binding sites was generated and inserted into the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. The full-length WT 3′UTR, containing the predicted miR-27a-3p targeting site, and the MUT 3′UTR of FOXO1 were amplified and cloned into the psi-CHECK-2 vector.

Article Title: Long intergenic noncoding RNA 00707 promotes colorectal cancer cell proliferation and metastasis by sponging miR-206
Article Snippet: .. Luciferase reporter assay LINC00707 wild-type (WT) with potential miR-206-binding sites or mutant (MUT) of each sites were generated and fused to a luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA) to analyze the interaction between LINC00707 and miR-206. .. The binding site of miR-206 on LINC00707 was mutated into AGATAGGA.

Article Title: Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9
Article Snippet: All genomic fragments (Supplementary Table ) were amplified from human genomic DNA using Phusion polymerase (NEB) and cloned into the luciferase reporter vector pGL4.10 (Promega) containing the human beta-globin minimal promoter between bgIII and HindIII sites in the multiple cloning site. .. Briefly, cells were seeded at 80–90% confluency in 96 well plate for 2–4 h. Each well was transfected with a combination of enhancer reporter plasmid pgl4-beta-globin (75 ng) and transcription factors in pcDNA3.1 (100 ng) with 15 ng of Renilla luciferase pLR-TK (Promega) as a control using lipofecamine 2000 for 24 h. The cells were lysed using 1.25x passive lysis buffer (Promega) and luciferase was determined using Dual-Luciferase® Reporter Assay System (Promega) as previously described .

TA Cloning:

Article Title: Dopamine-2 Receptor Activation Suppresses PACAP Expression in Gonadotrophs
Article Snippet: A PCR-generated DNA fragment (−1218/+36) of the mouse PACAP promoter was cloned into the PSTBlue-1 Acceptor Vector (Novagen) by TA cloning. .. The PACAP promoter DNA was excised with the restriction enzymes Kpn I and Xho I and cloned into the luciferase reporter vector PGL-3 Basic (Promega Corp) in the correct orientation.

Construct:

Article Title: Dopamine-2 Receptor Activation Suppresses PACAP Expression in Gonadotrophs
Article Snippet: Paragraph title: DNA constructs and transfection. ... The PACAP promoter DNA was excised with the restriction enzymes Kpn I and Xho I and cloned into the luciferase reporter vector PGL-3 Basic (Promega Corp) in the correct orientation.

Article Title: Smad2 and PEA3 cooperatively regulate transcription of response gene to complement 32 in TGF-?-induced smooth muscle cell differentiation of neural crest cells
Article Snippet: RGC-32 promoter DNA were amplified from mouse genomic DNA (Promega) by PCR and cloned into luciferase reporter vector pGL3-basic (Promega) at the Kpn I/ Bgl II restriction site. .. Three RGC-32 promoter reporter plasmids were constructed as follows: p-2107/+844RGCluc contains the regulatory region from nucleotide (nt) −2107 to +844 bp of RGC-32 gene; p-2107RGCluc and p-1500RGCluc contain the regulatory regions from nt −2107 and −1500 to +22 bp of the RGC-32 gene, respectively.

Article Title: Structure and transcription of the Helicoverpa armigera densovirus (HaDV2) genome and its expression strategy in LD652 cells
Article Snippet: NS protein expression and subcellular localization To characterize the expression of HaDV2 NS proteins in insect cells, we constructed two plasmids. .. Firstly, the HaDV2 NS promoter was amplified by primers NSPF/NSPR (Additional file : Table S1), digested with restriction endonuclease Kpn I/Hind II and cloned into a luciferase reporter vector pGL-3 Basic (Promega).

Article Title: Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9
Article Snippet: Paragraph title: Reporter constructs and luciferase assays ... All genomic fragments (Supplementary Table ) were amplified from human genomic DNA using Phusion polymerase (NEB) and cloned into the luciferase reporter vector pGL4.10 (Promega) containing the human beta-globin minimal promoter between bgIII and HindIII sites in the multiple cloning site.

Amplification:

Article Title: The lncRNA CRNDE promotes colorectal cancer cell proliferation and chemoresistance via miR-181a-5p-mediated regulation of Wnt/β-catenin signaling
Article Snippet: Luciferase assay CRNDE wild type with potential miR-181a-5p binding sites or mutant of each sites were generated and fused to the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. The full-length wild-type (WT) 3′ untranslated region (UTR) containing the predicted miR-181a-5p targeting site, and mutant (MUT) 3′-UTR of β-catenin and TCF4 were amplified and cloned into the psi-CHECK-2 vector.

Article Title: Ascl2 activation by YAP1/KLF5 ensures the self-renewability of colon cancer progenitor cells
Article Snippet: .. Transfection and luciferase assays The Ascl2 5′-flanking sequence (-2588/+620 bp region), amplified using PCR from intestinal tissue DNA, was inserted into the luciferase reporter vector pGL3-Basic (Promega, Madison, WI, USA). ..

Article Title: Pu-erh Tea Protects the Nervous System by Inhibiting the Expression of Metabotropic Glutamate Receptor 5
Article Snippet: .. Luciferase Assay Promoter regions were amplified by PCR amplification and subcloned into the KpnI/HindIII sites of the luciferase reporter vector pGL3-Basic (Promega) upstream of the firefly luciferase gene (Fig. ). .. SH-SY5Y and HEK293T cells were transfected with mGluR5 promoter-pGL3 recombinant plasmid by electroporation according to the manufacturer’s instructions.

Article Title: Smad2 and PEA3 cooperatively regulate transcription of response gene to complement 32 in TGF-?-induced smooth muscle cell differentiation of neural crest cells
Article Snippet: .. RGC-32 promoter DNA were amplified from mouse genomic DNA (Promega) by PCR and cloned into luciferase reporter vector pGL3-basic (Promega) at the Kpn I/ Bgl II restriction site. .. Three RGC-32 promoter reporter plasmids were constructed as follows: p-2107/+844RGCluc contains the regulatory region from nucleotide (nt) −2107 to +844 bp of RGC-32 gene; p-2107RGCluc and p-1500RGCluc contain the regulatory regions from nt −2107 and −1500 to +22 bp of the RGC-32 gene, respectively.

Article Title: CtBP2 is an independent prognostic marker that promotes GLI1 induced epithelial-mesenchymal transition in hepatocellular carcinoma
Article Snippet: .. Luciferase reporter assay Four CtBP2 promoter fragments (−2000/−1 bp, −1307/−1 bp, −675/−1 bp and −1350/−652 bp from the transcription start site) were isolated by PCR amplification and ligated into the luciferase reporter vector pGL3-Basic from Promega (Fitchburg, WI, USA). .. Huh7 cells were plated on 24-well plates one day prior to the transfection.

Article Title: LncRNA ADAMTS9-AS2 inhibits cell proliferation and decreases chemoresistance in clear cell renal cell carcinoma via the miR-27a-3p/FOXO1 axis
Article Snippet: Dual-luciferase reporter assay A fragment of WT ADAMTS9-AS2 with potential miR-27a-3p binding sites or MUT ADAMTS9-AS2 with nonfunctional binding sites was generated and inserted into the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. The full-length WT 3′UTR, containing the predicted miR-27a-3p targeting site, and the MUT 3′UTR of FOXO1 were amplified and cloned into the psi-CHECK-2 vector.

Article Title: Structure and transcription of the Helicoverpa armigera densovirus (HaDV2) genome and its expression strategy in LD652 cells
Article Snippet: .. Firstly, the HaDV2 NS promoter was amplified by primers NSPF/NSPR (Additional file : Table S1), digested with restriction endonuclease Kpn I/Hind II and cloned into a luciferase reporter vector pGL-3 Basic (Promega). .. This created the pNSP-Luc plasmid, in which the luciferase gene was under the control of the HaDV2 promoter.

Article Title: Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9
Article Snippet: .. All genomic fragments (Supplementary Table ) were amplified from human genomic DNA using Phusion polymerase (NEB) and cloned into the luciferase reporter vector pGL4.10 (Promega) containing the human beta-globin minimal promoter between bgIII and HindIII sites in the multiple cloning site. .. Transcription factor consensus binding sites (SF1, SOX9 etc) were identified using JASPER motif analysis software and mutated using the Quikchange site directed mutagenesis kit II XL (Agilent) (see Supplementary Table for primers).

Activity Assay:

Article Title: Ascl2 activation by YAP1/KLF5 ensures the self-renewability of colon cancer progenitor cells
Article Snippet: Transfection and luciferase assays The Ascl2 5′-flanking sequence (-2588/+620 bp region), amplified using PCR from intestinal tissue DNA, was inserted into the luciferase reporter vector pGL3-Basic (Promega, Madison, WI, USA). .. Luciferase activity was measured 48 h post-transfection using the Dual-Luciferase Reporter Assay System (Promega) as described in our previous report [ ].

Article Title: LINC01354 interacting with hnRNP-D contributes to the proliferation and metastasis in colorectal cancer through activating Wnt/β-catenin signaling pathway
Article Snippet: Luciferase reporter gene assays CTNNB1 wild type with potential LINC01354 binding sites or relative mutant sites was generated by Sangon Biotech (Shanghai, China) and fused with the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. After 48 h of transfection, dual luciferase assay kit (Promega, Madison, WI, USA) was applied to measure the luciferase activity.

Article Title: Linc-POU3F3 is overexpressed in in-stent restenosis patients and induces VSMC phenotypic transformation via POU3F3/miR-449a/KLF4 signaling pathway
Article Snippet: A fragment of KLF-4 3’-UTR and the mutated variants of the predicted miR-449a binding site on KLF-4 3’-UTR were PCR-amplified and fused to the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. After 48 h of transfection, the luciferase activity was evaluated using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).

Article Title: CtBP2 is an independent prognostic marker that promotes GLI1 induced epithelial-mesenchymal transition in hepatocellular carcinoma
Article Snippet: Luciferase reporter assay Four CtBP2 promoter fragments (−2000/−1 bp, −1307/−1 bp, −675/−1 bp and −1350/−652 bp from the transcription start site) were isolated by PCR amplification and ligated into the luciferase reporter vector pGL3-Basic from Promega (Fitchburg, WI, USA). .. After 48 hours, luciferase activity was examined using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol.

Expressing:

Article Title: CtBP2 is an independent prognostic marker that promotes GLI1 induced epithelial-mesenchymal transition in hepatocellular carcinoma
Article Snippet: Luciferase reporter assay Four CtBP2 promoter fragments (−2000/−1 bp, −1307/−1 bp, −675/−1 bp and −1350/−652 bp from the transcription start site) were isolated by PCR amplification and ligated into the luciferase reporter vector pGL3-Basic from Promega (Fitchburg, WI, USA). .. Huh7 cells were co-transfected with luciferase promoter reporter vectors and GLI1 expressing plasmids.

Article Title: Structure and transcription of the Helicoverpa armigera densovirus (HaDV2) genome and its expression strategy in LD652 cells
Article Snippet: Paragraph title: NS protein expression and subcellular localization ... Firstly, the HaDV2 NS promoter was amplified by primers NSPF/NSPR (Additional file : Table S1), digested with restriction endonuclease Kpn I/Hind II and cloned into a luciferase reporter vector pGL-3 Basic (Promega).

Electroporation:

Article Title: Pu-erh Tea Protects the Nervous System by Inhibiting the Expression of Metabotropic Glutamate Receptor 5
Article Snippet: Luciferase Assay Promoter regions were amplified by PCR amplification and subcloned into the KpnI/HindIII sites of the luciferase reporter vector pGL3-Basic (Promega) upstream of the firefly luciferase gene (Fig. ). .. SH-SY5Y and HEK293T cells were transfected with mGluR5 promoter-pGL3 recombinant plasmid by electroporation according to the manufacturer’s instructions.

Transfection:

Article Title: Long non‐coding RNA NNT‐ AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer, et al. Long non‐coding RNA NNT‐AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer
Article Snippet: 2.7 Luciferase reporter assay For Luciferase assays, NNT‐AS1 wild type with potential miR‐424 binding sites or mutant sites was generated by Sangon Biotech (Shanghai, China), and then fused with the luciferase reporter vector psi‐CHECK‐2 (Promega, Madison, WI, USA). .. Cells were cultured in 12‐well plates and transfected with 5 ng plasmid and 5 ng Renilla using Dual‐Luciferase Reporter Assay System (Promega).

Article Title: Dopamine-2 Receptor Activation Suppresses PACAP Expression in Gonadotrophs
Article Snippet: Paragraph title: DNA constructs and transfection. ... The PACAP promoter DNA was excised with the restriction enzymes Kpn I and Xho I and cloned into the luciferase reporter vector PGL-3 Basic (Promega Corp) in the correct orientation.

Article Title: Ascl2 activation by YAP1/KLF5 ensures the self-renewability of colon cancer progenitor cells
Article Snippet: .. Transfection and luciferase assays The Ascl2 5′-flanking sequence (-2588/+620 bp region), amplified using PCR from intestinal tissue DNA, was inserted into the luciferase reporter vector pGL3-Basic (Promega, Madison, WI, USA). ..

Article Title: LINC01354 interacting with hnRNP-D contributes to the proliferation and metastasis in colorectal cancer through activating Wnt/β-catenin signaling pathway
Article Snippet: Luciferase reporter gene assays CTNNB1 wild type with potential LINC01354 binding sites or relative mutant sites was generated by Sangon Biotech (Shanghai, China) and fused with the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. After 48 h of transfection, dual luciferase assay kit (Promega, Madison, WI, USA) was applied to measure the luciferase activity.

Article Title: Pu-erh Tea Protects the Nervous System by Inhibiting the Expression of Metabotropic Glutamate Receptor 5
Article Snippet: Luciferase Assay Promoter regions were amplified by PCR amplification and subcloned into the KpnI/HindIII sites of the luciferase reporter vector pGL3-Basic (Promega) upstream of the firefly luciferase gene (Fig. ). .. SH-SY5Y and HEK293T cells were transfected with mGluR5 promoter-pGL3 recombinant plasmid by electroporation according to the manufacturer’s instructions.

Article Title: Linc-POU3F3 is overexpressed in in-stent restenosis patients and induces VSMC phenotypic transformation via POU3F3/miR-449a/KLF4 signaling pathway
Article Snippet: A fragment of KLF-4 3’-UTR and the mutated variants of the predicted miR-449a binding site on KLF-4 3’-UTR were PCR-amplified and fused to the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. VSMCs were transfected with luciferase plasmid and miR-449a mimic or control using Lipofectamine 3000 (Invitrogen, USA).

Article Title: Long non‐coding RNA NNT‐ AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer, et al. Long non‐coding RNA NNT‐AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer
Article Snippet: For Luciferase assays, NNT‐AS1 wild type with potential miR‐424 binding sites or mutant sites was generated by Sangon Biotech (Shanghai, China), and then fused with the luciferase reporter vector psi‐CHECK‐2 (Promega, Madison, WI, USA). .. Cells were cultured in 12‐well plates and transfected with 5 ng plasmid and 5 ng Renilla using Dual‐Luciferase Reporter Assay System (Promega).

Article Title: CtBP2 is an independent prognostic marker that promotes GLI1 induced epithelial-mesenchymal transition in hepatocellular carcinoma
Article Snippet: Luciferase reporter assay Four CtBP2 promoter fragments (−2000/−1 bp, −1307/−1 bp, −675/−1 bp and −1350/−652 bp from the transcription start site) were isolated by PCR amplification and ligated into the luciferase reporter vector pGL3-Basic from Promega (Fitchburg, WI, USA). .. Huh7 cells were plated on 24-well plates one day prior to the transfection.

Article Title: Structure and transcription of the Helicoverpa armigera densovirus (HaDV2) genome and its expression strategy in LD652 cells
Article Snippet: Firstly, the HaDV2 NS promoter was amplified by primers NSPF/NSPR (Additional file : Table S1), digested with restriction endonuclease Kpn I/Hind II and cloned into a luciferase reporter vector pGL-3 Basic (Promega). .. LD652 cells transfected with pHaDNV-T plasmid were then analyzed using 12% SDS-PAGE and transferred onto PVDF membranes.

Article Title: Long intergenic noncoding RNA 00707 promotes colorectal cancer cell proliferation and metastasis by sponging miR-206
Article Snippet: Luciferase reporter assay LINC00707 wild-type (WT) with potential miR-206-binding sites or mutant (MUT) of each sites were generated and fused to a luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA) to analyze the interaction between LINC00707 and miR-206. .. After 48 hrs transfection, luciferase activities were detected with a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.

Article Title: Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9
Article Snippet: All genomic fragments (Supplementary Table ) were amplified from human genomic DNA using Phusion polymerase (NEB) and cloned into the luciferase reporter vector pGL4.10 (Promega) containing the human beta-globin minimal promoter between bgIII and HindIII sites in the multiple cloning site. .. Briefly, cells were seeded at 80–90% confluency in 96 well plate for 2–4 h. Each well was transfected with a combination of enhancer reporter plasmid pgl4-beta-globin (75 ng) and transcription factors in pcDNA3.1 (100 ng) with 15 ng of Renilla luciferase pLR-TK (Promega) as a control using lipofecamine 2000 for 24 h. The cells were lysed using 1.25x passive lysis buffer (Promega) and luciferase was determined using Dual-Luciferase® Reporter Assay System (Promega) as previously described .

Cell Culture:

Article Title: Long non‐coding RNA NNT‐ AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer, et al. Long non‐coding RNA NNT‐AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer
Article Snippet: 2.7 Luciferase reporter assay For Luciferase assays, NNT‐AS1 wild type with potential miR‐424 binding sites or mutant sites was generated by Sangon Biotech (Shanghai, China), and then fused with the luciferase reporter vector psi‐CHECK‐2 (Promega, Madison, WI, USA). .. Cells were cultured in 12‐well plates and transfected with 5 ng plasmid and 5 ng Renilla using Dual‐Luciferase Reporter Assay System (Promega).

Article Title: Genetic variation at an YY-1 response site regulates the transcription of cyclin-dependent kinase inhibitor p18INK4C transcript in lupus-prone mice
Article Snippet: The luciferase reporter vector pGL-4 basic and the assay kit were purchased from Promega. .. FCS was obtained from Atlanta biotech; and cell culture plasticware was purchased from Corning.

Article Title: Long non‐coding RNA NNT‐ AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer, et al. Long non‐coding RNA NNT‐AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer
Article Snippet: For Luciferase assays, NNT‐AS1 wild type with potential miR‐424 binding sites or mutant sites was generated by Sangon Biotech (Shanghai, China), and then fused with the luciferase reporter vector psi‐CHECK‐2 (Promega, Madison, WI, USA). .. Cells were cultured in 12‐well plates and transfected with 5 ng plasmid and 5 ng Renilla using Dual‐Luciferase Reporter Assay System (Promega).

Generated:

Article Title: The lncRNA CRNDE promotes colorectal cancer cell proliferation and chemoresistance via miR-181a-5p-mediated regulation of Wnt/β-catenin signaling
Article Snippet: .. Luciferase assay CRNDE wild type with potential miR-181a-5p binding sites or mutant of each sites were generated and fused to the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. The full-length wild-type (WT) 3′ untranslated region (UTR) containing the predicted miR-181a-5p targeting site, and mutant (MUT) 3′-UTR of β-catenin and TCF4 were amplified and cloned into the psi-CHECK-2 vector.

Article Title: Long non‐coding RNA NNT‐ AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer, et al. Long non‐coding RNA NNT‐AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer
Article Snippet: .. 2.7 Luciferase reporter assay For Luciferase assays, NNT‐AS1 wild type with potential miR‐424 binding sites or mutant sites was generated by Sangon Biotech (Shanghai, China), and then fused with the luciferase reporter vector psi‐CHECK‐2 (Promega, Madison, WI, USA). ..

Article Title: LINC01354 interacting with hnRNP-D contributes to the proliferation and metastasis in colorectal cancer through activating Wnt/β-catenin signaling pathway
Article Snippet: .. Luciferase reporter gene assays CTNNB1 wild type with potential LINC01354 binding sites or relative mutant sites was generated by Sangon Biotech (Shanghai, China) and fused with the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. Cells were co-transfected with luciferase plasmids and siLINC01354 or siNC using the Lipofectamine 2000 under the manufacturer’s protocols.

Article Title: Long non‐coding RNA NNT‐ AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer, et al. Long non‐coding RNA NNT‐AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer
Article Snippet: .. For Luciferase assays, NNT‐AS1 wild type with potential miR‐424 binding sites or mutant sites was generated by Sangon Biotech (Shanghai, China), and then fused with the luciferase reporter vector psi‐CHECK‐2 (Promega, Madison, WI, USA). ..

Article Title: LncRNA ADAMTS9-AS2 inhibits cell proliferation and decreases chemoresistance in clear cell renal cell carcinoma via the miR-27a-3p/FOXO1 axis
Article Snippet: .. Dual-luciferase reporter assay A fragment of WT ADAMTS9-AS2 with potential miR-27a-3p binding sites or MUT ADAMTS9-AS2 with nonfunctional binding sites was generated and inserted into the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. The full-length WT 3′UTR, containing the predicted miR-27a-3p targeting site, and the MUT 3′UTR of FOXO1 were amplified and cloned into the psi-CHECK-2 vector.

Article Title: Long intergenic noncoding RNA 00707 promotes colorectal cancer cell proliferation and metastasis by sponging miR-206
Article Snippet: .. Luciferase reporter assay LINC00707 wild-type (WT) with potential miR-206-binding sites or mutant (MUT) of each sites were generated and fused to a luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA) to analyze the interaction between LINC00707 and miR-206. .. The binding site of miR-206 on LINC00707 was mutated into AGATAGGA.

Polymerase Chain Reaction:

Article Title: Dopamine-2 Receptor Activation Suppresses PACAP Expression in Gonadotrophs
Article Snippet: A PCR-generated DNA fragment (−1218/+36) of the mouse PACAP promoter was cloned into the PSTBlue-1 Acceptor Vector (Novagen) by TA cloning. .. The PACAP promoter DNA was excised with the restriction enzymes Kpn I and Xho I and cloned into the luciferase reporter vector PGL-3 Basic (Promega Corp) in the correct orientation.

Article Title: Ascl2 activation by YAP1/KLF5 ensures the self-renewability of colon cancer progenitor cells
Article Snippet: .. Transfection and luciferase assays The Ascl2 5′-flanking sequence (-2588/+620 bp region), amplified using PCR from intestinal tissue DNA, was inserted into the luciferase reporter vector pGL3-Basic (Promega, Madison, WI, USA). ..

Article Title: Pu-erh Tea Protects the Nervous System by Inhibiting the Expression of Metabotropic Glutamate Receptor 5
Article Snippet: .. Luciferase Assay Promoter regions were amplified by PCR amplification and subcloned into the KpnI/HindIII sites of the luciferase reporter vector pGL3-Basic (Promega) upstream of the firefly luciferase gene (Fig. ). .. SH-SY5Y and HEK293T cells were transfected with mGluR5 promoter-pGL3 recombinant plasmid by electroporation according to the manufacturer’s instructions.

Article Title: Linc-POU3F3 is overexpressed in in-stent restenosis patients and induces VSMC phenotypic transformation via POU3F3/miR-449a/KLF4 signaling pathway
Article Snippet: .. A fragment of KLF-4 3’-UTR and the mutated variants of the predicted miR-449a binding site on KLF-4 3’-UTR were PCR-amplified and fused to the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. VSMCs were transfected with luciferase plasmid and miR-449a mimic or control using Lipofectamine 3000 (Invitrogen, USA).

Article Title: Smad2 and PEA3 cooperatively regulate transcription of response gene to complement 32 in TGF-?-induced smooth muscle cell differentiation of neural crest cells
Article Snippet: .. RGC-32 promoter DNA were amplified from mouse genomic DNA (Promega) by PCR and cloned into luciferase reporter vector pGL3-basic (Promega) at the Kpn I/ Bgl II restriction site. .. Three RGC-32 promoter reporter plasmids were constructed as follows: p-2107/+844RGCluc contains the regulatory region from nucleotide (nt) −2107 to +844 bp of RGC-32 gene; p-2107RGCluc and p-1500RGCluc contain the regulatory regions from nt −2107 and −1500 to +22 bp of the RGC-32 gene, respectively.

Article Title: CtBP2 is an independent prognostic marker that promotes GLI1 induced epithelial-mesenchymal transition in hepatocellular carcinoma
Article Snippet: .. Luciferase reporter assay Four CtBP2 promoter fragments (−2000/−1 bp, −1307/−1 bp, −675/−1 bp and −1350/−652 bp from the transcription start site) were isolated by PCR amplification and ligated into the luciferase reporter vector pGL3-Basic from Promega (Fitchburg, WI, USA). .. Huh7 cells were plated on 24-well plates one day prior to the transfection.

Binding Assay:

Article Title: The lncRNA CRNDE promotes colorectal cancer cell proliferation and chemoresistance via miR-181a-5p-mediated regulation of Wnt/β-catenin signaling
Article Snippet: .. Luciferase assay CRNDE wild type with potential miR-181a-5p binding sites or mutant of each sites were generated and fused to the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. The full-length wild-type (WT) 3′ untranslated region (UTR) containing the predicted miR-181a-5p targeting site, and mutant (MUT) 3′-UTR of β-catenin and TCF4 were amplified and cloned into the psi-CHECK-2 vector.

Article Title: Long non‐coding RNA NNT‐ AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer, et al. Long non‐coding RNA NNT‐AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer
Article Snippet: .. 2.7 Luciferase reporter assay For Luciferase assays, NNT‐AS1 wild type with potential miR‐424 binding sites or mutant sites was generated by Sangon Biotech (Shanghai, China), and then fused with the luciferase reporter vector psi‐CHECK‐2 (Promega, Madison, WI, USA). ..

Article Title: LINC01354 interacting with hnRNP-D contributes to the proliferation and metastasis in colorectal cancer through activating Wnt/β-catenin signaling pathway
Article Snippet: .. Luciferase reporter gene assays CTNNB1 wild type with potential LINC01354 binding sites or relative mutant sites was generated by Sangon Biotech (Shanghai, China) and fused with the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. Cells were co-transfected with luciferase plasmids and siLINC01354 or siNC using the Lipofectamine 2000 under the manufacturer’s protocols.

Article Title: Linc-POU3F3 is overexpressed in in-stent restenosis patients and induces VSMC phenotypic transformation via POU3F3/miR-449a/KLF4 signaling pathway
Article Snippet: .. A fragment of KLF-4 3’-UTR and the mutated variants of the predicted miR-449a binding site on KLF-4 3’-UTR were PCR-amplified and fused to the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. VSMCs were transfected with luciferase plasmid and miR-449a mimic or control using Lipofectamine 3000 (Invitrogen, USA).

Article Title: Long non‐coding RNA NNT‐ AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer, et al. Long non‐coding RNA NNT‐AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer
Article Snippet: .. For Luciferase assays, NNT‐AS1 wild type with potential miR‐424 binding sites or mutant sites was generated by Sangon Biotech (Shanghai, China), and then fused with the luciferase reporter vector psi‐CHECK‐2 (Promega, Madison, WI, USA). ..

Article Title: LncRNA ADAMTS9-AS2 inhibits cell proliferation and decreases chemoresistance in clear cell renal cell carcinoma via the miR-27a-3p/FOXO1 axis
Article Snippet: .. Dual-luciferase reporter assay A fragment of WT ADAMTS9-AS2 with potential miR-27a-3p binding sites or MUT ADAMTS9-AS2 with nonfunctional binding sites was generated and inserted into the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. The full-length WT 3′UTR, containing the predicted miR-27a-3p targeting site, and the MUT 3′UTR of FOXO1 were amplified and cloned into the psi-CHECK-2 vector.

Article Title: Long intergenic noncoding RNA 00707 promotes colorectal cancer cell proliferation and metastasis by sponging miR-206
Article Snippet: Luciferase reporter assay LINC00707 wild-type (WT) with potential miR-206-binding sites or mutant (MUT) of each sites were generated and fused to a luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA) to analyze the interaction between LINC00707 and miR-206. .. The binding site of miR-206 on LINC00707 was mutated into AGATAGGA.

Article Title: Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9
Article Snippet: The eALDI enhancer was located using targeted bioinformatic screening of the upstream region of SOX9 using multiple datasets, ENCODE , RoadMap , transcription factor binding site and vertebrate conservation , focusing on regions that showed enhancer function in all three datasets. .. All genomic fragments (Supplementary Table ) were amplified from human genomic DNA using Phusion polymerase (NEB) and cloned into the luciferase reporter vector pGL4.10 (Promega) containing the human beta-globin minimal promoter between bgIII and HindIII sites in the multiple cloning site.

Mutagenesis:

Article Title: The lncRNA CRNDE promotes colorectal cancer cell proliferation and chemoresistance via miR-181a-5p-mediated regulation of Wnt/β-catenin signaling
Article Snippet: .. Luciferase assay CRNDE wild type with potential miR-181a-5p binding sites or mutant of each sites were generated and fused to the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. The full-length wild-type (WT) 3′ untranslated region (UTR) containing the predicted miR-181a-5p targeting site, and mutant (MUT) 3′-UTR of β-catenin and TCF4 were amplified and cloned into the psi-CHECK-2 vector.

Article Title: Long non‐coding RNA NNT‐ AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer, et al. Long non‐coding RNA NNT‐AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer
Article Snippet: .. 2.7 Luciferase reporter assay For Luciferase assays, NNT‐AS1 wild type with potential miR‐424 binding sites or mutant sites was generated by Sangon Biotech (Shanghai, China), and then fused with the luciferase reporter vector psi‐CHECK‐2 (Promega, Madison, WI, USA). ..

Article Title: LINC01354 interacting with hnRNP-D contributes to the proliferation and metastasis in colorectal cancer through activating Wnt/β-catenin signaling pathway
Article Snippet: .. Luciferase reporter gene assays CTNNB1 wild type with potential LINC01354 binding sites or relative mutant sites was generated by Sangon Biotech (Shanghai, China) and fused with the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. Cells were co-transfected with luciferase plasmids and siLINC01354 or siNC using the Lipofectamine 2000 under the manufacturer’s protocols.

Article Title: Long non‐coding RNA NNT‐ AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer, et al. Long non‐coding RNA NNT‐AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer
Article Snippet: .. For Luciferase assays, NNT‐AS1 wild type with potential miR‐424 binding sites or mutant sites was generated by Sangon Biotech (Shanghai, China), and then fused with the luciferase reporter vector psi‐CHECK‐2 (Promega, Madison, WI, USA). ..

Article Title: Long intergenic noncoding RNA 00707 promotes colorectal cancer cell proliferation and metastasis by sponging miR-206
Article Snippet: .. Luciferase reporter assay LINC00707 wild-type (WT) with potential miR-206-binding sites or mutant (MUT) of each sites were generated and fused to a luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA) to analyze the interaction between LINC00707 and miR-206. .. The binding site of miR-206 on LINC00707 was mutated into AGATAGGA.

Article Title: Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9
Article Snippet: All genomic fragments (Supplementary Table ) were amplified from human genomic DNA using Phusion polymerase (NEB) and cloned into the luciferase reporter vector pGL4.10 (Promega) containing the human beta-globin minimal promoter between bgIII and HindIII sites in the multiple cloning site. .. Transcription factor consensus binding sites (SF1, SOX9 etc) were identified using JASPER motif analysis software and mutated using the Quikchange site directed mutagenesis kit II XL (Agilent) (see Supplementary Table for primers).

Isolation:

Article Title: CtBP2 is an independent prognostic marker that promotes GLI1 induced epithelial-mesenchymal transition in hepatocellular carcinoma
Article Snippet: .. Luciferase reporter assay Four CtBP2 promoter fragments (−2000/−1 bp, −1307/−1 bp, −675/−1 bp and −1350/−652 bp from the transcription start site) were isolated by PCR amplification and ligated into the luciferase reporter vector pGL3-Basic from Promega (Fitchburg, WI, USA). .. Huh7 cells were plated on 24-well plates one day prior to the transfection.

Sequencing:

Article Title: Ascl2 activation by YAP1/KLF5 ensures the self-renewability of colon cancer progenitor cells
Article Snippet: .. Transfection and luciferase assays The Ascl2 5′-flanking sequence (-2588/+620 bp region), amplified using PCR from intestinal tissue DNA, was inserted into the luciferase reporter vector pGL3-Basic (Promega, Madison, WI, USA). ..

Article Title: Smad2 and PEA3 cooperatively regulate transcription of response gene to complement 32 in TGF-?-induced smooth muscle cell differentiation of neural crest cells
Article Snippet: RGC-32 promoter DNA were amplified from mouse genomic DNA (Promega) by PCR and cloned into luciferase reporter vector pGL3-basic (Promega) at the Kpn I/ Bgl II restriction site. .. The transcription start site was determined based on RGC-32 gene sequence (accession number ).

SDS Page:

Article Title: Structure and transcription of the Helicoverpa armigera densovirus (HaDV2) genome and its expression strategy in LD652 cells
Article Snippet: Firstly, the HaDV2 NS promoter was amplified by primers NSPF/NSPR (Additional file : Table S1), digested with restriction endonuclease Kpn I/Hind II and cloned into a luciferase reporter vector pGL-3 Basic (Promega). .. LD652 cells transfected with pHaDNV-T plasmid were then analyzed using 12% SDS-PAGE and transferred onto PVDF membranes.

Plasmid Preparation:

Article Title: The lncRNA CRNDE promotes colorectal cancer cell proliferation and chemoresistance via miR-181a-5p-mediated regulation of Wnt/β-catenin signaling
Article Snippet: .. Luciferase assay CRNDE wild type with potential miR-181a-5p binding sites or mutant of each sites were generated and fused to the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. The full-length wild-type (WT) 3′ untranslated region (UTR) containing the predicted miR-181a-5p targeting site, and mutant (MUT) 3′-UTR of β-catenin and TCF4 were amplified and cloned into the psi-CHECK-2 vector.

Article Title: Long non‐coding RNA NNT‐ AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer, et al. Long non‐coding RNA NNT‐AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer
Article Snippet: .. 2.7 Luciferase reporter assay For Luciferase assays, NNT‐AS1 wild type with potential miR‐424 binding sites or mutant sites was generated by Sangon Biotech (Shanghai, China), and then fused with the luciferase reporter vector psi‐CHECK‐2 (Promega, Madison, WI, USA). ..

Article Title: Dopamine-2 Receptor Activation Suppresses PACAP Expression in Gonadotrophs
Article Snippet: .. The PACAP promoter DNA was excised with the restriction enzymes Kpn I and Xho I and cloned into the luciferase reporter vector PGL-3 Basic (Promega Corp) in the correct orientation. .. Activation of the cAMP response element (CRE) sequences was studied using pCRE-LUC from Clontech Laboratories, Inc, that contains the 2 copies of the canonical cAMP-responsive element (TGACGTCA) placed upstream of the LUC reporter.

Article Title: Genetic variation at an YY-1 response site regulates the transcription of cyclin-dependent kinase inhibitor p18INK4C transcript in lupus-prone mice
Article Snippet: .. The luciferase reporter vector pGL-4 basic and the assay kit were purchased from Promega. .. RPMI, DMEM-high glucose and DMEM-F-12 culture media were purchased from Cellgro.

Article Title: Ascl2 activation by YAP1/KLF5 ensures the self-renewability of colon cancer progenitor cells
Article Snippet: .. Transfection and luciferase assays The Ascl2 5′-flanking sequence (-2588/+620 bp region), amplified using PCR from intestinal tissue DNA, was inserted into the luciferase reporter vector pGL3-Basic (Promega, Madison, WI, USA). ..

Article Title: LINC01354 interacting with hnRNP-D contributes to the proliferation and metastasis in colorectal cancer through activating Wnt/β-catenin signaling pathway
Article Snippet: .. Luciferase reporter gene assays CTNNB1 wild type with potential LINC01354 binding sites or relative mutant sites was generated by Sangon Biotech (Shanghai, China) and fused with the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. Cells were co-transfected with luciferase plasmids and siLINC01354 or siNC using the Lipofectamine 2000 under the manufacturer’s protocols.

Article Title: Pu-erh Tea Protects the Nervous System by Inhibiting the Expression of Metabotropic Glutamate Receptor 5
Article Snippet: .. Luciferase Assay Promoter regions were amplified by PCR amplification and subcloned into the KpnI/HindIII sites of the luciferase reporter vector pGL3-Basic (Promega) upstream of the firefly luciferase gene (Fig. ). .. SH-SY5Y and HEK293T cells were transfected with mGluR5 promoter-pGL3 recombinant plasmid by electroporation according to the manufacturer’s instructions.

Article Title: Linc-POU3F3 is overexpressed in in-stent restenosis patients and induces VSMC phenotypic transformation via POU3F3/miR-449a/KLF4 signaling pathway
Article Snippet: .. A fragment of KLF-4 3’-UTR and the mutated variants of the predicted miR-449a binding site on KLF-4 3’-UTR were PCR-amplified and fused to the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. VSMCs were transfected with luciferase plasmid and miR-449a mimic or control using Lipofectamine 3000 (Invitrogen, USA).

Article Title: Smad2 and PEA3 cooperatively regulate transcription of response gene to complement 32 in TGF-?-induced smooth muscle cell differentiation of neural crest cells
Article Snippet: .. RGC-32 promoter DNA were amplified from mouse genomic DNA (Promega) by PCR and cloned into luciferase reporter vector pGL3-basic (Promega) at the Kpn I/ Bgl II restriction site. .. Three RGC-32 promoter reporter plasmids were constructed as follows: p-2107/+844RGCluc contains the regulatory region from nucleotide (nt) −2107 to +844 bp of RGC-32 gene; p-2107RGCluc and p-1500RGCluc contain the regulatory regions from nt −2107 and −1500 to +22 bp of the RGC-32 gene, respectively.

Article Title: Long non‐coding RNA NNT‐ AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer, et al. Long non‐coding RNA NNT‐AS1 sponges miR‐424/E2F1 to promote the tumorigenesis and cell cycle progression of gastric cancer
Article Snippet: .. For Luciferase assays, NNT‐AS1 wild type with potential miR‐424 binding sites or mutant sites was generated by Sangon Biotech (Shanghai, China), and then fused with the luciferase reporter vector psi‐CHECK‐2 (Promega, Madison, WI, USA). ..

Article Title: CtBP2 is an independent prognostic marker that promotes GLI1 induced epithelial-mesenchymal transition in hepatocellular carcinoma
Article Snippet: .. Luciferase reporter assay Four CtBP2 promoter fragments (−2000/−1 bp, −1307/−1 bp, −675/−1 bp and −1350/−652 bp from the transcription start site) were isolated by PCR amplification and ligated into the luciferase reporter vector pGL3-Basic from Promega (Fitchburg, WI, USA). .. Huh7 cells were plated on 24-well plates one day prior to the transfection.

Article Title: LncRNA ADAMTS9-AS2 inhibits cell proliferation and decreases chemoresistance in clear cell renal cell carcinoma via the miR-27a-3p/FOXO1 axis
Article Snippet: .. Dual-luciferase reporter assay A fragment of WT ADAMTS9-AS2 with potential miR-27a-3p binding sites or MUT ADAMTS9-AS2 with nonfunctional binding sites was generated and inserted into the luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA). .. The full-length WT 3′UTR, containing the predicted miR-27a-3p targeting site, and the MUT 3′UTR of FOXO1 were amplified and cloned into the psi-CHECK-2 vector.

Article Title: Structure and transcription of the Helicoverpa armigera densovirus (HaDV2) genome and its expression strategy in LD652 cells
Article Snippet: .. Firstly, the HaDV2 NS promoter was amplified by primers NSPF/NSPR (Additional file : Table S1), digested with restriction endonuclease Kpn I/Hind II and cloned into a luciferase reporter vector pGL-3 Basic (Promega). .. This created the pNSP-Luc plasmid, in which the luciferase gene was under the control of the HaDV2 promoter.

Article Title: Long intergenic noncoding RNA 00707 promotes colorectal cancer cell proliferation and metastasis by sponging miR-206
Article Snippet: .. Luciferase reporter assay LINC00707 wild-type (WT) with potential miR-206-binding sites or mutant (MUT) of each sites were generated and fused to a luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA) to analyze the interaction between LINC00707 and miR-206. .. The binding site of miR-206 on LINC00707 was mutated into AGATAGGA.

Article Title: Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9
Article Snippet: .. All genomic fragments (Supplementary Table ) were amplified from human genomic DNA using Phusion polymerase (NEB) and cloned into the luciferase reporter vector pGL4.10 (Promega) containing the human beta-globin minimal promoter between bgIII and HindIII sites in the multiple cloning site. .. Transcription factor consensus binding sites (SF1, SOX9 etc) were identified using JASPER motif analysis software and mutated using the Quikchange site directed mutagenesis kit II XL (Agilent) (see Supplementary Table for primers).

Software:

Article Title: Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9
Article Snippet: All genomic fragments (Supplementary Table ) were amplified from human genomic DNA using Phusion polymerase (NEB) and cloned into the luciferase reporter vector pGL4.10 (Promega) containing the human beta-globin minimal promoter between bgIII and HindIII sites in the multiple cloning site. .. Transcription factor consensus binding sites (SF1, SOX9 etc) were identified using JASPER motif analysis software and mutated using the Quikchange site directed mutagenesis kit II XL (Agilent) (see Supplementary Table for primers).

Negative Control:

Article Title: Long intergenic noncoding RNA 00707 promotes colorectal cancer cell proliferation and metastasis by sponging miR-206
Article Snippet: Luciferase reporter assay LINC00707 wild-type (WT) with potential miR-206-binding sites or mutant (MUT) of each sites were generated and fused to a luciferase reporter vector psi-CHECK-2 (Promega, Madison, WI, USA) to analyze the interaction between LINC00707 and miR-206. .. HEK293T cells were co-transfected with luciferase plasmids and miR-206 mimic or miRNA negative control.

Recombinant:

Article Title: Pu-erh Tea Protects the Nervous System by Inhibiting the Expression of Metabotropic Glutamate Receptor 5
Article Snippet: Luciferase Assay Promoter regions were amplified by PCR amplification and subcloned into the KpnI/HindIII sites of the luciferase reporter vector pGL3-Basic (Promega) upstream of the firefly luciferase gene (Fig. ). .. SH-SY5Y and HEK293T cells were transfected with mGluR5 promoter-pGL3 recombinant plasmid by electroporation according to the manufacturer’s instructions.

Activation Assay:

Article Title: Dopamine-2 Receptor Activation Suppresses PACAP Expression in Gonadotrophs
Article Snippet: The PACAP promoter DNA was excised with the restriction enzymes Kpn I and Xho I and cloned into the luciferase reporter vector PGL-3 Basic (Promega Corp) in the correct orientation. .. Activation of the cAMP response element (CRE) sequences was studied using pCRE-LUC from Clontech Laboratories, Inc, that contains the 2 copies of the canonical cAMP-responsive element (TGACGTCA) placed upstream of the LUC reporter.

Lysis:

Article Title: Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9
Article Snippet: All genomic fragments (Supplementary Table ) were amplified from human genomic DNA using Phusion polymerase (NEB) and cloned into the luciferase reporter vector pGL4.10 (Promega) containing the human beta-globin minimal promoter between bgIII and HindIII sites in the multiple cloning site. .. Briefly, cells were seeded at 80–90% confluency in 96 well plate for 2–4 h. Each well was transfected with a combination of enhancer reporter plasmid pgl4-beta-globin (75 ng) and transcription factors in pcDNA3.1 (100 ng) with 15 ng of Renilla luciferase pLR-TK (Promega) as a control using lipofecamine 2000 for 24 h. The cells were lysed using 1.25x passive lysis buffer (Promega) and luciferase was determined using Dual-Luciferase® Reporter Assay System (Promega) as previously described .

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    Promega luciferase reporter vector pgl4 10
    Duplication or deletion of the human SOX9 testicular enhancer eSR-A is associated with DSD. a The 600 kb genomic region upstream of human SOX9 showing the XYSR, RevSex and TESCO candidate regulatory regions. b The XYSR region was defined by deletions in two previously published 46,XY DSD patients (blue) 17 . Two novel duplications in 46,XX DSD patients (grey), allowed us to redefine the minimal overlap to 5.2 kb (green). c Sub-cloning strategy, dark blue lines indicate sub-clones analysed for enhancer activity in the <t>pGL4.10</t> Beta-globin (βg) plasmid using luciferase assays. Predicted transcription factor binding motifs for SF1 (SF1-a and SF1-b) and SOX9 are shown with vertical light blue and magenta lines. Bioinformatic tracks from the UCSC genome browser are shown including the ENCODE track of enhancers present in human mammary epithelial cells (HMEC) (yellow denotes a weak enhancer) 28 and DNaseI hypersensitivity data from human foetal testis and ovary (ROADMap) 23 . This shows a testis-specific peak over the a4 fragment. The 100-vertebrate conservation track shows a spike of conservation beneath the a4 fragment DNaseI peak. d Enhancer activities of sub-cloned fragments a1-a5 as measured by luciferase assays transfected with SF1 and SOX9 ( n = 4). e Mutation of the SF1-a (eSR-A ΔSF1-a), and SOX9 (eSR-A ΔSOX9) binding motifs either separately or together results in a loss of enhancer activity compared to non-mutated eSR-A as assessed by luciferase assays with co-transfection of SF1 and SOX9 ( n = 4). f . Co-transfection of FOXL2 with SF1 + SOX9 shows a repression of eSR-A activity compared to SF1 + SOX9 ( n = 3). All luciferase assays carried out in COS7 cells. Error bars are s.e.m. P -values (two-tailed t tests): * P ≤ 0.05. ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are provided as a Source Data file.
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    Duplication or deletion of the human SOX9 testicular enhancer eSR-A is associated with DSD. a The 600 kb genomic region upstream of human SOX9 showing the XYSR, RevSex and TESCO candidate regulatory regions. b The XYSR region was defined by deletions in two previously published 46,XY DSD patients (blue) 17 . Two novel duplications in 46,XX DSD patients (grey), allowed us to redefine the minimal overlap to 5.2 kb (green). c Sub-cloning strategy, dark blue lines indicate sub-clones analysed for enhancer activity in the pGL4.10 Beta-globin (βg) plasmid using luciferase assays. Predicted transcription factor binding motifs for SF1 (SF1-a and SF1-b) and SOX9 are shown with vertical light blue and magenta lines. Bioinformatic tracks from the UCSC genome browser are shown including the ENCODE track of enhancers present in human mammary epithelial cells (HMEC) (yellow denotes a weak enhancer) 28 and DNaseI hypersensitivity data from human foetal testis and ovary (ROADMap) 23 . This shows a testis-specific peak over the a4 fragment. The 100-vertebrate conservation track shows a spike of conservation beneath the a4 fragment DNaseI peak. d Enhancer activities of sub-cloned fragments a1-a5 as measured by luciferase assays transfected with SF1 and SOX9 ( n = 4). e Mutation of the SF1-a (eSR-A ΔSF1-a), and SOX9 (eSR-A ΔSOX9) binding motifs either separately or together results in a loss of enhancer activity compared to non-mutated eSR-A as assessed by luciferase assays with co-transfection of SF1 and SOX9 ( n = 4). f . Co-transfection of FOXL2 with SF1 + SOX9 shows a repression of eSR-A activity compared to SF1 + SOX9 ( n = 3). All luciferase assays carried out in COS7 cells. Error bars are s.e.m. P -values (two-tailed t tests): * P ≤ 0.05. ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9

    doi: 10.1038/s41467-018-07784-9

    Figure Lengend Snippet: Duplication or deletion of the human SOX9 testicular enhancer eSR-A is associated with DSD. a The 600 kb genomic region upstream of human SOX9 showing the XYSR, RevSex and TESCO candidate regulatory regions. b The XYSR region was defined by deletions in two previously published 46,XY DSD patients (blue) 17 . Two novel duplications in 46,XX DSD patients (grey), allowed us to redefine the minimal overlap to 5.2 kb (green). c Sub-cloning strategy, dark blue lines indicate sub-clones analysed for enhancer activity in the pGL4.10 Beta-globin (βg) plasmid using luciferase assays. Predicted transcription factor binding motifs for SF1 (SF1-a and SF1-b) and SOX9 are shown with vertical light blue and magenta lines. Bioinformatic tracks from the UCSC genome browser are shown including the ENCODE track of enhancers present in human mammary epithelial cells (HMEC) (yellow denotes a weak enhancer) 28 and DNaseI hypersensitivity data from human foetal testis and ovary (ROADMap) 23 . This shows a testis-specific peak over the a4 fragment. The 100-vertebrate conservation track shows a spike of conservation beneath the a4 fragment DNaseI peak. d Enhancer activities of sub-cloned fragments a1-a5 as measured by luciferase assays transfected with SF1 and SOX9 ( n = 4). e Mutation of the SF1-a (eSR-A ΔSF1-a), and SOX9 (eSR-A ΔSOX9) binding motifs either separately or together results in a loss of enhancer activity compared to non-mutated eSR-A as assessed by luciferase assays with co-transfection of SF1 and SOX9 ( n = 4). f . Co-transfection of FOXL2 with SF1 + SOX9 shows a repression of eSR-A activity compared to SF1 + SOX9 ( n = 3). All luciferase assays carried out in COS7 cells. Error bars are s.e.m. P -values (two-tailed t tests): * P ≤ 0.05. ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are provided as a Source Data file.

    Article Snippet: All genomic fragments (Supplementary Table ) were amplified from human genomic DNA using Phusion polymerase (NEB) and cloned into the luciferase reporter vector pGL4.10 (Promega) containing the human beta-globin minimal promoter between bgIII and HindIII sites in the multiple cloning site.

    Techniques: Electron Paramagnetic Resonance, Subcloning, Clone Assay, Activity Assay, Plasmid Preparation, Luciferase, Binding Assay, Transfection, Mutagenesis, Cotransfection, Two Tailed Test