Structured Review

Promega mir155hg promoter region
BCL6 binds to regulatory regions in <t>MIR155HG</t> and CHM loci and transcriptionally regulates miR-155 and miR-361 expression. (a) Schematic representation of the MIR155HG locus and relative fold enrichment as detected by ChIP for BCL6 followed by quantitative PCR (qChIP) analyses. The bound regions, A and B, are located in the promoter and in intron 2 of the MIR155HG locus, respectively; a region of not binding in the same locus (region C) was used as a negative control. The results are displayed as means and SD of two independent experiments, each performed in triplicates. Region A, P = 0.0006; region B, P = 0.005, Student’s t test. (b) Schematic representation of the CHM locus and relative fold enrichment as detected by qChIP analysis. The bound regions, D and E, are located in the promoter and in intron 9 of the CHM locus, respectively; a region of not binding in the same locus (region F) was used as a negative control. BCL6 binding to its own promoter (BCL6) and in the actin locus were used as positive and negative controls, respectively. The results are displayed as means and SD of two independent experiments, each performed in triplicates. Region D, P = 0.02; region B, P = 0.003, Student’s t test. (c and d) Promoter luciferase assays were performed to test the responsiveness to BCL6 repression of the bound regulatory regions in the MIR155HG (c) and in the CHM genes (d). The results are displayed as relative luciferase activity of the reporter constructs in presence of increasing amount of wild-type BCL6, or the maximum amount of its mutants (BCL6-ZF and BCL6ΔZF), compared with the reporter basal activity, normalized to renilla activity. The displayed means and SD are from three independent experiments, each performed in duplicates. Statistically significant changes were measured for the reporter constructs subjected to the highest dose of BCL6 compared with the reporter vector lacking the tested regions (empty) and to the same setting using the BCL6 mutants (region A vs. empty, P = 0.04, vs. BCL6ZF, P = 0.01, vs. BCL6ΔZF, P = 0.02; region B vs. empty, P = 0.03, vs. BCL6ZF P = 0.0004, vs. BCL6ΔZF, P = 0.0008; region D vs. empty, P = 0.5 n.s., vs. BCL6ZF, P = 0.006, vs. BCL6ΔZF, P = 0.02; region E vs. empty, P = 0.002, vs. BCL6ZF, P = 0.0004, vs. BCL6ΔZF, P = 0.001, Student’s t test). (e) miR-155 qRT-PCR performed in Raji cells engineered to inducibly (upon doxycycline treatment, Dox) express an empty vector (EV) or the BCL6ΔPEST mutant and treated as indicated. The results are displayed as miR-155 fold change relative to the αIgM/αCD40-untreated cells (mean ± SD, n = 3). The miR-155 expression changes upon stimulation are significantly different in presence of BCL6ΔPEST (P = 1.5 × 10 −7 , Student’s t test). (f) Detection of BCL6 (endogenous, endo; exogenous, exo) and β-actin by immunoblotting in the BCL6ΔPEST inducible cell lines displayed in e.
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1) Product Images from "BCL6 positively regulates AID and germinal center gene expression via repression of miR-155"

Article Title: BCL6 positively regulates AID and germinal center gene expression via repression of miR-155

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20121387

BCL6 binds to regulatory regions in MIR155HG and CHM loci and transcriptionally regulates miR-155 and miR-361 expression. (a) Schematic representation of the MIR155HG locus and relative fold enrichment as detected by ChIP for BCL6 followed by quantitative PCR (qChIP) analyses. The bound regions, A and B, are located in the promoter and in intron 2 of the MIR155HG locus, respectively; a region of not binding in the same locus (region C) was used as a negative control. The results are displayed as means and SD of two independent experiments, each performed in triplicates. Region A, P = 0.0006; region B, P = 0.005, Student’s t test. (b) Schematic representation of the CHM locus and relative fold enrichment as detected by qChIP analysis. The bound regions, D and E, are located in the promoter and in intron 9 of the CHM locus, respectively; a region of not binding in the same locus (region F) was used as a negative control. BCL6 binding to its own promoter (BCL6) and in the actin locus were used as positive and negative controls, respectively. The results are displayed as means and SD of two independent experiments, each performed in triplicates. Region D, P = 0.02; region B, P = 0.003, Student’s t test. (c and d) Promoter luciferase assays were performed to test the responsiveness to BCL6 repression of the bound regulatory regions in the MIR155HG (c) and in the CHM genes (d). The results are displayed as relative luciferase activity of the reporter constructs in presence of increasing amount of wild-type BCL6, or the maximum amount of its mutants (BCL6-ZF and BCL6ΔZF), compared with the reporter basal activity, normalized to renilla activity. The displayed means and SD are from three independent experiments, each performed in duplicates. Statistically significant changes were measured for the reporter constructs subjected to the highest dose of BCL6 compared with the reporter vector lacking the tested regions (empty) and to the same setting using the BCL6 mutants (region A vs. empty, P = 0.04, vs. BCL6ZF, P = 0.01, vs. BCL6ΔZF, P = 0.02; region B vs. empty, P = 0.03, vs. BCL6ZF P = 0.0004, vs. BCL6ΔZF, P = 0.0008; region D vs. empty, P = 0.5 n.s., vs. BCL6ZF, P = 0.006, vs. BCL6ΔZF, P = 0.02; region E vs. empty, P = 0.002, vs. BCL6ZF, P = 0.0004, vs. BCL6ΔZF, P = 0.001, Student’s t test). (e) miR-155 qRT-PCR performed in Raji cells engineered to inducibly (upon doxycycline treatment, Dox) express an empty vector (EV) or the BCL6ΔPEST mutant and treated as indicated. The results are displayed as miR-155 fold change relative to the αIgM/αCD40-untreated cells (mean ± SD, n = 3). The miR-155 expression changes upon stimulation are significantly different in presence of BCL6ΔPEST (P = 1.5 × 10 −7 , Student’s t test). (f) Detection of BCL6 (endogenous, endo; exogenous, exo) and β-actin by immunoblotting in the BCL6ΔPEST inducible cell lines displayed in e.
Figure Legend Snippet: BCL6 binds to regulatory regions in MIR155HG and CHM loci and transcriptionally regulates miR-155 and miR-361 expression. (a) Schematic representation of the MIR155HG locus and relative fold enrichment as detected by ChIP for BCL6 followed by quantitative PCR (qChIP) analyses. The bound regions, A and B, are located in the promoter and in intron 2 of the MIR155HG locus, respectively; a region of not binding in the same locus (region C) was used as a negative control. The results are displayed as means and SD of two independent experiments, each performed in triplicates. Region A, P = 0.0006; region B, P = 0.005, Student’s t test. (b) Schematic representation of the CHM locus and relative fold enrichment as detected by qChIP analysis. The bound regions, D and E, are located in the promoter and in intron 9 of the CHM locus, respectively; a region of not binding in the same locus (region F) was used as a negative control. BCL6 binding to its own promoter (BCL6) and in the actin locus were used as positive and negative controls, respectively. The results are displayed as means and SD of two independent experiments, each performed in triplicates. Region D, P = 0.02; region B, P = 0.003, Student’s t test. (c and d) Promoter luciferase assays were performed to test the responsiveness to BCL6 repression of the bound regulatory regions in the MIR155HG (c) and in the CHM genes (d). The results are displayed as relative luciferase activity of the reporter constructs in presence of increasing amount of wild-type BCL6, or the maximum amount of its mutants (BCL6-ZF and BCL6ΔZF), compared with the reporter basal activity, normalized to renilla activity. The displayed means and SD are from three independent experiments, each performed in duplicates. Statistically significant changes were measured for the reporter constructs subjected to the highest dose of BCL6 compared with the reporter vector lacking the tested regions (empty) and to the same setting using the BCL6 mutants (region A vs. empty, P = 0.04, vs. BCL6ZF, P = 0.01, vs. BCL6ΔZF, P = 0.02; region B vs. empty, P = 0.03, vs. BCL6ZF P = 0.0004, vs. BCL6ΔZF, P = 0.0008; region D vs. empty, P = 0.5 n.s., vs. BCL6ZF, P = 0.006, vs. BCL6ΔZF, P = 0.02; region E vs. empty, P = 0.002, vs. BCL6ZF, P = 0.0004, vs. BCL6ΔZF, P = 0.001, Student’s t test). (e) miR-155 qRT-PCR performed in Raji cells engineered to inducibly (upon doxycycline treatment, Dox) express an empty vector (EV) or the BCL6ΔPEST mutant and treated as indicated. The results are displayed as miR-155 fold change relative to the αIgM/αCD40-untreated cells (mean ± SD, n = 3). The miR-155 expression changes upon stimulation are significantly different in presence of BCL6ΔPEST (P = 1.5 × 10 −7 , Student’s t test). (f) Detection of BCL6 (endogenous, endo; exogenous, exo) and β-actin by immunoblotting in the BCL6ΔPEST inducible cell lines displayed in e.

Techniques Used: Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Negative Control, Luciferase, Activity Assay, Construct, Plasmid Preparation, Quantitative RT-PCR, Mutagenesis

2) Product Images from "The Transcription Factors Sox10 and Myrf Define an Essential Regulatory Network Module in Differentiating Oligodendrocytes"

Article Title: The Transcription Factors Sox10 and Myrf Define an Essential Regulatory Network Module in Differentiating Oligodendrocytes

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003907

Sox10 binds to intron 1 of the Myrf gene both in vivo and in vitro. ( A ) Schematic representation of the location of regions from the Myrf locus probed by PCR in ChIP studies including the Myrf minimal promoter (myrf), ECR9, ECR11/12 and additional control regions from the 5′ (fl5) and 3′ (3fl) flanking regions of the Myrf gene and from within the adjacent Dagla gene (dagla). ( B–E ) ChIP was performed on 33B cells ( B,C ), rat primary oligodendroglial cells kept in proliferation medium (OPC) or differentiation medium (OL) ( D ) and P14 spinal cord from wildtype (wt) and Sox10 ΔCNS (ko) mice ( E ) using antibodies directed against Sox10 (α-Sox10) ( B , D , E ) or Sox8 (α-Sox8) ( C ) and control preimmune serum (PI). Quantitative PCR was applied on the immunoprecipitate. Values for each fragment correspond to the percentage of material precipitated from the input and represent the mean ± SEM of at least 3 biological replicates. ( F ) EMSA was performed with radiolabelled double-stranded oligonucleotides 3 and 4 in wildtype (3, 4) and mutant (3a, 3b, 3c, 3bc, 4a) versions as indicated below the gels. Oligonucleotides were incubated in the absence (−), or presence (control, Sox10) of protein extracts before gel electrophoresis as indicated above the lanes. Extracts were from mock-transfected HEK293 cells (control) or HEK293 cells expressing full length Sox10 (Sox10). Oligonucleotides with site B and site C/C′ from the Mpz promoter [29] served as positive control for Sox10 binding and as marker for the mobility of complexes containing either Sox10 monomers (m) or dimers (d). ( G ) ECR9_myrf-luc reporter plasmids were co-transfected in wildtype (wt) or mutant (3bc, 4a, mt) version in 33B cells with empty shRNA expression vector or vectors coding for Sox10-specific shRNA. The mt version corresponds to a combination of the 3bc and 4a mutations. Luciferase activities were determined and the activity of the wildtype ECR9_myrf-luc reporter in the presence of empty shRNA expression vector was arbitrarily set to 1. All other activities were calculated relative to this value and are presented as mean ± SEM. All experiments were performed three times in quadruplicates.
Figure Legend Snippet: Sox10 binds to intron 1 of the Myrf gene both in vivo and in vitro. ( A ) Schematic representation of the location of regions from the Myrf locus probed by PCR in ChIP studies including the Myrf minimal promoter (myrf), ECR9, ECR11/12 and additional control regions from the 5′ (fl5) and 3′ (3fl) flanking regions of the Myrf gene and from within the adjacent Dagla gene (dagla). ( B–E ) ChIP was performed on 33B cells ( B,C ), rat primary oligodendroglial cells kept in proliferation medium (OPC) or differentiation medium (OL) ( D ) and P14 spinal cord from wildtype (wt) and Sox10 ΔCNS (ko) mice ( E ) using antibodies directed against Sox10 (α-Sox10) ( B , D , E ) or Sox8 (α-Sox8) ( C ) and control preimmune serum (PI). Quantitative PCR was applied on the immunoprecipitate. Values for each fragment correspond to the percentage of material precipitated from the input and represent the mean ± SEM of at least 3 biological replicates. ( F ) EMSA was performed with radiolabelled double-stranded oligonucleotides 3 and 4 in wildtype (3, 4) and mutant (3a, 3b, 3c, 3bc, 4a) versions as indicated below the gels. Oligonucleotides were incubated in the absence (−), or presence (control, Sox10) of protein extracts before gel electrophoresis as indicated above the lanes. Extracts were from mock-transfected HEK293 cells (control) or HEK293 cells expressing full length Sox10 (Sox10). Oligonucleotides with site B and site C/C′ from the Mpz promoter [29] served as positive control for Sox10 binding and as marker for the mobility of complexes containing either Sox10 monomers (m) or dimers (d). ( G ) ECR9_myrf-luc reporter plasmids were co-transfected in wildtype (wt) or mutant (3bc, 4a, mt) version in 33B cells with empty shRNA expression vector or vectors coding for Sox10-specific shRNA. The mt version corresponds to a combination of the 3bc and 4a mutations. Luciferase activities were determined and the activity of the wildtype ECR9_myrf-luc reporter in the presence of empty shRNA expression vector was arbitrarily set to 1. All other activities were calculated relative to this value and are presented as mean ± SEM. All experiments were performed three times in quadruplicates.

Techniques Used: In Vivo, In Vitro, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Mouse Assay, Real-time Polymerase Chain Reaction, Mutagenesis, Incubation, Nucleic Acid Electrophoresis, Transfection, Expressing, Positive Control, Binding Assay, Marker, shRNA, Plasmid Preparation, Luciferase, Activity Assay

Myrf is a Sox10 target gene in OL. ( A–C ) Primary rat OPC were transfected with expression vectors for scrambled (shSCR) or Sox10-specific shRNAs (shSox10) and GFP, and replated in differentiation medium. One day later, transfected cells were visualized by GFP expression (green) and analyzed for their expression of Sox10 ( A ), Myrf ( B ) and Mbp ( C ) (all in red) as indicated. The yellow color in the merged pictures indicates co-expression. Scale bar, 25 µm. ( D,E ) Neural tube electroporations were carried out in HH11-stage chicken embryos using expression vectors for GFP ( D ) and a combination of Sox10 and GFP ( E ). Analysis was one day after electroporation and the electroporated right side is visualized by GFP expression (green). Sections were simultaneously probed for the occurrence of Sox10 (white) and Myrf (red). Scale bars, 25 µm. ( F ) Several ECR ( ECR1-ECR13 ) are localized in the Myrf genomic interval on mouse chromosome 19 between the adjacent Dagla and 1810006K21 Rik genes. ECR locations relative to introns and exons of the Myrf gene are shown. ECR1-ECR6 and ECR13 (marked in green) are conserved among mammals, ECR7-ECR12 (marked in pink) additionally in birds. ( G, H ) The Myrf ECR were tested in 33B cells after transient transfection for their ability to increase expression of a luciferase reporter under control of a minimal promoter (mp). The minimal promoter was taken from the Hsp68 gene ( hsp68-luc ) ( G ) or the Myrf gene ( myrf-luc ) ( H ). Luciferase activities in extracts from transfected cells were determined 48 hours post-transfection in three experiments each performed in quadruplicates. The luciferase activity obtained for a reporter plasmid containing only the minimal promoter (−) was arbitrarily set to 1. Activities in the presence of ECRs were calculated relative to minimal promoter activity and are presented as mean ± SEM. A reporter in which the minimal promoter was combined with Mbp regulatory regions served as positive control. ( I, J ) Transfections of the ECR containing hsp68-luc ( I ) and myrf-luc ( J ) reporters were carried out in the presence of Sox10-specific shRNA (shSox10) and scrambled (shSCR) shRNA. Luciferase activities were determined and the ratio of activities in the presence of Sox10-specific shRNA versus scrambled shRNA was calculated. Normalized values are presented as mean ± SEM. Experiments were performed three times in quadruplicates. shSox10-dependent downregulation of the activity of ECR9-containing luciferase reporters was statistically significant (P≤0.05, determined by Student's t test).
Figure Legend Snippet: Myrf is a Sox10 target gene in OL. ( A–C ) Primary rat OPC were transfected with expression vectors for scrambled (shSCR) or Sox10-specific shRNAs (shSox10) and GFP, and replated in differentiation medium. One day later, transfected cells were visualized by GFP expression (green) and analyzed for their expression of Sox10 ( A ), Myrf ( B ) and Mbp ( C ) (all in red) as indicated. The yellow color in the merged pictures indicates co-expression. Scale bar, 25 µm. ( D,E ) Neural tube electroporations were carried out in HH11-stage chicken embryos using expression vectors for GFP ( D ) and a combination of Sox10 and GFP ( E ). Analysis was one day after electroporation and the electroporated right side is visualized by GFP expression (green). Sections were simultaneously probed for the occurrence of Sox10 (white) and Myrf (red). Scale bars, 25 µm. ( F ) Several ECR ( ECR1-ECR13 ) are localized in the Myrf genomic interval on mouse chromosome 19 between the adjacent Dagla and 1810006K21 Rik genes. ECR locations relative to introns and exons of the Myrf gene are shown. ECR1-ECR6 and ECR13 (marked in green) are conserved among mammals, ECR7-ECR12 (marked in pink) additionally in birds. ( G, H ) The Myrf ECR were tested in 33B cells after transient transfection for their ability to increase expression of a luciferase reporter under control of a minimal promoter (mp). The minimal promoter was taken from the Hsp68 gene ( hsp68-luc ) ( G ) or the Myrf gene ( myrf-luc ) ( H ). Luciferase activities in extracts from transfected cells were determined 48 hours post-transfection in three experiments each performed in quadruplicates. The luciferase activity obtained for a reporter plasmid containing only the minimal promoter (−) was arbitrarily set to 1. Activities in the presence of ECRs were calculated relative to minimal promoter activity and are presented as mean ± SEM. A reporter in which the minimal promoter was combined with Mbp regulatory regions served as positive control. ( I, J ) Transfections of the ECR containing hsp68-luc ( I ) and myrf-luc ( J ) reporters were carried out in the presence of Sox10-specific shRNA (shSox10) and scrambled (shSCR) shRNA. Luciferase activities were determined and the ratio of activities in the presence of Sox10-specific shRNA versus scrambled shRNA was calculated. Normalized values are presented as mean ± SEM. Experiments were performed three times in quadruplicates. shSox10-dependent downregulation of the activity of ECR9-containing luciferase reporters was statistically significant (P≤0.05, determined by Student's t test).

Techniques Used: Transfection, Expressing, Electroporation, Luciferase, Activity Assay, Plasmid Preparation, Positive Control, shRNA

Sox10 and Myrf interact physically and functionally. ( A ) Co-immunoprecipitation (IP) of endogenous Myrf with anti-Sox10 antiserum (αSox10) or preimmune serum (PI) from OLN93 cell extracts. The upper panel shows western blot (WB) detection of Sox10, while the lower panel probes the presence of Myrf in the precipitate using antibodies specifically directed against the carboxyterminal part of the protein. Input corresponds to one tenth of the amount of the protein used in the assay. ( B ) Schematic representation of the Myrf isoform identified by [19] (upper bar, NCBI accession number Q3UR85.2), the splice variant used in this study (lower bar, NCBI accession number AAI57943.1) and various fragments used in interaction studies. Numbers represent amino acid positions. The DNA-binding Ntd80 domain is marked in grey. ( C ) Pulldown assays were performed with Sox10 fragments immobilized as GST-fusions on glutathione sepharose beads and the Myc-tagged Myrf fragments produced in HEK293 cell extracts. Detection of Myrf fragments was by western blot using an antibody directed against the Myc tag. Sox10 regions fused to GST included the dimerization and HMG domains (Dim/HMG), the K2 region and the transactivation domain (TA). ( D–K ) Transient transfections were performed in N2a cells with a luciferase reporter under control of the 727 bp Cx-47 1b promoter ( D ), the 416 bp Cx-32 P2 promoter ( E ), the 626 bp Mag promoter ( F ), the 1.2 kb WmN1 Plp enhancer ( G ), the 3 kb upstream region of the Mbp gene ( H ), a 631 bp conserved region 17 kb upstream of the Mbp gene ( I ), the 415 bp Mpz promoter ( J ) and the 1.3 kb MSE Krox20 enhancer ( K ). Empty pCMV5 expression plasmids (−) or expression plasmids for Sox10 and Myrf were co-transfected as indicated below the bars. Luciferase activities in extracts from transfected cells were determined in at least four experiments each performed in triplicates. The activity obtained for the luciferase reporter in the absence of ectopic transcription factor was arbitrarily set to 1. Fold inductions in the presence of transcription factors were calculated and are presented as mean ± SEM.
Figure Legend Snippet: Sox10 and Myrf interact physically and functionally. ( A ) Co-immunoprecipitation (IP) of endogenous Myrf with anti-Sox10 antiserum (αSox10) or preimmune serum (PI) from OLN93 cell extracts. The upper panel shows western blot (WB) detection of Sox10, while the lower panel probes the presence of Myrf in the precipitate using antibodies specifically directed against the carboxyterminal part of the protein. Input corresponds to one tenth of the amount of the protein used in the assay. ( B ) Schematic representation of the Myrf isoform identified by [19] (upper bar, NCBI accession number Q3UR85.2), the splice variant used in this study (lower bar, NCBI accession number AAI57943.1) and various fragments used in interaction studies. Numbers represent amino acid positions. The DNA-binding Ntd80 domain is marked in grey. ( C ) Pulldown assays were performed with Sox10 fragments immobilized as GST-fusions on glutathione sepharose beads and the Myc-tagged Myrf fragments produced in HEK293 cell extracts. Detection of Myrf fragments was by western blot using an antibody directed against the Myc tag. Sox10 regions fused to GST included the dimerization and HMG domains (Dim/HMG), the K2 region and the transactivation domain (TA). ( D–K ) Transient transfections were performed in N2a cells with a luciferase reporter under control of the 727 bp Cx-47 1b promoter ( D ), the 416 bp Cx-32 P2 promoter ( E ), the 626 bp Mag promoter ( F ), the 1.2 kb WmN1 Plp enhancer ( G ), the 3 kb upstream region of the Mbp gene ( H ), a 631 bp conserved region 17 kb upstream of the Mbp gene ( I ), the 415 bp Mpz promoter ( J ) and the 1.3 kb MSE Krox20 enhancer ( K ). Empty pCMV5 expression plasmids (−) or expression plasmids for Sox10 and Myrf were co-transfected as indicated below the bars. Luciferase activities in extracts from transfected cells were determined in at least four experiments each performed in triplicates. The activity obtained for the luciferase reporter in the absence of ectopic transcription factor was arbitrarily set to 1. Fold inductions in the presence of transcription factors were calculated and are presented as mean ± SEM.

Techniques Used: Immunoprecipitation, Western Blot, Variant Assay, Binding Assay, Produced, Transfection, Luciferase, Plasmid Purification, Expressing, Activity Assay

3) Product Images from "Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function"

Article Title: Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks451

A 3′ ss can mimic a promoter in the pGL4 system by inducing production of spliced readthrough transcripts. ( A ) Expression of luciferase from pGL4.10 and pGL4.17 with and without the globin 3′ ss inserted into the MCS. Error bars, SD. ( B ) 5′ RACE products from pGL4.17 containing the globin insert.
Figure Legend Snippet: A 3′ ss can mimic a promoter in the pGL4 system by inducing production of spliced readthrough transcripts. ( A ) Expression of luciferase from pGL4.10 and pGL4.17 with and without the globin 3′ ss inserted into the MCS. Error bars, SD. ( B ) 5′ RACE products from pGL4.17 containing the globin insert.

Techniques Used: Expressing, Luciferase

4) Product Images from "Determinants of Cell- and Gene-Specific Transcriptional Regulation by the Glucocorticoid Receptor"

Article Title: Determinants of Cell- and Gene-Specific Transcriptional Regulation by the Glucocorticoid Receptor

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.0030094

Some GREs Are Conserved Nongenic Sequences That Exhibit Increased DNAse I Accessibility Upon GR Occupancy (A) Dex induces increased DNAse I accessibility. Nuclei from A549 cells treated with EtOH or dex for 1 h were isolated, treated with DNAse I, and harvested for DNA. The relative amount of the DNA at the corresponding region were assessed by qPCR and presented as percent cleavage, with standard error of mean averaged among at least three independent experiments. Controls #1 and #2 correspond to regions near AMOTL2 and CDH17 genes, respectively, which do not exhibit dex-induced GR occupancy (unpublished data). (B) Human–mouse sequence conservation within GREs is presented. The mouse sequences aligned with 500-bp human GRE sequences were obtained from UCSC Genome Browser. The lengths of the continuous GRE sequences without gaps between human and mouse are shown, and the percent identity of these regions were calculated as number of matched base pairs divided by length of fragment.
Figure Legend Snippet: Some GREs Are Conserved Nongenic Sequences That Exhibit Increased DNAse I Accessibility Upon GR Occupancy (A) Dex induces increased DNAse I accessibility. Nuclei from A549 cells treated with EtOH or dex for 1 h were isolated, treated with DNAse I, and harvested for DNA. The relative amount of the DNA at the corresponding region were assessed by qPCR and presented as percent cleavage, with standard error of mean averaged among at least three independent experiments. Controls #1 and #2 correspond to regions near AMOTL2 and CDH17 genes, respectively, which do not exhibit dex-induced GR occupancy (unpublished data). (B) Human–mouse sequence conservation within GREs is presented. The mouse sequences aligned with 500-bp human GRE sequences were obtained from UCSC Genome Browser. The lengths of the continuous GRE sequences without gaps between human and mouse are shown, and the percent identity of these regions were calculated as number of matched base pairs divided by length of fragment.

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Sequencing

Percentage of Genes Associated with One or More GBRs in A549 Cells A549-specific dex-responsive genes are regulated by GR in A549 cells but not U2OS cells. U2OS-specific dex-responsive genes are regulated by GR in U2OS cells but not in A549 cells. The 34 genes regulated by GR in both A549 and U2OS cells, 12 of which associated with an A549 GBR, were excluded from the analysis shown. Genes responsive to glucocorticoids or androgens in cells other than A549 and U2OS are denoted as “other cells steroid responsive.” Lastly, additional genes that were wholly or partially included in our ChIP-chip arrays due to the extensive sampling of regions around all the genes mentioned above are represented as “genes included in arrays.”
Figure Legend Snippet: Percentage of Genes Associated with One or More GBRs in A549 Cells A549-specific dex-responsive genes are regulated by GR in A549 cells but not U2OS cells. U2OS-specific dex-responsive genes are regulated by GR in U2OS cells but not in A549 cells. The 34 genes regulated by GR in both A549 and U2OS cells, 12 of which associated with an A549 GBR, were excluded from the analysis shown. Genes responsive to glucocorticoids or androgens in cells other than A549 and U2OS are denoted as “other cells steroid responsive.” Lastly, additional genes that were wholly or partially included in our ChIP-chip arrays due to the extensive sampling of regions around all the genes mentioned above are represented as “genes included in arrays.”

Techniques Used: Chromatin Immunoprecipitation, Sampling

Sequence of GREs as Determinants of Gene-Specific Transcriptional Regulation by GR (A) Binding of GR at mouse orthologs of primary GR target genes from human A549 cells is shown. ChIP experiments were performed to monitor GR binding in EtOH and dex-treated C3H10T1/2 cells at genes shown. Immunoprecipitated DNA samples were analyzed with qPCR and normalized to a region near the mouse Hsp70 gene. The nomenclature mGRE represents GRE sequences detected in the mouse genome. (B) Genes adjacent to GREs are regulated by GR in C3H10T1/2 cells. Reverse transcribed RNA samples (cDNA) from C3H10T1/2 cells treated with EtOH or 100 nM dex were subjected to qPCR and normalized to mouse Rpl19 transcripts. (C) Core GR binding sequences are highly conserved. GR binding sequences from human (h), mouse (m), rat (r), and dog (d) are shown. Red sequences represent bases that are identical to that of human. Note that GREs 6.1 and 16.1 each contain two GR binding sites. ( D) Comparative sequence conservation across individual GREs. Sequence identities between human and mouse of GRE X.1 and X.2 were obtained using the same calculation as Figure 3 C. The coordinates represent bp positions with 0 defined as the center of core GR binding sites.
Figure Legend Snippet: Sequence of GREs as Determinants of Gene-Specific Transcriptional Regulation by GR (A) Binding of GR at mouse orthologs of primary GR target genes from human A549 cells is shown. ChIP experiments were performed to monitor GR binding in EtOH and dex-treated C3H10T1/2 cells at genes shown. Immunoprecipitated DNA samples were analyzed with qPCR and normalized to a region near the mouse Hsp70 gene. The nomenclature mGRE represents GRE sequences detected in the mouse genome. (B) Genes adjacent to GREs are regulated by GR in C3H10T1/2 cells. Reverse transcribed RNA samples (cDNA) from C3H10T1/2 cells treated with EtOH or 100 nM dex were subjected to qPCR and normalized to mouse Rpl19 transcripts. (C) Core GR binding sequences are highly conserved. GR binding sequences from human (h), mouse (m), rat (r), and dog (d) are shown. Red sequences represent bases that are identical to that of human. Note that GREs 6.1 and 16.1 each contain two GR binding sites. ( D) Comparative sequence conservation across individual GREs. Sequence identities between human and mouse of GRE X.1 and X.2 were obtained using the same calculation as Figure 3 C. The coordinates represent bp positions with 0 defined as the center of core GR binding sites.

Techniques Used: Sequencing, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

Native Chromosomal GREs Are Composite Elements (A) GBRs confer glucocorticoid responsiveness. A549 cells transfected with luciferase reporter genes linked to 500 bp GBRs were treated with EtOH or 100 nM dex for 5–7 h, harvested, and measured for luciferase activity. Fold dex inductions are plotted for wildtype (white) reporters and mutant (black) reporters with singly (mutGR) or doubly mutated (dmutGR) GR binding sites; standard errors of mean over at least three independent experiments are shown. The 13 mutated GR binding sites were randomly chosen. The GREs that harbor these GR binding sites represent a range of enriched GBRs, ranging from ~6- to 40-fold dex-induced GR occupancy as assessed by ChIP-qPCR (unpublished data). (B) Identification of enriched motifs within GBRs is shown. Top panel: Sequence logo, generated using WebLogo [ 58 ], represents all the compiled sequences resembling GR binding sites identified through computational analysis. Bottom panel shows other enriched motifs (displayed in IUPAC symbols) found in the GRE sequences. Motifs resembling AP-1, HNF4, and C/EBP binding sites were identified using BioProspector whereas motifs similar to ETS and SP1 binding sites were found with MobyDick. The p -values of the enriched motifs represent the random probability of these motifs occurring within the GREs. (C) Conservation analysis of GREs. The identity of the human and mouse sequences was calculated as number of bp matches minus the number of bp deletions or insertions, divided by a 50-bp window. Shown are the average identities for each window across 50 GREs. The background level was calculated as the average of all conservation scores across the 4-kb region. The abscissa shows bp positions with 0 defined as the center of core GR binding sites for GREs.
Figure Legend Snippet: Native Chromosomal GREs Are Composite Elements (A) GBRs confer glucocorticoid responsiveness. A549 cells transfected with luciferase reporter genes linked to 500 bp GBRs were treated with EtOH or 100 nM dex for 5–7 h, harvested, and measured for luciferase activity. Fold dex inductions are plotted for wildtype (white) reporters and mutant (black) reporters with singly (mutGR) or doubly mutated (dmutGR) GR binding sites; standard errors of mean over at least three independent experiments are shown. The 13 mutated GR binding sites were randomly chosen. The GREs that harbor these GR binding sites represent a range of enriched GBRs, ranging from ~6- to 40-fold dex-induced GR occupancy as assessed by ChIP-qPCR (unpublished data). (B) Identification of enriched motifs within GBRs is shown. Top panel: Sequence logo, generated using WebLogo [ 58 ], represents all the compiled sequences resembling GR binding sites identified through computational analysis. Bottom panel shows other enriched motifs (displayed in IUPAC symbols) found in the GRE sequences. Motifs resembling AP-1, HNF4, and C/EBP binding sites were identified using BioProspector whereas motifs similar to ETS and SP1 binding sites were found with MobyDick. The p -values of the enriched motifs represent the random probability of these motifs occurring within the GREs. (C) Conservation analysis of GREs. The identity of the human and mouse sequences was calculated as number of bp matches minus the number of bp deletions or insertions, divided by a 50-bp window. Shown are the average identities for each window across 50 GREs. The background level was calculated as the average of all conservation scores across the 4-kb region. The abscissa shows bp positions with 0 defined as the center of core GR binding sites for GREs.

Techniques Used: Transfection, Luciferase, Activity Assay, Mutagenesis, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Sequencing, Generated

Location and Position of GREs (A) Locations of GREs relative to the TSS of target genes. The number of GREs resident in 10-kb increments relative to the TSS of the target gene are plotted. White bars and black bars represent GREs upstream and downstream of the TSS, respectively. (B) Distribution of GREs relative to target gene transcription start site is shown. The chart presents percentage of GREs at various positions upstream and downstream of target genes. Note that only 64 of the 73 GREs detected in A549 cells were included in these analyses; the remaining nine GREs did not associate with a dex-responsive gene in these cells. The GREs were assigned to the nearest gene regulated by GR in A549 cells from the final list of genes that were included or impinged upon by the ChIP-chip arrays . Coordinates of TSSs were obtained from UCSC Genome Browser based on RefSeq. Similar results were obtained when we used TSS coordinates that were experimentally determined (DataBase of Transcriptional Start Sites) through 5′ end cloning (unpublished data) [ 59 ]. The TSS of the longest transcript was used for genes that have multiple alternative TSSs. Similar results were obtained if the GREs were assigned the closest TSS of the associated dex-responsive gene: 38% of GREs were located downstream from TSS; 58% of GREs were positioned farther than 10 kb from the assigned TSSs.
Figure Legend Snippet: Location and Position of GREs (A) Locations of GREs relative to the TSS of target genes. The number of GREs resident in 10-kb increments relative to the TSS of the target gene are plotted. White bars and black bars represent GREs upstream and downstream of the TSS, respectively. (B) Distribution of GREs relative to target gene transcription start site is shown. The chart presents percentage of GREs at various positions upstream and downstream of target genes. Note that only 64 of the 73 GREs detected in A549 cells were included in these analyses; the remaining nine GREs did not associate with a dex-responsive gene in these cells. The GREs were assigned to the nearest gene regulated by GR in A549 cells from the final list of genes that were included or impinged upon by the ChIP-chip arrays . Coordinates of TSSs were obtained from UCSC Genome Browser based on RefSeq. Similar results were obtained when we used TSS coordinates that were experimentally determined (DataBase of Transcriptional Start Sites) through 5′ end cloning (unpublished data) [ 59 ]. The TSS of the longest transcript was used for genes that have multiple alternative TSSs. Similar results were obtained if the GREs were assigned the closest TSS of the associated dex-responsive gene: 38% of GREs were located downstream from TSS; 58% of GREs were positioned farther than 10 kb from the assigned TSSs.

Techniques Used: Chromatin Immunoprecipitation, Clone Assay

5) Product Images from "Retinoic Acid-Related Orphan Receptor ? (ROR?): A Novel Participant in the Diurnal Regulation of Hepatic Gluconeogenesis and Insulin Sensitivity"

Article Title: Retinoic Acid-Related Orphan Receptor ? (ROR?): A Novel Participant in the Diurnal Regulation of Hepatic Gluconeogenesis and Insulin Sensitivity

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1004331

RORγ regulates the circadian expression of genes involved in gluconeogenesis and glycolysis pathways. ( A ) Circadian expression pattern of G6pase , Pepck , Glut2 , Pklr , Gck , Gckr , Gys2 , Pparδ , and Dlat in liver of WT(ND) and RORγ −/− (ND) mice (n = 4). RNA was isolated every 4 h over a period of 24 h. ( B ) Pklr protein levels at ZT4 and ZT16 in whole liver lysates prepared from WT and RORγ −/− mice fed either a ND or HFD (n = 2–3). Pklr was examined by Western blot analysis. ( C ) Differential expression of several metabolic genes in liver of WT(HFD) and RORγ −/− (HFD) mice collected at ZT0 and ZT12 (n = 5). ( D ) Differential expression of Pcx and Klf15 in WT and RORγ −/− livers collected at ZT12. ( E ) Adenovirus mediated over-expressing of RORγ in RORγ −/− liver enhanced the expression of several glucose metabolic genes. ( F ) G6pase , Pepck , Gck , Glut2 , and Gys2 expression in primary hepatocytes isolated from RORγ −/− mice (n = 3) infected with either empty or RORγ lentivirus. Data represent mean ±SD, * P
Figure Legend Snippet: RORγ regulates the circadian expression of genes involved in gluconeogenesis and glycolysis pathways. ( A ) Circadian expression pattern of G6pase , Pepck , Glut2 , Pklr , Gck , Gckr , Gys2 , Pparδ , and Dlat in liver of WT(ND) and RORγ −/− (ND) mice (n = 4). RNA was isolated every 4 h over a period of 24 h. ( B ) Pklr protein levels at ZT4 and ZT16 in whole liver lysates prepared from WT and RORγ −/− mice fed either a ND or HFD (n = 2–3). Pklr was examined by Western blot analysis. ( C ) Differential expression of several metabolic genes in liver of WT(HFD) and RORγ −/− (HFD) mice collected at ZT0 and ZT12 (n = 5). ( D ) Differential expression of Pcx and Klf15 in WT and RORγ −/− livers collected at ZT12. ( E ) Adenovirus mediated over-expressing of RORγ in RORγ −/− liver enhanced the expression of several glucose metabolic genes. ( F ) G6pase , Pepck , Gck , Glut2 , and Gys2 expression in primary hepatocytes isolated from RORγ −/− mice (n = 3) infected with either empty or RORγ lentivirus. Data represent mean ±SD, * P

Techniques Used: Expressing, Mouse Assay, Isolation, Western Blot, Infection

Transcriptional regulation of glucose metabolic genes by RORγ. ( A ) Sequence and activation of the RORγ binding region of the G6pase (−500/+58) proximal promoter. The ROREs and PPRE are indicated in bold. Activation of the G6pase promoter by RORγ was examined by transfecting Huh-7 cells as indicated with pCMV-β-Gal, pCMV10-3xFlag-RORγ, -RORα or -PPARα (with 10 µM Wy14,643) expression vectors and a pGL4.10 reporter driven by G6Pase (−500/+58) or the promoter in which the RORE and PPRE were mutated. Luciferase activities were normalized to the control transfected with the empty expression vector. ( B ) Inhibition of the activation of the G6pase (−500/+58) promoter by RORγ-selective antagonist “A”. ( C ) Activation of the Pparδ regulatory region by RORγ. Sequence of the RORγ binding region in intron 2 of Pparδ . The three potential ROREs are indicated in bold. Huh-7 cells were co-transfected with pCMV-β-Gal, pCMV10-3xFlag-RORγ or -RORα expression vector, and the pGL4.27 reporter plasmid containing the Pparδ (intron 2) or the intron in which the ROREs are mutated. ( D ) Inhibition of the activation of the Pparδ (intron 2) by the RORγ-selective antagonist. Data represent mean ±SEM, * P
Figure Legend Snippet: Transcriptional regulation of glucose metabolic genes by RORγ. ( A ) Sequence and activation of the RORγ binding region of the G6pase (−500/+58) proximal promoter. The ROREs and PPRE are indicated in bold. Activation of the G6pase promoter by RORγ was examined by transfecting Huh-7 cells as indicated with pCMV-β-Gal, pCMV10-3xFlag-RORγ, -RORα or -PPARα (with 10 µM Wy14,643) expression vectors and a pGL4.10 reporter driven by G6Pase (−500/+58) or the promoter in which the RORE and PPRE were mutated. Luciferase activities were normalized to the control transfected with the empty expression vector. ( B ) Inhibition of the activation of the G6pase (−500/+58) promoter by RORγ-selective antagonist “A”. ( C ) Activation of the Pparδ regulatory region by RORγ. Sequence of the RORγ binding region in intron 2 of Pparδ . The three potential ROREs are indicated in bold. Huh-7 cells were co-transfected with pCMV-β-Gal, pCMV10-3xFlag-RORγ or -RORα expression vector, and the pGL4.27 reporter plasmid containing the Pparδ (intron 2) or the intron in which the ROREs are mutated. ( D ) Inhibition of the activation of the Pparδ (intron 2) by the RORγ-selective antagonist. Data represent mean ±SEM, * P

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

6) Product Images from "Retinoic Acid-Related Orphan Receptor ? (ROR?): A Novel Participant in the Diurnal Regulation of Hepatic Gluconeogenesis and Insulin Sensitivity"

Article Title: Retinoic Acid-Related Orphan Receptor ? (ROR?): A Novel Participant in the Diurnal Regulation of Hepatic Gluconeogenesis and Insulin Sensitivity

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1004331

RORγ regulates the circadian expression of genes involved in gluconeogenesis and glycolysis pathways. ( A ) Circadian expression pattern of G6pase , Pepck , Glut2 , Pklr , Gck , Gckr , Gys2 , Pparδ , and Dlat in liver of WT(ND) and RORγ −/− (ND) mice (n = 4). RNA was isolated every 4 h over a period of 24 h. ( B ) Pklr protein levels at ZT4 and ZT16 in whole liver lysates prepared from WT and RORγ −/− mice fed either a ND or HFD (n = 2–3). Pklr was examined by Western blot analysis. ( C ) Differential expression of several metabolic genes in liver of WT(HFD) and RORγ −/− (HFD) mice collected at ZT0 and ZT12 (n = 5). ( D ) Differential expression of Pcx and Klf15 in WT and RORγ −/− livers collected at ZT12. ( E ) Adenovirus mediated over-expressing of RORγ in RORγ −/− liver enhanced the expression of several glucose metabolic genes. ( F ) G6pase , Pepck , Gck , Glut2 , and Gys2 expression in primary hepatocytes isolated from RORγ −/− mice (n = 3) infected with either empty or RORγ lentivirus. Data represent mean ±SD, * P
Figure Legend Snippet: RORγ regulates the circadian expression of genes involved in gluconeogenesis and glycolysis pathways. ( A ) Circadian expression pattern of G6pase , Pepck , Glut2 , Pklr , Gck , Gckr , Gys2 , Pparδ , and Dlat in liver of WT(ND) and RORγ −/− (ND) mice (n = 4). RNA was isolated every 4 h over a period of 24 h. ( B ) Pklr protein levels at ZT4 and ZT16 in whole liver lysates prepared from WT and RORγ −/− mice fed either a ND or HFD (n = 2–3). Pklr was examined by Western blot analysis. ( C ) Differential expression of several metabolic genes in liver of WT(HFD) and RORγ −/− (HFD) mice collected at ZT0 and ZT12 (n = 5). ( D ) Differential expression of Pcx and Klf15 in WT and RORγ −/− livers collected at ZT12. ( E ) Adenovirus mediated over-expressing of RORγ in RORγ −/− liver enhanced the expression of several glucose metabolic genes. ( F ) G6pase , Pepck , Gck , Glut2 , and Gys2 expression in primary hepatocytes isolated from RORγ −/− mice (n = 3) infected with either empty or RORγ lentivirus. Data represent mean ±SD, * P

Techniques Used: Expressing, Mouse Assay, Isolation, Western Blot, Infection

Transcriptional regulation of glucose metabolic genes by RORγ. ( A ) Sequence and activation of the RORγ binding region of the G6pase (−500/+58) proximal promoter. The ROREs and PPRE are indicated in bold. Activation of the G6pase promoter by RORγ was examined by transfecting Huh-7 cells as indicated with pCMV-β-Gal, pCMV10-3xFlag-RORγ, -RORα or -PPARα (with 10 µM Wy14,643) expression vectors and a pGL4.10 reporter driven by G6Pase (−500/+58) or the promoter in which the RORE and PPRE were mutated. Luciferase activities were normalized to the control transfected with the empty expression vector. ( B ) Inhibition of the activation of the G6pase (−500/+58) promoter by RORγ-selective antagonist “A”. ( C ) Activation of the Pparδ regulatory region by RORγ. Sequence of the RORγ binding region in intron 2 of Pparδ . The three potential ROREs are indicated in bold. Huh-7 cells were co-transfected with pCMV-β-Gal, pCMV10-3xFlag-RORγ or -RORα expression vector, and the pGL4.27 reporter plasmid containing the Pparδ (intron 2) or the intron in which the ROREs are mutated. ( D ) Inhibition of the activation of the Pparδ (intron 2) by the RORγ-selective antagonist. Data represent mean ±SEM, * P
Figure Legend Snippet: Transcriptional regulation of glucose metabolic genes by RORγ. ( A ) Sequence and activation of the RORγ binding region of the G6pase (−500/+58) proximal promoter. The ROREs and PPRE are indicated in bold. Activation of the G6pase promoter by RORγ was examined by transfecting Huh-7 cells as indicated with pCMV-β-Gal, pCMV10-3xFlag-RORγ, -RORα or -PPARα (with 10 µM Wy14,643) expression vectors and a pGL4.10 reporter driven by G6Pase (−500/+58) or the promoter in which the RORE and PPRE were mutated. Luciferase activities were normalized to the control transfected with the empty expression vector. ( B ) Inhibition of the activation of the G6pase (−500/+58) promoter by RORγ-selective antagonist “A”. ( C ) Activation of the Pparδ regulatory region by RORγ. Sequence of the RORγ binding region in intron 2 of Pparδ . The three potential ROREs are indicated in bold. Huh-7 cells were co-transfected with pCMV-β-Gal, pCMV10-3xFlag-RORγ or -RORα expression vector, and the pGL4.27 reporter plasmid containing the Pparδ (intron 2) or the intron in which the ROREs are mutated. ( D ) Inhibition of the activation of the Pparδ (intron 2) by the RORγ-selective antagonist. Data represent mean ±SEM, * P

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

7) Product Images from "Retinoic Acid-Related Orphan Receptor ? (ROR?): A Novel Participant in the Diurnal Regulation of Hepatic Gluconeogenesis and Insulin Sensitivity"

Article Title: Retinoic Acid-Related Orphan Receptor ? (ROR?): A Novel Participant in the Diurnal Regulation of Hepatic Gluconeogenesis and Insulin Sensitivity

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1004331

Transcriptional regulation of glucose metabolic genes by RORγ. ( A ) Sequence and activation of the RORγ binding region of the G6pase (−500/+58) proximal promoter. The ROREs and PPRE are indicated in bold. Activation of the G6pase promoter by RORγ was examined by transfecting Huh-7 cells as indicated with pCMV-β-Gal, pCMV10-3xFlag-RORγ, -RORα or -PPARα (with 10 µM Wy14,643) expression vectors and a pGL4.10 reporter driven by G6Pase (−500/+58) or the promoter in which the RORE and PPRE were mutated. Luciferase activities were normalized to the control transfected with the empty expression vector. ( B ) Inhibition of the activation of the G6pase (−500/+58) promoter by RORγ-selective antagonist “A”. ( C ) Activation of the Pparδ regulatory region by RORγ. Sequence of the RORγ binding region in intron 2 of Pparδ . The three potential ROREs are indicated in bold. Huh-7 cells were co-transfected with pCMV-β-Gal, pCMV10-3xFlag-RORγ or -RORα expression vector, and the pGL4.27 reporter plasmid containing the Pparδ (intron 2) or the intron in which the ROREs are mutated. ( D ) Inhibition of the activation of the Pparδ (intron 2) by the RORγ-selective antagonist. Data represent mean ±SEM, * P
Figure Legend Snippet: Transcriptional regulation of glucose metabolic genes by RORγ. ( A ) Sequence and activation of the RORγ binding region of the G6pase (−500/+58) proximal promoter. The ROREs and PPRE are indicated in bold. Activation of the G6pase promoter by RORγ was examined by transfecting Huh-7 cells as indicated with pCMV-β-Gal, pCMV10-3xFlag-RORγ, -RORα or -PPARα (with 10 µM Wy14,643) expression vectors and a pGL4.10 reporter driven by G6Pase (−500/+58) or the promoter in which the RORE and PPRE were mutated. Luciferase activities were normalized to the control transfected with the empty expression vector. ( B ) Inhibition of the activation of the G6pase (−500/+58) promoter by RORγ-selective antagonist “A”. ( C ) Activation of the Pparδ regulatory region by RORγ. Sequence of the RORγ binding region in intron 2 of Pparδ . The three potential ROREs are indicated in bold. Huh-7 cells were co-transfected with pCMV-β-Gal, pCMV10-3xFlag-RORγ or -RORα expression vector, and the pGL4.27 reporter plasmid containing the Pparδ (intron 2) or the intron in which the ROREs are mutated. ( D ) Inhibition of the activation of the Pparδ (intron 2) by the RORγ-selective antagonist. Data represent mean ±SEM, * P

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

8) Product Images from "Cap-independent translation through the p27 5?-UTR"

Article Title: Cap-independent translation through the p27 5?-UTR

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm512

Effect of an IRE on expression of bicistronic reporters carrying the p27 5′-UTR. Top: Diagram of potential mRNA products resulting from transcription of a bicistronic reporter in which an IRE has been inserted just downstream of the transcriptional start site within the TK promoter. The top diagram shows the expected transcript if there is no cryptic splice site within the 5′-UTR. In this case, only the upstream cistron (Renilla luciferase) would be affected by iron chelation. The bottom diagram shows the expected transcript if the 5′-UTR has a cryptic splice acceptor site. The IRE would be linked to a monocistronic mRNA encoding only firefly luciferase and iron chelation would inhibit expression of this enzyme. Bottom: NIH3T3 cells were transfected with bicistronic reporters pTKLL-472 and pTKLL-472-IRE. These constructs are identical except that pTKLL-472-IRE has an IRE inserted 35 nt downstream of the TK transcriptional start site. The transfected cells were treated with (+, gray bars) or without (−, black bars) the iron chelator DFO. The cells were harvested and Renilla (R) and firefly (F) luciferase activities were determined. For each transfection, the value of luciferase activity in the absence of DFO was set to 1.
Figure Legend Snippet: Effect of an IRE on expression of bicistronic reporters carrying the p27 5′-UTR. Top: Diagram of potential mRNA products resulting from transcription of a bicistronic reporter in which an IRE has been inserted just downstream of the transcriptional start site within the TK promoter. The top diagram shows the expected transcript if there is no cryptic splice site within the 5′-UTR. In this case, only the upstream cistron (Renilla luciferase) would be affected by iron chelation. The bottom diagram shows the expected transcript if the 5′-UTR has a cryptic splice acceptor site. The IRE would be linked to a monocistronic mRNA encoding only firefly luciferase and iron chelation would inhibit expression of this enzyme. Bottom: NIH3T3 cells were transfected with bicistronic reporters pTKLL-472 and pTKLL-472-IRE. These constructs are identical except that pTKLL-472-IRE has an IRE inserted 35 nt downstream of the TK transcriptional start site. The transfected cells were treated with (+, gray bars) or without (−, black bars) the iron chelator DFO. The cells were harvested and Renilla (R) and firefly (F) luciferase activities were determined. For each transfection, the value of luciferase activity in the absence of DFO was set to 1.

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

Transfection of pGL4-based monocistronic vectors into MCF7 cells. ( A ) Diagram of monocistronic constructs. ( B ) Firefly luciferase activity after transfection of each of the constructs as shown in A. The number above each bar represents the mean of three separate transfections (in RLU × 10 −6 ). The error bars represent SD.
Figure Legend Snippet: Transfection of pGL4-based monocistronic vectors into MCF7 cells. ( A ) Diagram of monocistronic constructs. ( B ) Firefly luciferase activity after transfection of each of the constructs as shown in A. The number above each bar represents the mean of three separate transfections (in RLU × 10 −6 ). The error bars represent SD.

Techniques Used: Transfection, Construct, Luciferase, Activity Assay

9) Product Images from "Vitamin D Analogs Differentially Control Antimicrobial Peptide/"Alarmin"Expression in Psoriasis"

Article Title: Vitamin D Analogs Differentially Control Antimicrobial Peptide/"Alarmin"Expression in Psoriasis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0006340

Vitamin D analogs enhance cathelicidin promoter activity and induce expression in primary human epidermal keratinocytes. (A) To analyze the effects of vitamin D analogs on the cathelicidin promoter a 5 kb fragment of the 5′ UTR of the cathelicidin gene CAMP was cloned into a luciferase reporter plasmid and transfected into HaCaT keratinocytes. Cells were stimulated with 1,25D3, ZK159222, ZK191784, ZK203278 and calcipotriol (all at 10 −7 M) and luciferase activity was assayed (** P
Figure Legend Snippet: Vitamin D analogs enhance cathelicidin promoter activity and induce expression in primary human epidermal keratinocytes. (A) To analyze the effects of vitamin D analogs on the cathelicidin promoter a 5 kb fragment of the 5′ UTR of the cathelicidin gene CAMP was cloned into a luciferase reporter plasmid and transfected into HaCaT keratinocytes. Cells were stimulated with 1,25D3, ZK159222, ZK191784, ZK203278 and calcipotriol (all at 10 −7 M) and luciferase activity was assayed (** P

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

10) Product Images from "Species-specific difference in expression and splice-site choice in Inpp5b, an inositol polyphosphate 5-phosphatase paralogous to the enzyme deficient in Lowe Syndrome"

Article Title: Species-specific difference in expression and splice-site choice in Inpp5b, an inositol polyphosphate 5-phosphatase paralogous to the enzyme deficient in Lowe Syndrome

Journal: Mammalian Genome

doi: 10.1007/s00335-010-9281-7

a Alignment of segments of human and mouse Inpp5b enzyme encoded by exons 7 and 8. The segments corresponding to exons 7 and 8 are shaded as indicated. b Alignment of human and mouse exons 7, the portion of human intron 7 homologous to mouse exon 7, and exon 8. Segments are shaded with the same patterns used in a , indicating the various portions of exon 7 and exon 8. The location of primers discussed in the text is shown. The bent arrow indicates the approximate location of the transcriptional start of the transcripts generated from the internal promoter in mouse exon 7 and the analogous region in human intron 7
Figure Legend Snippet: a Alignment of segments of human and mouse Inpp5b enzyme encoded by exons 7 and 8. The segments corresponding to exons 7 and 8 are shaded as indicated. b Alignment of human and mouse exons 7, the portion of human intron 7 homologous to mouse exon 7, and exon 8. Segments are shaded with the same patterns used in a , indicating the various portions of exon 7 and exon 8. The location of primers discussed in the text is shown. The bent arrow indicates the approximate location of the transcriptional start of the transcripts generated from the internal promoter in mouse exon 7 and the analogous region in human intron 7

Techniques Used: Generated

a Semiquantitative reverse transcriptase PCR of murine brain (B) and kidney (K) RNA using primers MusF1 and MusR for 25, 30, and 35 cycles. A 50-bp DNA ladder is shown on the left with the 200- and 350-bp fragments labeled for orientation purposes. Only a 297-bp fragment corresponding to splicing at the GT splice site was seen with RT-PCR of mouse Inpp5b using MusF1 and MusR primers. b Reverse transcriptase PCR of human brain RNA using primers HsaF1, HsaF2, and HsaF3 and reverse primer HsaR1. A 50-bp DNA ladder is shown on the left with the 200- and 350-bp fragments labeled for orientation purposes. A negative control in which the reverse transcriptase was omitted (-RT) is shown. A 192-bp fragment corresponding to splicing at the GC splice site was seen with HsaF1 and HsaR1 primers, while a 146-bp fragment was seen with HsaF3 and HsaR1. PCR of an artificial DNA construct, ex7 + ex8, which consists of a segment of genomic DNA containing human exon 7 and the first 240 bp of intron 7 ligated to a segment of genomic DNA containing exon 8, is shown as a positive control for all three forward human primers, particularly HsaF2. c Same as B except kidney RNA was used
Figure Legend Snippet: a Semiquantitative reverse transcriptase PCR of murine brain (B) and kidney (K) RNA using primers MusF1 and MusR for 25, 30, and 35 cycles. A 50-bp DNA ladder is shown on the left with the 200- and 350-bp fragments labeled for orientation purposes. Only a 297-bp fragment corresponding to splicing at the GT splice site was seen with RT-PCR of mouse Inpp5b using MusF1 and MusR primers. b Reverse transcriptase PCR of human brain RNA using primers HsaF1, HsaF2, and HsaF3 and reverse primer HsaR1. A 50-bp DNA ladder is shown on the left with the 200- and 350-bp fragments labeled for orientation purposes. A negative control in which the reverse transcriptase was omitted (-RT) is shown. A 192-bp fragment corresponding to splicing at the GC splice site was seen with HsaF1 and HsaR1 primers, while a 146-bp fragment was seen with HsaF3 and HsaR1. PCR of an artificial DNA construct, ex7 + ex8, which consists of a segment of genomic DNA containing human exon 7 and the first 240 bp of intron 7 ligated to a segment of genomic DNA containing exon 8, is shown as a positive control for all three forward human primers, particularly HsaF2. c Same as B except kidney RNA was used

Techniques Used: Polymerase Chain Reaction, Labeling, Reverse Transcription Polymerase Chain Reaction, Negative Control, Construct, Positive Control

11) Product Images from "Intestinal Alkaline Phosphatase Inhibits the Translocation of Bacteria of Gut-Origin in Mice with Peritonitis: Mechanism of Action"

Article Title: Intestinal Alkaline Phosphatase Inhibits the Translocation of Bacteria of Gut-Origin in Mice with Peritonitis: Mechanism of Action

Journal: PLoS ONE

doi: 10.1371/journal.pone.0124835

Activity of the Claudin-2 promoter in Caco-2 cells treated with IAP. Caco-2 cells were treated with varying concentrations of IAP. Cells were transfected with the Claudin-2 promoter linked to a luciferase reporter and a Renilla luciferase reporter, as a control, for 24 h and were treated with varying dose of IAP over the following 24 h. Luciferase reporter activities were examined. The activity of the firefly reporter in the IAP-treated cells was divided by it’s the activity of the Renilla luciferase reporter in control cells. Values represent the mean ± SEM of data from three separate experiments. **P
Figure Legend Snippet: Activity of the Claudin-2 promoter in Caco-2 cells treated with IAP. Caco-2 cells were treated with varying concentrations of IAP. Cells were transfected with the Claudin-2 promoter linked to a luciferase reporter and a Renilla luciferase reporter, as a control, for 24 h and were treated with varying dose of IAP over the following 24 h. Luciferase reporter activities were examined. The activity of the firefly reporter in the IAP-treated cells was divided by it’s the activity of the Renilla luciferase reporter in control cells. Values represent the mean ± SEM of data from three separate experiments. **P

Techniques Used: Activity Assay, Transfection, Luciferase

Activity of the VEGF promoter and the Claudin-2 promoter in Caco-2 cells treated with IAP. A: The DNA binding domains of SP-1 and Cdx-1 respectively. B: The ChIP assay was performed to examine the DNA-binding activity of SP1 to the VEGF promoter and of Cdx-2 to the Claudin-2 promoter in Caco-2 cells receiving varying IAP treatments. C: The quantification of SP1 and VEGF promoter PCR data; the data were analyzed using the Image J software.
Figure Legend Snippet: Activity of the VEGF promoter and the Claudin-2 promoter in Caco-2 cells treated with IAP. A: The DNA binding domains of SP-1 and Cdx-1 respectively. B: The ChIP assay was performed to examine the DNA-binding activity of SP1 to the VEGF promoter and of Cdx-2 to the Claudin-2 promoter in Caco-2 cells receiving varying IAP treatments. C: The quantification of SP1 and VEGF promoter PCR data; the data were analyzed using the Image J software.

Techniques Used: Activity Assay, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Software

IAP inhibited VEGF and Claudin-2 expression in Caco-2 cells. Caco-2 cells treated with IAP were used for immunohistochemical analyses of VEGF and Claudin-2. Photomicrographs were taken at 20x magnification.
Figure Legend Snippet: IAP inhibited VEGF and Claudin-2 expression in Caco-2 cells. Caco-2 cells treated with IAP were used for immunohistochemical analyses of VEGF and Claudin-2. Photomicrographs were taken at 20x magnification.

Techniques Used: Expressing, Immunohistochemistry

ERK phosphorylation levels and expression of related proteins following treatment with a phosphatase inhibitor in Caco-2 cells pretreated with IAP. A: Caco-2 cells were treated with varying concentrations of IAP and sodium orthovanadate. Fresh protein samples were extracted from pretreated Caco-2 cells and were subsequently processed for Western blotting assays. Caco-2 cells were treated with increasing concentrations of IAP for 24 h (0, 4, 16 mIU) and with 15mM sodium orthovanadate. Whole-cell proteins were extracted and subjected to Western blotting. Primary antibodies of p-ERK, SP-1, VEGF, Cdx-2 or Claudin-2 were used for the blotting assays. β-actin immunoblotting was performed as an internal loading control. B: The quantification of the proteins Western blotting data; blots were analyzed using the Image J software.
Figure Legend Snippet: ERK phosphorylation levels and expression of related proteins following treatment with a phosphatase inhibitor in Caco-2 cells pretreated with IAP. A: Caco-2 cells were treated with varying concentrations of IAP and sodium orthovanadate. Fresh protein samples were extracted from pretreated Caco-2 cells and were subsequently processed for Western blotting assays. Caco-2 cells were treated with increasing concentrations of IAP for 24 h (0, 4, 16 mIU) and with 15mM sodium orthovanadate. Whole-cell proteins were extracted and subjected to Western blotting. Primary antibodies of p-ERK, SP-1, VEGF, Cdx-2 or Claudin-2 were used for the blotting assays. β-actin immunoblotting was performed as an internal loading control. B: The quantification of the proteins Western blotting data; blots were analyzed using the Image J software.

Techniques Used: Expressing, Western Blot, Software

Changes in protein expression and the time course of the changes in the ERK phosphorylation in Caco-2 cells pretreated with IAP. A: Caco-2 cells were treated with increasing concentrations of IAP for 48 h (0, 4, 16 mIU). Whole-cell protein were extracted and subjected to Western blotting. Primary antibodies of ERK, p-ERK, SP-1, VEGF, Cdx-2 or Claudin-2 were used for the blotting assays. β-Actin immunoblotting was performed as an internal loading control. B: Caco-2 cells were treated with 16 mIU IAP for varying lengths of time (0, 0.5, 2 or 4 h). Upper figure, levels of phosphorylated ERK detected by Western blotting. C: The quantification of ERK and p-ERK Western blotting data of the dose-cause; blots were analyzed using the Image J software. D: The quantification of other proteins Western blotting data of the dose-cause; blots were analyzed using the Image J software. E: The quantification of ERK and p-ERK Western blotting data of the time-cause; blots were analyzed using the Image J software.
Figure Legend Snippet: Changes in protein expression and the time course of the changes in the ERK phosphorylation in Caco-2 cells pretreated with IAP. A: Caco-2 cells were treated with increasing concentrations of IAP for 48 h (0, 4, 16 mIU). Whole-cell protein were extracted and subjected to Western blotting. Primary antibodies of ERK, p-ERK, SP-1, VEGF, Cdx-2 or Claudin-2 were used for the blotting assays. β-Actin immunoblotting was performed as an internal loading control. B: Caco-2 cells were treated with 16 mIU IAP for varying lengths of time (0, 0.5, 2 or 4 h). Upper figure, levels of phosphorylated ERK detected by Western blotting. C: The quantification of ERK and p-ERK Western blotting data of the dose-cause; blots were analyzed using the Image J software. D: The quantification of other proteins Western blotting data of the dose-cause; blots were analyzed using the Image J software. E: The quantification of ERK and p-ERK Western blotting data of the time-cause; blots were analyzed using the Image J software.

Techniques Used: Expressing, Western Blot, Software

12) 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

Genetic inhibition of CEBPA impairs HK3 and KLF5 upregulation during neutrophil differentiation of APL cells. (a) NB4 or HT93 APL cell lines were stably transduced with pLKO.1 lentiviral vectors expressing non-targeting or two independent CEBPA -targeting shRNAs. APL control and CEBPA knockdown cells were differentiated with 1 μM ATRA for 4 days. Knockdown efficiency in NB4 (top panels) or HT93 (bottom panels) APL cells was validated by qPCR. CEBPA mRNA expression was normalized to the housekeeping gene HMBS and is shown as n-fold changes compared to untreated SHC002 control cells. HK3 (b) and KLF5 (c) mRNA expression in NB4 or HT93 APL cells was determined by qPCR and analyzed as in 2a. Impairment in granulocytic differentiation of CEBPA knockdown cells was shown by a reduction of the neutrophil marker granulocyte colony-stimulating factor receptor (GCSFR or CSF3R) (d) Data represent the mean ± s.d. of at least three independent experiments. (e), (f) CEBPA Knockdown efficiency at the protein level in NB4 and HT93 APL cells was confirmed by western blotting. GAPDH is shown as a loading control. MWU: *p
Figure Legend Snippet: Genetic inhibition of CEBPA impairs HK3 and KLF5 upregulation during neutrophil differentiation of APL cells. (a) NB4 or HT93 APL cell lines were stably transduced with pLKO.1 lentiviral vectors expressing non-targeting or two independent CEBPA -targeting shRNAs. APL control and CEBPA knockdown cells were differentiated with 1 μM ATRA for 4 days. Knockdown efficiency in NB4 (top panels) or HT93 (bottom panels) APL cells was validated by qPCR. CEBPA mRNA expression was normalized to the housekeeping gene HMBS and is shown as n-fold changes compared to untreated SHC002 control cells. HK3 (b) and KLF5 (c) mRNA expression in NB4 or HT93 APL cells was determined by qPCR and analyzed as in 2a. Impairment in granulocytic differentiation of CEBPA knockdown cells was shown by a reduction of the neutrophil marker granulocyte colony-stimulating factor receptor (GCSFR or CSF3R) (d) Data represent the mean ± s.d. of at least three independent experiments. (e), (f) CEBPA Knockdown efficiency at the protein level in NB4 and HT93 APL cells was confirmed by western blotting. GAPDH is shown as a loading control. MWU: *p

Techniques Used: Inhibition, Stable Transfection, Transduction, Expressing, Real-time Polymerase Chain Reaction, Marker, Western Blot

HK3 and KLF5 expression is significantly downregulated in CEBPA -mutated AML patients. HK3 (a) and KLF5 (b) mRNA levels were measured by qPCR in total RNA extracted from primary AML (FAB M0-M7) blasts, CD34 + samples or granulocytes from healthy donors. Patient characteristics are summarized in Supplementary Table 1 . HK3 (c) and KLF5 (d) levels in 154 patients from the Taskesen cohort with normal karyotype, expressing wild type CEBPA (WT), one allele mutated (SM) or two alleles mutated (DM). MWU: *p
Figure Legend Snippet: HK3 and KLF5 expression is significantly downregulated in CEBPA -mutated AML patients. HK3 (a) and KLF5 (b) mRNA levels were measured by qPCR in total RNA extracted from primary AML (FAB M0-M7) blasts, CD34 + samples or granulocytes from healthy donors. Patient characteristics are summarized in Supplementary Table 1 . HK3 (c) and KLF5 (d) levels in 154 patients from the Taskesen cohort with normal karyotype, expressing wild type CEBPA (WT), one allele mutated (SM) or two alleles mutated (DM). MWU: *p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

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

Ectopic expression of CEBPA activates HK3 and KLF5 transcription. (a–e) HT93 cells were transiently transfected with pcDNA3.1 empty control or a CEBPA expression vector. HK3 (a) and KLF5 (b) mRNA expression was quantified by qPCR. Data were normalized to HMBS and are shown as n-fold regulation as compared to control transfected cells. Induction of CEBPE mRNA, a direct target gene of CEBPA , was measured as a positive control for CEBPA activity (c). Results are the means ± s.d. of at least triplicate transfections. CEBPA transfection efficiency was measured by qPCR (d) and western blotting (e). GAPDH is shown as a loading control. (f–g) Different CEBPA-ER fusion constructs were induced by treating the respective K562 cell lines with 5 μM Tamoxifen for 24 h. HK3 (f) or KLF5 (g) mRNA expression was quantified by qPCR as in 2a. Expression of the CEBPA target CEBPE was measured as positive control for CEBPA activation in wildtype CEBPA p42 expressing K562 cells. MWU: *p
Figure Legend Snippet: Ectopic expression of CEBPA activates HK3 and KLF5 transcription. (a–e) HT93 cells were transiently transfected with pcDNA3.1 empty control or a CEBPA expression vector. HK3 (a) and KLF5 (b) mRNA expression was quantified by qPCR. Data were normalized to HMBS and are shown as n-fold regulation as compared to control transfected cells. Induction of CEBPE mRNA, a direct target gene of CEBPA , was measured as a positive control for CEBPA activity (c). Results are the means ± s.d. of at least triplicate transfections. CEBPA transfection efficiency was measured by qPCR (d) and western blotting (e). GAPDH is shown as a loading control. (f–g) Different CEBPA-ER fusion constructs were induced by treating the respective K562 cell lines with 5 μM Tamoxifen for 24 h. HK3 (f) or KLF5 (g) mRNA expression was quantified by qPCR as in 2a. Expression of the CEBPA target CEBPE was measured as positive control for CEBPA activation in wildtype CEBPA p42 expressing K562 cells. MWU: *p

Techniques Used: Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Positive Control, Activity Assay, Western Blot, Construct, Activation Assay

13) Product Images from "Cell Cycle-Dependent Expression of Dub3, Nanog and the p160 Family of Nuclear Receptor Coactivators (NCoAs) in Mouse Embryonic Stem Cells"

Article Title: Cell Cycle-Dependent Expression of Dub3, Nanog and the p160 Family of Nuclear Receptor Coactivators (NCoAs) in Mouse Embryonic Stem Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0093663

Transcriptional regulation of Dub3 by Esrrb is dependent on coactivator recruitment. (A) Schematic representation of the proximal promoter of the mouse Dub3 gene and reporter construct is shown. Transcriptional activity of the ERRs family of orphan receptors on the Dub3 promoter. CV1 cells were transfected with equal amount of plasmid DNA and luciferase activity was measured 48 hours after transfection. Data is shown as average of fold induction of six biological replicates and the error bars indicate the standard deviation. Two asterisks indicate that 0.001
Figure Legend Snippet: Transcriptional regulation of Dub3 by Esrrb is dependent on coactivator recruitment. (A) Schematic representation of the proximal promoter of the mouse Dub3 gene and reporter construct is shown. Transcriptional activity of the ERRs family of orphan receptors on the Dub3 promoter. CV1 cells were transfected with equal amount of plasmid DNA and luciferase activity was measured 48 hours after transfection. Data is shown as average of fold induction of six biological replicates and the error bars indicate the standard deviation. Two asterisks indicate that 0.001

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

Direct interaction between NCoA1 splice variants and Esrrb. (A) CV1 cells were transfected with equal amount of plasmid DNA (50 ng reporter/450 ng Esrrb/450 ng NCoA) and luciferase activity was measured 48 hours after transfection. Data were normalised to pGL4.10 empty vector. Data is shown as average of fold induction of six biological replicates and the error bars indicate the standard deviation. Four asterisks indicate that P
Figure Legend Snippet: Direct interaction between NCoA1 splice variants and Esrrb. (A) CV1 cells were transfected with equal amount of plasmid DNA (50 ng reporter/450 ng Esrrb/450 ng NCoA) and luciferase activity was measured 48 hours after transfection. Data were normalised to pGL4.10 empty vector. Data is shown as average of fold induction of six biological replicates and the error bars indicate the standard deviation. Four asterisks indicate that P

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

14) Product Images from "Development of a Tet-On inducible expression system for the anhydrobiotic cell line, Pv11"

Article Title: Development of a Tet-On inducible expression system for the anhydrobiotic cell line, Pv11

Journal: bioRxiv

doi: 10.1101/2020.05.29.123570

The 137 bp-fragment from the 121-promoter and its secondary structure. (A) Optical density (OD) of E. coli transformed with pTetO-137bp-Nluc and pTetO-202bp-Nluc. The transformants were cultured in LB medium containing ampicillin at 37°C for 16 h, then OD 600 was measured. (B) Yields of the pTetO-137bp-Nluc and pTetO-202bp-Nluc plasmids. The plasmids were purified, and the concentration was measured. Normalized values are expressed as means ± standard deviations (SD). **** p
Figure Legend Snippet: The 137 bp-fragment from the 121-promoter and its secondary structure. (A) Optical density (OD) of E. coli transformed with pTetO-137bp-Nluc and pTetO-202bp-Nluc. The transformants were cultured in LB medium containing ampicillin at 37°C for 16 h, then OD 600 was measured. (B) Yields of the pTetO-137bp-Nluc and pTetO-202bp-Nluc plasmids. The plasmids were purified, and the concentration was measured. Normalized values are expressed as means ± standard deviations (SD). **** p

Techniques Used: Transformation Assay, Cell Culture, Purification, Concentration Assay

15) Product Images from "Elements at the 5′ end of Xist harbor SPEN-independent transcriptional antiterminator activity"

Article Title: Elements at the 5′ end of Xist harbor SPEN-independent transcriptional antiterminator activity

Journal: bioRxiv

doi: 10.1101/2020.05.13.090506

As single-copy transgenes, 5′ fragments of Xist cause transcriptional readthrough beyond their polyadenylation sites. ( A ) Diagram of recombination-mediated cassette exchange (RMCE) expression cassette inserted into the Rosa26 locus on the B6 allele of chromosome 6 in B6/Cast F1 hybrid ES cells. Full-length Xist and three mutant transgene cargos were inserted into this expression cassette under a doxycycline-inducible promoter. Xist -5.5kb is missing a portion of Repeat B (Supplementary Figure S6A). HygroR , constitutively expressed hygromycin resistance gene. ( B ) Stellaris single-molecule RNA FISH for Xist RNA (red) in cells expressing Xist -2kb, Xist -5.5kb, Xist -2kb+6,7 or full-length Xist inserted at Rosa26 , treated with or without doxycycline. DAPI-stained nuclei are blue. Scale bar = 10 μm. ( C ) Xist expression in each cell line following 3 d doxycycline treatment, determined via RNA-seq. Reads per kilobase per million (RPKM) values were calculated using reads aligned to the endogenous Xist locus divided by length of inserted transcript in kb divided by total aligned reads in millions for each dataset. ( D ) RNA-seq reads were aligned to a custom-made IGV build containing the single-insertion Xist / HygroR cassette within the Rosa26 locus on chromosome 6. Top panel depicts this cassette upstream of the endogenous Setd5 gene. Dashed lines within the Xist locus represent sequences not present in each version of the RMCE cassette. The bottom panel shows a closer view of RNA-seq reads extending beyond the SV40 polyadenylation sequence at the 3′ end of Xist , with regions of clear transcriptional readthrough highlighted in grey. ( E ) Nuclear ratio of reads mapping to HygroR following doxycycline treatment. ( F ) Nuclear ratio of allele-specific reads mapping to Setd5 following doxycycline treatment. Note the difference in y-axis compared to (E). ***P
Figure Legend Snippet: As single-copy transgenes, 5′ fragments of Xist cause transcriptional readthrough beyond their polyadenylation sites. ( A ) Diagram of recombination-mediated cassette exchange (RMCE) expression cassette inserted into the Rosa26 locus on the B6 allele of chromosome 6 in B6/Cast F1 hybrid ES cells. Full-length Xist and three mutant transgene cargos were inserted into this expression cassette under a doxycycline-inducible promoter. Xist -5.5kb is missing a portion of Repeat B (Supplementary Figure S6A). HygroR , constitutively expressed hygromycin resistance gene. ( B ) Stellaris single-molecule RNA FISH for Xist RNA (red) in cells expressing Xist -2kb, Xist -5.5kb, Xist -2kb+6,7 or full-length Xist inserted at Rosa26 , treated with or without doxycycline. DAPI-stained nuclei are blue. Scale bar = 10 μm. ( C ) Xist expression in each cell line following 3 d doxycycline treatment, determined via RNA-seq. Reads per kilobase per million (RPKM) values were calculated using reads aligned to the endogenous Xist locus divided by length of inserted transcript in kb divided by total aligned reads in millions for each dataset. ( D ) RNA-seq reads were aligned to a custom-made IGV build containing the single-insertion Xist / HygroR cassette within the Rosa26 locus on chromosome 6. Top panel depicts this cassette upstream of the endogenous Setd5 gene. Dashed lines within the Xist locus represent sequences not present in each version of the RMCE cassette. The bottom panel shows a closer view of RNA-seq reads extending beyond the SV40 polyadenylation sequence at the 3′ end of Xist , with regions of clear transcriptional readthrough highlighted in grey. ( E ) Nuclear ratio of reads mapping to HygroR following doxycycline treatment. ( F ) Nuclear ratio of allele-specific reads mapping to Setd5 following doxycycline treatment. Note the difference in y-axis compared to (E). ***P

Techniques Used: Expressing, Mutagenesis, Fluorescence In Situ Hybridization, Staining, RNA Sequencing Assay, Sequencing

Xist -2kb binds SPEN and RBM15, but these proteins are not required for transcriptional readthrough. ( A ) Diagram of Xist -2kb showing locations of qPCR primer pairs. ( B ) Four different cell identities (cells without TETRIS cargo, TETRISv1 Xist -2kb cells treated with or without doxycycline, and TETRISv1 Xist -2kb ΔrA234 cells treated with doxycycline) were used for RNA immunoprecipitation with SPEN, RBM15, or IgG control antibodies, followed by random-primed RT and qPCR with primer pairs shown in (A). Values are relative to Xist -2kb + dox input. ( C ) Luminescence assay in wild-type and Δ Spen cells harboring TETRISv1 Xist -2kb cargo. ( D ) Nuclear RNA from Δ Spen cells harboring TETRISv1 Xist -2kb cargo was reverse-transcribed with random primers and analyzed by qPCR with primer pairs targeting regions shown in Figure 1A . ( E ) Western blots demonstrating successful CRISPR-Cas9 targeting of Ezh2 , Rbm15 , Rbm15b , and both Rbm15 and Rbm15b in cells harboring TETRISv1 Xist -2kb cargo. ( F ) Luminescence assays with CRISPR knockdown TETRISv1 Xist -2kb cells shown in (E). See Supplementary Figure S4 for characterization of Δ Spen cells and for locations of CRISPR gRNAs. Dots represent individual technical replicate measurements, and bars represent the average value. See Supplementary Table S1 for information regarding experimental replicates and Supplementary Table S2 for oligo sequences.
Figure Legend Snippet: Xist -2kb binds SPEN and RBM15, but these proteins are not required for transcriptional readthrough. ( A ) Diagram of Xist -2kb showing locations of qPCR primer pairs. ( B ) Four different cell identities (cells without TETRIS cargo, TETRISv1 Xist -2kb cells treated with or without doxycycline, and TETRISv1 Xist -2kb ΔrA234 cells treated with doxycycline) were used for RNA immunoprecipitation with SPEN, RBM15, or IgG control antibodies, followed by random-primed RT and qPCR with primer pairs shown in (A). Values are relative to Xist -2kb + dox input. ( C ) Luminescence assay in wild-type and Δ Spen cells harboring TETRISv1 Xist -2kb cargo. ( D ) Nuclear RNA from Δ Spen cells harboring TETRISv1 Xist -2kb cargo was reverse-transcribed with random primers and analyzed by qPCR with primer pairs targeting regions shown in Figure 1A . ( E ) Western blots demonstrating successful CRISPR-Cas9 targeting of Ezh2 , Rbm15 , Rbm15b , and both Rbm15 and Rbm15b in cells harboring TETRISv1 Xist -2kb cargo. ( F ) Luminescence assays with CRISPR knockdown TETRISv1 Xist -2kb cells shown in (E). See Supplementary Figure S4 for characterization of Δ Spen cells and for locations of CRISPR gRNAs. Dots represent individual technical replicate measurements, and bars represent the average value. See Supplementary Table S1 for information regarding experimental replicates and Supplementary Table S2 for oligo sequences.

Techniques Used: Real-time Polymerase Chain Reaction, Immunoprecipitation, Random Primed, Luminescence Assay, Western Blot, CRISPR

Xist -2kb induces mild levels of local transcriptional silencing in a SPEN-independent manner. ( A ) Diagram of TETRISv3 DNA cassette. ( B) RT-qPCR showing levels of Spen mRNA in wild-type and Δ Spen cells harboring the TETRISv3 empty and Xist -2kb cassettes. See Supplementary Figure S4 for additional characterization of Δ Spen cells. ( C ) Luminescence assay showing relative luciferase protein levels in TETRISv3 empty and Xist -2kb cells treated with or without doxycycline for two days. ( D ) The Xist -2kb values in (C) are shown normalized to the corresponding empty values. ( E-F ) Random-primed reverse transcription of nuclear and cytoplasmic RNA from each cell line was followed by qPCR targeting either luciferase (E) or PuroR (F). ( G-H ) The sum of nuclear and cytoplasmic qPCR signal for each cell line in (E-F) was normalized to one to depict nucleocytoplasmic ratios. Dots represent individual technical replicate measurements, and bars represent the average value. See Supplementary Table S1 for information regarding experimental replicates and Supplementary Table S2 for oligo sequences.
Figure Legend Snippet: Xist -2kb induces mild levels of local transcriptional silencing in a SPEN-independent manner. ( A ) Diagram of TETRISv3 DNA cassette. ( B) RT-qPCR showing levels of Spen mRNA in wild-type and Δ Spen cells harboring the TETRISv3 empty and Xist -2kb cassettes. See Supplementary Figure S4 for additional characterization of Δ Spen cells. ( C ) Luminescence assay showing relative luciferase protein levels in TETRISv3 empty and Xist -2kb cells treated with or without doxycycline for two days. ( D ) The Xist -2kb values in (C) are shown normalized to the corresponding empty values. ( E-F ) Random-primed reverse transcription of nuclear and cytoplasmic RNA from each cell line was followed by qPCR targeting either luciferase (E) or PuroR (F). ( G-H ) The sum of nuclear and cytoplasmic qPCR signal for each cell line in (E-F) was normalized to one to depict nucleocytoplasmic ratios. Dots represent individual technical replicate measurements, and bars represent the average value. See Supplementary Table S1 for information regarding experimental replicates and Supplementary Table S2 for oligo sequences.

Techniques Used: Quantitative RT-PCR, Luminescence Assay, Luciferase, Random Primed, Real-time Polymerase Chain Reaction

Xist -2kb represses expression of adjacent genes by reading through multiple polyadenylation sequences. ( A ) Schematics of TETRISv1 DNA cassettes. ITR, inverted tandem repeat recognized by piggyBac transposase; HS4, chicken β-globin insulator sequence; TRE, tetracycline response element (doxycycline-inducible promoter); Xist -2kb, nucleotides 1-2016 of mouse Xist ; SV40, simian virus 40 (polyadenylation sequences); Luc , firefly luciferase gene; PGK and EF1α, constitutive promoters; PuroR , puromycin resistance gene. Numbered bars indicate regions targeted by qPCR primer pairs. ( B ) Luminescence assay (left) and western blot (right) showing effects of Hottip and Xist -2kb induction on luciferase protein expression. ( C ) Relative survival of TETRISv1 Xist -2kb cells grown for 24 or 48 h in the presence of puromycin after 48 h of treatment with or without doxycycline. Representative images show differences in cell survival after 48 h in puromycin. ( D ) Stellaris single-molecule RNA FISH shows Xist RNA (red) and Luc mRNA (green) in TETRISv1 Xist -2kb cells. DAPI-stained nuclei are blue. Scale bar = 10 μm. ( E-F ) RNA was prepared from TETRISv1 Xist -2kb cells directly (total) or following subcellular fractionation. Reverse transcription was performed with a pool of random oligos, and quantities of RNA corresponding to the Luc (E) or PuroR (F) gene in each sample were measured via qPCR. See Supplementary Figure S1A for fractionation/loading controls. ( G ) Total RNA from TETRISv1 Xist -2kb cells was reverse-transcribed using strand-specific primers targeting either the Luc mRNA or RNA produced from transcription occurring in the opposite direction ( Luc antisense RNA). Quantities of RNA in each sample were measured with the same qPCR primer pair (relative to the – dox average for each strand-specific RT). ( H ) Nuclear RNA from TETRISv1 Hottip cells was reverse transcribed with random primers, and doxycycline-dependent changes in RNA produced from the Hottip gene and from the Hottip-Luc and Luc - PuroR intergenic regions were measured via qPCR with the primer pairs shown in (A). ( I ) Analysis as in (H) but with nuclear RNA from TETRISv1 Xist -2kb cells. ( J ) Diagram of the TETRISv2 DNA cassette and regions targeted by qPCR primer pairs in (L). ( K ) Luminescence assay as in (B) but with TETRISv2 empty (no lncRNA cargo) or Xist -2kb cells. ( L ) Analysis as in (H-I) but with TETRISv2 Xist -2kb cells. For all panels, cells were treated with or without 1 μg/mL doxycycline for 48 h prior to assaying. Unless stated otherwise, numerical values are shown relative to the – dox average for each cell identity or qPCR primer pair, which is set to one. Dots represent individual technical replicate measurements from a minimum of biological duplicate experiments, and bars represent the average value. See Supplementary Table 1 for information regarding experimental replicates and Supplementary Table 2 for oligo sequences.
Figure Legend Snippet: Xist -2kb represses expression of adjacent genes by reading through multiple polyadenylation sequences. ( A ) Schematics of TETRISv1 DNA cassettes. ITR, inverted tandem repeat recognized by piggyBac transposase; HS4, chicken β-globin insulator sequence; TRE, tetracycline response element (doxycycline-inducible promoter); Xist -2kb, nucleotides 1-2016 of mouse Xist ; SV40, simian virus 40 (polyadenylation sequences); Luc , firefly luciferase gene; PGK and EF1α, constitutive promoters; PuroR , puromycin resistance gene. Numbered bars indicate regions targeted by qPCR primer pairs. ( B ) Luminescence assay (left) and western blot (right) showing effects of Hottip and Xist -2kb induction on luciferase protein expression. ( C ) Relative survival of TETRISv1 Xist -2kb cells grown for 24 or 48 h in the presence of puromycin after 48 h of treatment with or without doxycycline. Representative images show differences in cell survival after 48 h in puromycin. ( D ) Stellaris single-molecule RNA FISH shows Xist RNA (red) and Luc mRNA (green) in TETRISv1 Xist -2kb cells. DAPI-stained nuclei are blue. Scale bar = 10 μm. ( E-F ) RNA was prepared from TETRISv1 Xist -2kb cells directly (total) or following subcellular fractionation. Reverse transcription was performed with a pool of random oligos, and quantities of RNA corresponding to the Luc (E) or PuroR (F) gene in each sample were measured via qPCR. See Supplementary Figure S1A for fractionation/loading controls. ( G ) Total RNA from TETRISv1 Xist -2kb cells was reverse-transcribed using strand-specific primers targeting either the Luc mRNA or RNA produced from transcription occurring in the opposite direction ( Luc antisense RNA). Quantities of RNA in each sample were measured with the same qPCR primer pair (relative to the – dox average for each strand-specific RT). ( H ) Nuclear RNA from TETRISv1 Hottip cells was reverse transcribed with random primers, and doxycycline-dependent changes in RNA produced from the Hottip gene and from the Hottip-Luc and Luc - PuroR intergenic regions were measured via qPCR with the primer pairs shown in (A). ( I ) Analysis as in (H) but with nuclear RNA from TETRISv1 Xist -2kb cells. ( J ) Diagram of the TETRISv2 DNA cassette and regions targeted by qPCR primer pairs in (L). ( K ) Luminescence assay as in (B) but with TETRISv2 empty (no lncRNA cargo) or Xist -2kb cells. ( L ) Analysis as in (H-I) but with TETRISv2 Xist -2kb cells. For all panels, cells were treated with or without 1 μg/mL doxycycline for 48 h prior to assaying. Unless stated otherwise, numerical values are shown relative to the – dox average for each cell identity or qPCR primer pair, which is set to one. Dots represent individual technical replicate measurements from a minimum of biological duplicate experiments, and bars represent the average value. See Supplementary Table 1 for information regarding experimental replicates and Supplementary Table 2 for oligo sequences.

Techniques Used: Expressing, Sequencing, Luciferase, Real-time Polymerase Chain Reaction, Luminescence Assay, Western Blot, Fluorescence In Situ Hybridization, Staining, Fractionation, Produced

Transcriptional readthrough by Xist -2kb requires Repeat A and downstream sequence. ( A ) Diagram of Xist -2kb showing the location of Repeat A, Repeat F, stably structured elements (SS; ( 14 )), and mutants that reduce repressive activity in TETRIS ( 36 ). Sequences of each mutant can be found in ( 36 ). The deleted ss234 region corresponds to nt 733-1474 of Xist , chromosomal coordinates chrX:103481759-103482500 in mm10. ( B-D ) Nuclear RNA from cells harboring TETRISv1 Xist -2kb deletion mutants was reverse-transcribed with random primers and analyzed by qPCR with primer pairs targeting regions shown in Figure 1A . Numerical values are shown relative to the – dox average for each qPCR primer pair, which is set to one. Dots represent individual technical replicate measurements, and bars represent the average value. ( E-F ) Northern blot analysis of total RNA prepared from cells harboring the indicated TETRIS expression cassettes. Following membrane transfer, ethidium-stained RNA was imaged to visualize evenness of loading (18S rRNA in (E)) and migration of RNA size markers. Membrane was probed first with a single-stranded DNA oligo targeting Luc antisense RNA (F), stripped and imaged to ensure removal of signal (not shown), and re-probed with a single-stranded DNA oligo targeting Xist -2kb (E). See Supplementary Figure S2 for additional images. For all panels, cells were treated with or without 1 μg/mL doxycycline for 48 h prior to assaying. See Supplementary Table 1 for information regarding experimental replicates and Supplementary Table 2 for oligo sequences.
Figure Legend Snippet: Transcriptional readthrough by Xist -2kb requires Repeat A and downstream sequence. ( A ) Diagram of Xist -2kb showing the location of Repeat A, Repeat F, stably structured elements (SS; ( 14 )), and mutants that reduce repressive activity in TETRIS ( 36 ). Sequences of each mutant can be found in ( 36 ). The deleted ss234 region corresponds to nt 733-1474 of Xist , chromosomal coordinates chrX:103481759-103482500 in mm10. ( B-D ) Nuclear RNA from cells harboring TETRISv1 Xist -2kb deletion mutants was reverse-transcribed with random primers and analyzed by qPCR with primer pairs targeting regions shown in Figure 1A . Numerical values are shown relative to the – dox average for each qPCR primer pair, which is set to one. Dots represent individual technical replicate measurements, and bars represent the average value. ( E-F ) Northern blot analysis of total RNA prepared from cells harboring the indicated TETRIS expression cassettes. Following membrane transfer, ethidium-stained RNA was imaged to visualize evenness of loading (18S rRNA in (E)) and migration of RNA size markers. Membrane was probed first with a single-stranded DNA oligo targeting Luc antisense RNA (F), stripped and imaged to ensure removal of signal (not shown), and re-probed with a single-stranded DNA oligo targeting Xist -2kb (E). See Supplementary Figure S2 for additional images. For all panels, cells were treated with or without 1 μg/mL doxycycline for 48 h prior to assaying. See Supplementary Table 1 for information regarding experimental replicates and Supplementary Table 2 for oligo sequences.

Techniques Used: Sequencing, Stable Transfection, Activity Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Northern Blot, Expressing, Staining, Migration

16) Product Images from "Pax6 Exerts Regional Control of Cortical Progenitor Proliferation via Direct Repression of Cdk6 and Hypophosphorylation of pRb"

Article Title: Pax6 Exerts Regional Control of Cortical Progenitor Proliferation via Direct Repression of Cdk6 and Hypophosphorylation of pRb

Journal: Neuron

doi: 10.1016/j.neuron.2013.02.012

Evidence that Sites around the Cdk6 Gene Bind Pax6 In Vivo and Repress Gene Expression In Vitro (A) Sonication of cortical chromatin gave mainly 100–600 bp genomic fragments for ChIP with antibodies against either Pax6 (experiment) or IgG (control). (B) Results of qChIP on DNA from E12.5 dorsal telencephalon using anti-Pax6 and anti-IgG antibodies followed by qPCR to test for enrichment of each putative Pax6 binding site. For each binding site, the amount of qPCR product obtained with anti-Pax6 antibody was first expressed relative to that obtained with anti-IgG antibody. The resulting ratio was expressed relative to the average ratio obtained with primers for a sequence from the Syt8 gene that does not bind Pax6 (which was set to 1.0; Sansom et al., 2009 ). A Gab1 sequence known to bind Pax6 ( Sansom et al., 2009 ) was used as a positive control. Means ± SEM (n = 3); ∗ p
Figure Legend Snippet: Evidence that Sites around the Cdk6 Gene Bind Pax6 In Vivo and Repress Gene Expression In Vitro (A) Sonication of cortical chromatin gave mainly 100–600 bp genomic fragments for ChIP with antibodies against either Pax6 (experiment) or IgG (control). (B) Results of qChIP on DNA from E12.5 dorsal telencephalon using anti-Pax6 and anti-IgG antibodies followed by qPCR to test for enrichment of each putative Pax6 binding site. For each binding site, the amount of qPCR product obtained with anti-Pax6 antibody was first expressed relative to that obtained with anti-IgG antibody. The resulting ratio was expressed relative to the average ratio obtained with primers for a sequence from the Syt8 gene that does not bind Pax6 (which was set to 1.0; Sansom et al., 2009 ). A Gab1 sequence known to bind Pax6 ( Sansom et al., 2009 ) was used as a positive control. Means ± SEM (n = 3); ∗ p

Techniques Used: In Vivo, Expressing, In Vitro, Sonication, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Sequencing, Positive Control

17) Product Images from "Identification of regulatory elements flanking human XIST reveals species differences"

Article Title: Identification of regulatory elements flanking human XIST reveals species differences

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-11-20

Dual luciferase reporter assays examining promoter and enhancer activities for DNA fragments containing HS site . The pGL4.10 vector, which contains the promoterless synthetic firefly luc2 , was used in the promoter assay (left). The pGL4.23 vector, which contains the synthetic firefly luc2 driven by a minimal promoter, was used in the enhancer assay (right). The histograms show a summary of the ratio of luciferase activity (adjusted by dividing the firefly luciferase with the control Renilla luciferase) for each insert (from the HS sites shown in lower panel) relative to the luciferase activity for pGL4.10 or pGL4.23. Each fragment was tested in triplicate and experiments were carried out three times independently. Error bars represent the standard deviations of three trials. While fragments upstream of the XIST promoter containing HS sites showed background luciferase activity, fragment -65 displayed five fold and seven fold increases in promoter activity and ten fold and six fold increases in enhancer activity in the XIST and antisense orientation, respectively.
Figure Legend Snippet: Dual luciferase reporter assays examining promoter and enhancer activities for DNA fragments containing HS site . The pGL4.10 vector, which contains the promoterless synthetic firefly luc2 , was used in the promoter assay (left). The pGL4.23 vector, which contains the synthetic firefly luc2 driven by a minimal promoter, was used in the enhancer assay (right). The histograms show a summary of the ratio of luciferase activity (adjusted by dividing the firefly luciferase with the control Renilla luciferase) for each insert (from the HS sites shown in lower panel) relative to the luciferase activity for pGL4.10 or pGL4.23. Each fragment was tested in triplicate and experiments were carried out three times independently. Error bars represent the standard deviations of three trials. While fragments upstream of the XIST promoter containing HS sites showed background luciferase activity, fragment -65 displayed five fold and seven fold increases in promoter activity and ten fold and six fold increases in enhancer activity in the XIST and antisense orientation, respectively.

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

18) Product Images from "Joint Effect of MCP-1 Genotype GG and MMP-1 Genotype 2G/2G Increases the Likelihood of Developing Pulmonary Tuberculosis in BCG-Vaccinated Individuals"

Article Title: Joint Effect of MCP-1 Genotype GG and MMP-1 Genotype 2G/2G Increases the Likelihood of Developing Pulmonary Tuberculosis in BCG-Vaccinated Individuals

Journal: PLoS ONE

doi: 10.1371/journal.pone.0008881

MCP-1 increases MMP-1 expression inTHP-1 monocytic cells stimulated by M. tuberculosis-sonicate antigens . We measured the relative changes in MMP-1 gene expression by real-time PCR. Data are presented as the fold change in gene expression normalized to the endogenous reference gene PDHB and relative to untreated controls (RQ values). A, MMP-1 expression following 24 hours in vitro stimulation of THP-1 cells with the indicated amounts of M. tuberculosis- sonicate antigens. The effective dose 50 (ED50) of the M. tuberculosis -sonicate is indicated. B, MMP-1 expression data following 24 hours in vitro stimulations of THP-1 cells with the indicated amounts of human recombinant MCP-1 (rMCP-1). Graph A and B show data from one of three experiments. C, MMP-1 expression data for non-stimulated (−) and M. tuberculosis -sonicate antigen stimulated (+) THP-1 cells that were cultured for 24 hours with the indicated amounts of human rMCP-1. The results presented are from three independent experiments showing minimum and maximum RQ values. D, MMP-1 secretion levels for non-stimulated (−) and M. tuberculosis -sonicate antigen stimulated (+) THP-1 cells that were cultured for 48 hours in serum-free media with or without increasing amounts of human rMCP-1. Results presented are from three independent experiments and the bars indicate the standard deviation from the mean. The ED50 of M. tuberculosis -sonicate was used to stimulate the cells for the experiments shown in B, C, and D. The p- values from Wilcoxon-Mann-Whitney U-tests of comparisons of two independent variables are shown in C and D. Antigen = M. tuberculosis -sonicate 1000 ng/ml.
Figure Legend Snippet: MCP-1 increases MMP-1 expression inTHP-1 monocytic cells stimulated by M. tuberculosis-sonicate antigens . We measured the relative changes in MMP-1 gene expression by real-time PCR. Data are presented as the fold change in gene expression normalized to the endogenous reference gene PDHB and relative to untreated controls (RQ values). A, MMP-1 expression following 24 hours in vitro stimulation of THP-1 cells with the indicated amounts of M. tuberculosis- sonicate antigens. The effective dose 50 (ED50) of the M. tuberculosis -sonicate is indicated. B, MMP-1 expression data following 24 hours in vitro stimulations of THP-1 cells with the indicated amounts of human recombinant MCP-1 (rMCP-1). Graph A and B show data from one of three experiments. C, MMP-1 expression data for non-stimulated (−) and M. tuberculosis -sonicate antigen stimulated (+) THP-1 cells that were cultured for 24 hours with the indicated amounts of human rMCP-1. The results presented are from three independent experiments showing minimum and maximum RQ values. D, MMP-1 secretion levels for non-stimulated (−) and M. tuberculosis -sonicate antigen stimulated (+) THP-1 cells that were cultured for 48 hours in serum-free media with or without increasing amounts of human rMCP-1. Results presented are from three independent experiments and the bars indicate the standard deviation from the mean. The ED50 of M. tuberculosis -sonicate was used to stimulate the cells for the experiments shown in B, C, and D. The p- values from Wilcoxon-Mann-Whitney U-tests of comparisons of two independent variables are shown in C and D. Antigen = M. tuberculosis -sonicate 1000 ng/ml.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, In Vitro, Recombinant, Cell Culture, Standard Deviation, MANN-WHITNEY

Luciferase activity in response to stimulation with M. tuberculosis -sonicate antigens and human rMCP-1 is greater in THP-1 cells transfected with the -1607 MMP-1 2G variant than in those transfected with the MMP-1 1G promoter variant. The activities of promoters carrying the -1607 alleles 1G and 2G were compared using a dual-luciferase assay system (see Materials and Methods for details). Cells were stimulated overnight with or without M. tuberculosis -sonicate antigens and with the indicated amounts of human recombinant MCP-1. The data are presented as the ratio of Firefly Luciferase signal to Renilla Luciferase (control) signal. The error bars represent the standard deviation of means obtained from four independent experiments. Blue bars indicate cells transfected with plasmids containing the -1607 MMP-1 1G variant and red bars show data from cells transfected with plasmids containing the 2G variant. Addition of human rMCP-1 to cells stimulated with M. tuberculosis -sonicate antigens increased specific luciferase activity in a dose-dependent manner and to a level greater than that induced by M. tuberculosis antigens alone. The p- values from student t-tests are shown. In blue we show the p- values from comparisons of non-stimulated cells and cells stimulated with M. tuberculosis -sonicate antigens alone. Antigen = M. tuberculosis -sonicate 10 ng/ml.
Figure Legend Snippet: Luciferase activity in response to stimulation with M. tuberculosis -sonicate antigens and human rMCP-1 is greater in THP-1 cells transfected with the -1607 MMP-1 2G variant than in those transfected with the MMP-1 1G promoter variant. The activities of promoters carrying the -1607 alleles 1G and 2G were compared using a dual-luciferase assay system (see Materials and Methods for details). Cells were stimulated overnight with or without M. tuberculosis -sonicate antigens and with the indicated amounts of human recombinant MCP-1. The data are presented as the ratio of Firefly Luciferase signal to Renilla Luciferase (control) signal. The error bars represent the standard deviation of means obtained from four independent experiments. Blue bars indicate cells transfected with plasmids containing the -1607 MMP-1 1G variant and red bars show data from cells transfected with plasmids containing the 2G variant. Addition of human rMCP-1 to cells stimulated with M. tuberculosis -sonicate antigens increased specific luciferase activity in a dose-dependent manner and to a level greater than that induced by M. tuberculosis antigens alone. The p- values from student t-tests are shown. In blue we show the p- values from comparisons of non-stimulated cells and cells stimulated with M. tuberculosis -sonicate antigens alone. Antigen = M. tuberculosis -sonicate 10 ng/ml.

Techniques Used: Luciferase, Activity Assay, Transfection, Variant Assay, Recombinant, Standard Deviation

19) Product Images from "VILIP-1 Downregulation in Non-Small Cell Lung Carcinomas: Mechanisms and Prediction of Survival"

Article Title: VILIP-1 Downregulation in Non-Small Cell Lung Carcinomas: Mechanisms and Prediction of Survival

Journal: PLoS ONE

doi: 10.1371/journal.pone.0001698

MSP analysis of representative primary lung SCC. Bands in lanes M are methylated, bands in lanes U are unmethylated. NHBE and HOP92 cells were used as controls. VILIP-1 expression is indicated under each case, using the IHC score.
Figure Legend Snippet: MSP analysis of representative primary lung SCC. Bands in lanes M are methylated, bands in lanes U are unmethylated. NHBE and HOP92 cells were used as controls. VILIP-1 expression is indicated under each case, using the IHC score.

Techniques Used: Methylation, Expressing, Immunohistochemistry

Silencing of the VILIP-1 gene in human lung cancer cell lines. A. Western blot result from normal human bronchial epithelial (NHBE) cells and 12 NSCLC cell lines. VILIP-1 was identified as 22 kDa. Glyceraldehyde-3 phosphate dehydrogenase (GAPDH) was used as loading control. B. Northern blot of total RNA extracted from NHBE and 12 NSCLC cell lines probed with VILIP-1 full-length cDNA. Immunohistochemistry of VILIP-1 in subcutaneous tumors derived from the NSCLC cell lines, C: NCI-H520, D: Calu1, E: Calu6, and F: A549. ×100. Effect of methylation on VILIP-1 promoter activity (G). pGL4.10 vector containing in vitro methylated (filled circles) or nonmethylated (no circles) VILIP-1 promoter fragment was transfected into NCI-H520 or NCI-H522 cells. Transfection efficiency was normalized to the cotransfected pGL4.73 vector. The data presented as mean±SD (bars) of triplicate experiments.
Figure Legend Snippet: Silencing of the VILIP-1 gene in human lung cancer cell lines. A. Western blot result from normal human bronchial epithelial (NHBE) cells and 12 NSCLC cell lines. VILIP-1 was identified as 22 kDa. Glyceraldehyde-3 phosphate dehydrogenase (GAPDH) was used as loading control. B. Northern blot of total RNA extracted from NHBE and 12 NSCLC cell lines probed with VILIP-1 full-length cDNA. Immunohistochemistry of VILIP-1 in subcutaneous tumors derived from the NSCLC cell lines, C: NCI-H520, D: Calu1, E: Calu6, and F: A549. ×100. Effect of methylation on VILIP-1 promoter activity (G). pGL4.10 vector containing in vitro methylated (filled circles) or nonmethylated (no circles) VILIP-1 promoter fragment was transfected into NCI-H520 or NCI-H522 cells. Transfection efficiency was normalized to the cotransfected pGL4.73 vector. The data presented as mean±SD (bars) of triplicate experiments.

Techniques Used: Western Blot, Northern Blot, Immunohistochemistry, Derivative Assay, Methylation, Activity Assay, Plasmid Preparation, In Vitro, Transfection

Methylation status of VILIP-1 promoter in NSCLC cell lines. A. Schematic map of VILIP-1 2kb promoter and CpG islands around exon1. Filled boxes, CpG islands. Open boxes, exons. Vertical ticks, CpG sites on the expanded axis. Start of exon 1 is marked at +1. TSS, translation start site (ATG start codon). B. MSP analysis of cell lines. Bands in lanes M are methylated, bands in lanes U are unmethylated. H 2 O: water was added instead of DNA; Untreated DNA: genomic DNA without treatment with sodium bisulfite; NHBE 1 and NHBE 2: DNA from two different individuals. C. Representative results of bisulfite sequencing of the second CpG island of VILIP-1 promoter in VILIP-1-expressing cell lines (+) and VILIP-1-nonexpressing cell lines (−). Open and filled squares indicate unmethylated and methylated CpG sites, respectively. Six to eight clones of PCR products amplified from bisulfite-treated genomic DNA were sequenced for each cell line. D. Reactivation of VILIP-1 expression by 5′-Aza-dC treatment in cell lines. VILIP-1 protein expression was determined by immunoblot analysis. GAPDH was included as a control for equal loading.
Figure Legend Snippet: Methylation status of VILIP-1 promoter in NSCLC cell lines. A. Schematic map of VILIP-1 2kb promoter and CpG islands around exon1. Filled boxes, CpG islands. Open boxes, exons. Vertical ticks, CpG sites on the expanded axis. Start of exon 1 is marked at +1. TSS, translation start site (ATG start codon). B. MSP analysis of cell lines. Bands in lanes M are methylated, bands in lanes U are unmethylated. H 2 O: water was added instead of DNA; Untreated DNA: genomic DNA without treatment with sodium bisulfite; NHBE 1 and NHBE 2: DNA from two different individuals. C. Representative results of bisulfite sequencing of the second CpG island of VILIP-1 promoter in VILIP-1-expressing cell lines (+) and VILIP-1-nonexpressing cell lines (−). Open and filled squares indicate unmethylated and methylated CpG sites, respectively. Six to eight clones of PCR products amplified from bisulfite-treated genomic DNA were sequenced for each cell line. D. Reactivation of VILIP-1 expression by 5′-Aza-dC treatment in cell lines. VILIP-1 protein expression was determined by immunoblot analysis. GAPDH was included as a control for equal loading.

Techniques Used: Methylation, Methylation Sequencing, Expressing, Clone Assay, Polymerase Chain Reaction, Amplification

20) 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

21) 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

22) Product Images from "Latent Transforming Growth Factor ?-Binding Protein 4 Is Downregulated in Esophageal Cancer via Promoter Methylation"

Article Title: Latent Transforming Growth Factor ?-Binding Protein 4 Is Downregulated in Esophageal Cancer via Promoter Methylation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0065614

Effect of Gata1, SP1, SMAD3 and E2F4 on transcriptional activity of LTBP4L and LTBP4S promoter. HEK293 cells were transiently transfected with pGL4.10-LTBP4L or pGL4.10-LTBP4S and a Renilla luciferase expression vector for normalization. Cells were also co-transfected with different concentrations of A) a Gata1 expression vector, B) a SP1 expression vector, C) a SMAD3 expression vector or D) an E2F4 expression vector. After 24 h cells were lysed and luciferase activities were determined. Experiments were repeated at least 3 times and the mean value was calculated. Data are presented ± standard deviation.
Figure Legend Snippet: Effect of Gata1, SP1, SMAD3 and E2F4 on transcriptional activity of LTBP4L and LTBP4S promoter. HEK293 cells were transiently transfected with pGL4.10-LTBP4L or pGL4.10-LTBP4S and a Renilla luciferase expression vector for normalization. Cells were also co-transfected with different concentrations of A) a Gata1 expression vector, B) a SP1 expression vector, C) a SMAD3 expression vector or D) an E2F4 expression vector. After 24 h cells were lysed and luciferase activities were determined. Experiments were repeated at least 3 times and the mean value was calculated. Data are presented ± standard deviation.

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

23) Product Images from "Excessive mechanical loading promotes osteoarthritis through the gremlin-1–NF-κB pathway"

Article Title: Excessive mechanical loading promotes osteoarthritis through the gremlin-1–NF-κB pathway

Journal: Nature Communications

doi: 10.1038/s41467-019-09491-5

NF-κB signalling mediates the catabolic effects of gremlin-1. a Luciferase assay for screening of downstream signalling pathways of gremlin-1 using reporter vectors containing a response element for each pathway or transcription factor. * P
Figure Legend Snippet: NF-κB signalling mediates the catabolic effects of gremlin-1. a Luciferase assay for screening of downstream signalling pathways of gremlin-1 using reporter vectors containing a response element for each pathway or transcription factor. * P

Techniques Used: Luciferase

The ROS- NF-κB pathway enhances transcriptional induction of gremlin-1. a Fluorescence imaging time-course of ROS production in mouse primary chondrocytes after cyclic tensile strain loading. Nuclei were stained with DAPI (blue). Scale bars, 20 µm. b Luciferase assay using ATDC5 cells co-transfected with human GREM1 promoter (from −1970 to +20 bp relative to the transcriptional start site) and each expression vector. ## P
Figure Legend Snippet: The ROS- NF-κB pathway enhances transcriptional induction of gremlin-1. a Fluorescence imaging time-course of ROS production in mouse primary chondrocytes after cyclic tensile strain loading. Nuclei were stained with DAPI (blue). Scale bars, 20 µm. b Luciferase assay using ATDC5 cells co-transfected with human GREM1 promoter (from −1970 to +20 bp relative to the transcriptional start site) and each expression vector. ## P

Techniques Used: Fluorescence, Imaging, Staining, Luciferase, Transfection, Expressing, Plasmid Preparation

Gremlin-1 antagonizes the anabolic effects of BMPs. mRNA levels of Sox9 , Col2a1 and Acan in mouse primary chondrocytes treated with rhBMP2, 4, or 7, and rhGREM1 for 24 h. * P
Figure Legend Snippet: Gremlin-1 antagonizes the anabolic effects of BMPs. mRNA levels of Sox9 , Col2a1 and Acan in mouse primary chondrocytes treated with rhBMP2, 4, or 7, and rhGREM1 for 24 h. * P

Techniques Used:

Gremlin-1 induction by mechanical stress loading occurs through Rac1 activation. a Gremlin-1 mRNA levels in mouse primary chondrocytes treated with 10 µM inhibitors of FAK (PF-573228), ROCK (Y-27632), or RAC1 (NSC23766) 24 h after cyclic tensile strain loading. n = 3 biologically independent samples. * P
Figure Legend Snippet: Gremlin-1 induction by mechanical stress loading occurs through Rac1 activation. a Gremlin-1 mRNA levels in mouse primary chondrocytes treated with 10 µM inhibitors of FAK (PF-573228), ROCK (Y-27632), or RAC1 (NSC23766) 24 h after cyclic tensile strain loading. n = 3 biologically independent samples. * P

Techniques Used: Activation Assay

Induction of gremlin-1 by excessive mechanical loading. a Time-course of Mmp13 mRNA levels in mouse primary chondrocytes after tensile stress loading (stress+), or without loading (stress −). Cells were cultured up to 72 h after uniaxial cyclic tensile strain (10%, 0.5 Hz, 30 min). # P
Figure Legend Snippet: Induction of gremlin-1 by excessive mechanical loading. a Time-course of Mmp13 mRNA levels in mouse primary chondrocytes after tensile stress loading (stress+), or without loading (stress −). Cells were cultured up to 72 h after uniaxial cyclic tensile strain (10%, 0.5 Hz, 30 min). # P

Techniques Used: Cell Culture

Catabolic effects of gremlin-1 in cultured chondrocytes. a mRNA levels of marker genes in primary mouse articular chondrocytes treated with recombinant human gremlin-1 (rhGREM1) for 24 h. b Amount of glycosaminoglycans (GAG) released into the culture medium determined by dimethylmethylene blue assay of wild-type mouse femoral heads cultured with various amounts of rhGREM1 for 3 days. c mRNA levels of marker genes in mouse femoral heads cultured with 10 µg/mL rhGREM1 for 3 days. All data are expressed as mean ± SD of biologically independent three samples per group. * P
Figure Legend Snippet: Catabolic effects of gremlin-1 in cultured chondrocytes. a mRNA levels of marker genes in primary mouse articular chondrocytes treated with recombinant human gremlin-1 (rhGREM1) for 24 h. b Amount of glycosaminoglycans (GAG) released into the culture medium determined by dimethylmethylene blue assay of wild-type mouse femoral heads cultured with various amounts of rhGREM1 for 3 days. c mRNA levels of marker genes in mouse femoral heads cultured with 10 µg/mL rhGREM1 for 3 days. All data are expressed as mean ± SD of biologically independent three samples per group. * P

Techniques Used: Cell Culture, Marker, Recombinant, Dimethylmethylene Blue Assay

Regulation of osteoarthritis development by gremlin-1. a Safranin O staining and OARSI scores of mouse knee joints of Grem1 fl/fl (Cntl) and Col2a1-Cre ERT2 ;Grem1 fl/fl (cKO) mice 8 weeks after surgery. Tamoxifen induction was performed at 7 weeks. Both experimental groups consist of n = 9 biologically independent animals. Inset boxes indicate regions of immunofluorescence in ( c ). Scale bars, 100 µm. b TUNEL staining and rate of TUNEL-positive cells in mouse knee joints of Cntl and cKO mice 8 weeks after surgery. Nuclei were stained with DAPI (blue). Scale bars, 100 µm. n = 5 biologically independent experiments. c Safranin O staining and immunofluorescence of Mmp13, Adamts5, IκBα, phosphorylated IκBα (dual Ser32/36), HIF-2α, and gremlin-1 proteins in mouse knee joints of Cntl and cKO mice 8 weeks after surgery. Scale bars, 50 µm. The percentage of positive cells in the immunofluorescence is shown below. n = 5 biologically independent experiments. d Safranin O staining and OARSI scores of mouse knee joints of Cntl and cKO mice at 18 months of age. Tamoxifen induction was performed for 5 days at 8 weeks, 6 months, and 12 months. Both experimental groups consist of n = 8 biologically independent animals. Inset boxes indicate regions of immunofluorescence in ( f ). Scale bars, 100 µm. e TUNEL staining and rate of TUNEL-positive cells in mouse knee joints of Cntl and cKO mice at 18 months of age. Nuclei were stained with DAPI (blue). Scale bars, 100 µm. n = 5 biologically independent experiments. ( f ) Safranin O staining and immunofluorescence of Mmp13, Adamts5, IκBα, phosphorylated IκBα (dual Ser32/36), HIF-2α, and gremlin-1 proteins in mouse knee joints of Cntl and cKO mice at 18 months of age. Scale bars, 50 µm. The percentage of positive cells in the immunofluorescence is shown below. n = 5 biologically independent experiments. All data are expressed as mean ± SD. * P
Figure Legend Snippet: Regulation of osteoarthritis development by gremlin-1. a Safranin O staining and OARSI scores of mouse knee joints of Grem1 fl/fl (Cntl) and Col2a1-Cre ERT2 ;Grem1 fl/fl (cKO) mice 8 weeks after surgery. Tamoxifen induction was performed at 7 weeks. Both experimental groups consist of n = 9 biologically independent animals. Inset boxes indicate regions of immunofluorescence in ( c ). Scale bars, 100 µm. b TUNEL staining and rate of TUNEL-positive cells in mouse knee joints of Cntl and cKO mice 8 weeks after surgery. Nuclei were stained with DAPI (blue). Scale bars, 100 µm. n = 5 biologically independent experiments. c Safranin O staining and immunofluorescence of Mmp13, Adamts5, IκBα, phosphorylated IκBα (dual Ser32/36), HIF-2α, and gremlin-1 proteins in mouse knee joints of Cntl and cKO mice 8 weeks after surgery. Scale bars, 50 µm. The percentage of positive cells in the immunofluorescence is shown below. n = 5 biologically independent experiments. d Safranin O staining and OARSI scores of mouse knee joints of Cntl and cKO mice at 18 months of age. Tamoxifen induction was performed for 5 days at 8 weeks, 6 months, and 12 months. Both experimental groups consist of n = 8 biologically independent animals. Inset boxes indicate regions of immunofluorescence in ( f ). Scale bars, 100 µm. e TUNEL staining and rate of TUNEL-positive cells in mouse knee joints of Cntl and cKO mice at 18 months of age. Nuclei were stained with DAPI (blue). Scale bars, 100 µm. n = 5 biologically independent experiments. ( f ) Safranin O staining and immunofluorescence of Mmp13, Adamts5, IκBα, phosphorylated IκBα (dual Ser32/36), HIF-2α, and gremlin-1 proteins in mouse knee joints of Cntl and cKO mice at 18 months of age. Scale bars, 50 µm. The percentage of positive cells in the immunofluorescence is shown below. n = 5 biologically independent experiments. All data are expressed as mean ± SD. * P

Techniques Used: Staining, Mouse Assay, Immunofluorescence, TUNEL Assay

Effects of recombinant human gremlin-1 (rhGREM1) and gremlin-1 antibody in vivo. a Safranin O staining and OARSI scores of mouse knee joints after intra-articular administration (twice a week for 8 weeks) of 10 µL of 10 µg/mL rhGREM1 or vehicle. Both experimental groups consist of n = 8 biologically independent animals. Inset boxes indicate regions of immunofluorescence in ( c ). Scale bars, 100 µm. b TUNEL staining and rate of TUNEL-positive cells in mouse knee joints after intra-articular administration of rhGREM1 or vehicle. Nuclei were stained with DAPI (blue). Scale bars, 100 µm. n = 5 biologically independent experiments. c Safranin O staining and immunofluorescence of Mmp13, Adamts5, IκBα, phosphorylated IκBα (dual Ser32/36), and HIF-2α proteins in mouse knee joints after intra-articular administration of rhGREM1 or vehicle. Scale bars, 50 µm. The percentage of positive cells in the immunofluorescence is shown below. n = 5 biologically independent experiments. d Safranin O staining and OARSI scores of mouse knee joints after with intra-articular administration (twice a week for 8 weeks) of 10 µL of 10 µg/mL gremlin-1 antibody or vehicle. Both experimental groups consist of biologically independent animals: vehicle n = 8, antibody n = 10. Inset boxes indicate regions of immunofluorescence in ( f ). Scale bars, 100 µm. e TUNEL staining and rate of TUNEL-positive cells in mouse knee joints after intra-articular administration of gremlin-1 antibody or vehicle. Nuclei were stained with DAPI (blue). Scale bars, 100 µm. n = 5 biologically independent experiments. f Safranin O staining and immunofluorescence of Mmp13, Adamts5, IκBα, phosphorylated IκBα (dual Ser32/36), and HIF-2α proteins in mouse knee joints after intra-articular administration of gremlin-1 antibody or vehicle. Scale bars, 50 µm. The percentage of positive cells in the immunofluorescence is shown below. n = 5 biologically independent experiments. All data are expressed as mean ± SD. * P
Figure Legend Snippet: Effects of recombinant human gremlin-1 (rhGREM1) and gremlin-1 antibody in vivo. a Safranin O staining and OARSI scores of mouse knee joints after intra-articular administration (twice a week for 8 weeks) of 10 µL of 10 µg/mL rhGREM1 or vehicle. Both experimental groups consist of n = 8 biologically independent animals. Inset boxes indicate regions of immunofluorescence in ( c ). Scale bars, 100 µm. b TUNEL staining and rate of TUNEL-positive cells in mouse knee joints after intra-articular administration of rhGREM1 or vehicle. Nuclei were stained with DAPI (blue). Scale bars, 100 µm. n = 5 biologically independent experiments. c Safranin O staining and immunofluorescence of Mmp13, Adamts5, IκBα, phosphorylated IκBα (dual Ser32/36), and HIF-2α proteins in mouse knee joints after intra-articular administration of rhGREM1 or vehicle. Scale bars, 50 µm. The percentage of positive cells in the immunofluorescence is shown below. n = 5 biologically independent experiments. d Safranin O staining and OARSI scores of mouse knee joints after with intra-articular administration (twice a week for 8 weeks) of 10 µL of 10 µg/mL gremlin-1 antibody or vehicle. Both experimental groups consist of biologically independent animals: vehicle n = 8, antibody n = 10. Inset boxes indicate regions of immunofluorescence in ( f ). Scale bars, 100 µm. e TUNEL staining and rate of TUNEL-positive cells in mouse knee joints after intra-articular administration of gremlin-1 antibody or vehicle. Nuclei were stained with DAPI (blue). Scale bars, 100 µm. n = 5 biologically independent experiments. f Safranin O staining and immunofluorescence of Mmp13, Adamts5, IκBα, phosphorylated IκBα (dual Ser32/36), and HIF-2α proteins in mouse knee joints after intra-articular administration of gremlin-1 antibody or vehicle. Scale bars, 50 µm. The percentage of positive cells in the immunofluorescence is shown below. n = 5 biologically independent experiments. All data are expressed as mean ± SD. * P

Techniques Used: Recombinant, In Vivo, Staining, Immunofluorescence, TUNEL Assay

24) Product Images from "An Arf-Egr-C/EBPβ Pathway Linked to Ras-Induced Senescence and Cancer"

Article Title: An Arf-Egr-C/EBPβ Pathway Linked to Ras-Induced Senescence and Cancer

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01489-14

Egr proteins bind to the endogenous Cebpb promoter region. (A) Diagram of the Cebpb promoter and PCR primers spanning the E2 and E5 sites. Arrows indicate primer positions (F, forward primer; R, reverse primer). TSS, transcription start site. (B) Interaction
Figure Legend Snippet: Egr proteins bind to the endogenous Cebpb promoter region. (A) Diagram of the Cebpb promoter and PCR primers spanning the E2 and E5 sites. Arrows indicate primer positions (F, forward primer; R, reverse primer). TSS, transcription start site. (B) Interaction

Techniques Used: Polymerase Chain Reaction

25) Product Images from "Characterization of a Suppressive Cis-acting Element in the Epstein–Barr Virus LMP1 Promoter"

Article Title: Characterization of a Suppressive Cis-acting Element in the Epstein–Barr Virus LMP1 Promoter

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.02302

Reporter assays confirmed the importance of the E-box/Ikaros motif for LMP1 promoter activation. (A) Diagram of pLMP1/ED-L1-FLuc and its derivatives. The firefly luciferase gene is driven by the proximal wild-type LMP1 promoter (WT). The pLMP1/ED-L1(mEbox/Ikaros)-FLuc (mEbox/Ikaros), pLMP1/ED-L1(mEbox)-FLuc (mEbox), or pLMP1/ED-L1(mIkaros)-FLuc (mIkaros) plasmids carry the indicated mutations. (B,C) Results of the luciferase assays. AGS (B) and AGS-EBV (C) cells were transfected with pLMP1/ED-L1-FLuc (WT), pLMP1/ED-L1(mEbox/Ikaros)-FLuc (mEbox/Ikaros), pLMP1/ED-L1(mEbox)-FLuc (mEbox), or pLMP1/ED-L1(mIkaros)-FLuc (mIkaros), together with the pRL-null control vector. After 24 h, cells were lysed and subjected to luciferase assays. Relative firefly luciferase activity of mEbox after normalization to Renilla luciferase is shown as the fold change in activation over that of the WT. Each bar represents the mean and standard deviation of three independent transfections. Student’s t -test was performed. ∗ p
Figure Legend Snippet: Reporter assays confirmed the importance of the E-box/Ikaros motif for LMP1 promoter activation. (A) Diagram of pLMP1/ED-L1-FLuc and its derivatives. The firefly luciferase gene is driven by the proximal wild-type LMP1 promoter (WT). The pLMP1/ED-L1(mEbox/Ikaros)-FLuc (mEbox/Ikaros), pLMP1/ED-L1(mEbox)-FLuc (mEbox), or pLMP1/ED-L1(mIkaros)-FLuc (mIkaros) plasmids carry the indicated mutations. (B,C) Results of the luciferase assays. AGS (B) and AGS-EBV (C) cells were transfected with pLMP1/ED-L1-FLuc (WT), pLMP1/ED-L1(mEbox/Ikaros)-FLuc (mEbox/Ikaros), pLMP1/ED-L1(mEbox)-FLuc (mEbox), or pLMP1/ED-L1(mIkaros)-FLuc (mIkaros), together with the pRL-null control vector. After 24 h, cells were lysed and subjected to luciferase assays. Relative firefly luciferase activity of mEbox after normalization to Renilla luciferase is shown as the fold change in activation over that of the WT. Each bar represents the mean and standard deviation of three independent transfections. Student’s t -test was performed. ∗ p

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

26) Product Images from "Mutational Analysis of the Hypervariable Region of Hepatitis E Virus Reveals Its Involvement in the Efficiency of Viral RNA Replication ▿"

Article Title: Mutational Analysis of the Hypervariable Region of Hepatitis E Virus Reveals Its Involvement in the Efficiency of Viral RNA Replication ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00763-11

Generation of a genotype 1 human HEV luciferase replicon.
Figure Legend Snippet: Generation of a genotype 1 human HEV luciferase replicon.

Techniques Used: Luciferase

Replication kinetics of chimeric HEV luciferase replicons with swapped HVRs. (A) Replication kinetics of the wild-type genotype 1 HEV replicon pSK-REP, the chimeric replicon pSKHEV2-Sw-luc, and the mutant replicon pSKHEV2-ΔHVR-luc. The firefly
Figure Legend Snippet: Replication kinetics of chimeric HEV luciferase replicons with swapped HVRs. (A) Replication kinetics of the wild-type genotype 1 HEV replicon pSK-REP, the chimeric replicon pSKHEV2-Sw-luc, and the mutant replicon pSKHEV2-ΔHVR-luc. The firefly

Techniques Used: Luciferase, Mutagenesis

Schematic diagrams of the organization of HEV luciferase replicons and their derived HVR deletion mutants. (A) Subgenomic luciferase replicon of genotype 1 human HEV. The putative functional domains are indicated at the top. MT, methyltransferase; Y,
Figure Legend Snippet: Schematic diagrams of the organization of HEV luciferase replicons and their derived HVR deletion mutants. (A) Subgenomic luciferase replicon of genotype 1 human HEV. The putative functional domains are indicated at the top. MT, methyltransferase; Y,

Techniques Used: Luciferase, Derivative Assay, Functional Assay

27) Product Images from "GSK-3? and GSK-3? Proteins Are Involved in Early Stages of Chondrocyte Differentiation with Functional Redundancy through RelA Protein Phosphorylation *"

Article Title: GSK-3? and GSK-3? Proteins Are Involved in Early Stages of Chondrocyte Differentiation with Functional Redundancy through RelA Protein Phosphorylation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.372086

A , mRNA levels of Gsk3a , Gsk3b , and early ( Sox9 , Col2a1 , and Acan ) and later ( Col10a1 ) chondrocyte differentiation markers during culture of mouse chondrogenic ATDC5 cells in differentiation medium (ITS) for 3 weeks. Data are expressed as means ±
Figure Legend Snippet: A , mRNA levels of Gsk3a , Gsk3b , and early ( Sox9 , Col2a1 , and Acan ) and later ( Col10a1 ) chondrocyte differentiation markers during culture of mouse chondrogenic ATDC5 cells in differentiation medium (ITS) for 3 weeks. Data are expressed as means ±

Techniques Used:

A , luciferase activity after transfection with RelA or RelA mutants with replacement of putative phosphorylation sites with nonphosphorylatable residues or control GFP into stable lines of HeLa and ATDC5 cells containing either SOX9 gene reporter construct
Figure Legend Snippet: A , luciferase activity after transfection with RelA or RelA mutants with replacement of putative phosphorylation sites with nonphosphorylatable residues or control GFP into stable lines of HeLa and ATDC5 cells containing either SOX9 gene reporter construct

Techniques Used: Luciferase, Activity Assay, Transfection, Construct

A , mRNA levels of Sox9 , Col2a1 , Acan , and toluidine blue staining in stable lines of ATDC5 cells retrovirally transfected with GSK-3α, GSK-3β, a combination of both, or the control GFP and in nontransfected parental cells (−) after
Figure Legend Snippet: A , mRNA levels of Sox9 , Col2a1 , Acan , and toluidine blue staining in stable lines of ATDC5 cells retrovirally transfected with GSK-3α, GSK-3β, a combination of both, or the control GFP and in nontransfected parental cells (−) after

Techniques Used: Staining, Transfection

28) Product Images from "Vitamin D Regulation of OX40 Ligand in Immune Responses to Aspergillus fumigatus"

Article Title: Vitamin D Regulation of OX40 Ligand in Immune Responses to Aspergillus fumigatus

Journal: Infection and Immunity

doi: 10.1128/IAI.01345-12

NF-κB molecules regulate expression of OX40L. U937 cells were treated with vitamin D (0.1 μM), TNF-α (10 ng/ml), and/or TSLP (5 ng/ml). (A) ChIP assays show no NF-κB p50 or p65 binding in unstimulated cells. (B) ChIP assays
Figure Legend Snippet: NF-κB molecules regulate expression of OX40L. U937 cells were treated with vitamin D (0.1 μM), TNF-α (10 ng/ml), and/or TSLP (5 ng/ml). (A) ChIP assays show no NF-κB p50 or p65 binding in unstimulated cells. (B) ChIP assays

Techniques Used: Expressing, Chromatin Immunoprecipitation, Binding Assay

Vitamin D regulates expression of OX40L. U937 cells were treated with vitamin D (0.1 μM), TNF-α (10 ng/ml), and/or TSLP (5 ng/ml). ChIP assays show binding of VDR in the promoter region of OX40L when cells are stimulated with vitamin D
Figure Legend Snippet: Vitamin D regulates expression of OX40L. U937 cells were treated with vitamin D (0.1 μM), TNF-α (10 ng/ml), and/or TSLP (5 ng/ml). ChIP assays show binding of VDR in the promoter region of OX40L when cells are stimulated with vitamin D

Techniques Used: Expressing, Chromatin Immunoprecipitation, Binding Assay

29) Product Images from "Angiotensin II Triggers Expression of the Adrenal Gland Zona Glomerulosa-Specific 3β-Hydroxysteroid Dehydrogenase Isoenzyme through De Novo Protein Synthesis of the Orphan Nuclear Receptors NGFIB and NURR1"

Article Title: Angiotensin II Triggers Expression of the Adrenal Gland Zona Glomerulosa-Specific 3β-Hydroxysteroid Dehydrogenase Isoenzyme through De Novo Protein Synthesis of the Orphan Nuclear Receptors NGFIB and NURR1

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00852-14

AngII-induced NGFIB and NURR1 bind to the NBRE site of the HSD3B1 promoter. (A and B) EMSA analysis for NGFIB binding at the NBRE. A radiolabeled oligonucleotide probe for the HSD3B1 NBRE (positions −130 to −110) was incubated with the
Figure Legend Snippet: AngII-induced NGFIB and NURR1 bind to the NBRE site of the HSD3B1 promoter. (A and B) EMSA analysis for NGFIB binding at the NBRE. A radiolabeled oligonucleotide probe for the HSD3B1 NBRE (positions −130 to −110) was incubated with the

Techniques Used: Binding Assay, Incubation

30) Product Images from "Transforming Growth Factor-β (TGF-β) Inhibits the Expression of Factor VII-activating Protease (FSAP) in Hepatocytes *"

Article Title: Transforming Growth Factor-β (TGF-β) Inhibits the Expression of Factor VII-activating Protease (FSAP) in Hepatocytes *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.744631

Inhibition of Habp2 mRNA expression by TGF-β. A , murine AML12 hepatocyte cell line ( black bars ) and murine primary hepatocytes ( gray bars ) were stimulated with epidermal growth factor (EGF, 50 ng/ml), platelet-derived growth factor (PDGF-BB, 50 ng/ml), basic fibroblast growth factor (bFGF, 10 ng/ml), hepatocyte growth factor (HGF, 10 ng/ml), connective tissue growth factor (CTGF, 50 ng/ml), transforming growth factor-β (TGF-β, 100 ng/ml), interleukin-1β (IL-1β, 20 ng/ml), estrogen ( Estr ., 10 μg/ml), and progesterone (Prog., 10 μg/ml) for 24 h. Relative Habp2 mRNA levels were normalized to Gapdh and represent mean ± S.D. ( n = 3). B and C , AML12 cells (square) and primary hepatocytes ( circles ) were treated with 100 ng/ml of TGF-β for increasing time ( B ), or with increasing concentrations of TGF-β for 24 h ( C ) and Habp2 mRNA levels were quantified. Values are mean ± S.E. ( n = 3) of a single experiment and similar results were obtained in three independent experiments. Statistically significant results ( p
Figure Legend Snippet: Inhibition of Habp2 mRNA expression by TGF-β. A , murine AML12 hepatocyte cell line ( black bars ) and murine primary hepatocytes ( gray bars ) were stimulated with epidermal growth factor (EGF, 50 ng/ml), platelet-derived growth factor (PDGF-BB, 50 ng/ml), basic fibroblast growth factor (bFGF, 10 ng/ml), hepatocyte growth factor (HGF, 10 ng/ml), connective tissue growth factor (CTGF, 50 ng/ml), transforming growth factor-β (TGF-β, 100 ng/ml), interleukin-1β (IL-1β, 20 ng/ml), estrogen ( Estr ., 10 μg/ml), and progesterone (Prog., 10 μg/ml) for 24 h. Relative Habp2 mRNA levels were normalized to Gapdh and represent mean ± S.D. ( n = 3). B and C , AML12 cells (square) and primary hepatocytes ( circles ) were treated with 100 ng/ml of TGF-β for increasing time ( B ), or with increasing concentrations of TGF-β for 24 h ( C ) and Habp2 mRNA levels were quantified. Values are mean ± S.E. ( n = 3) of a single experiment and similar results were obtained in three independent experiments. Statistically significant results ( p

Techniques Used: Inhibition, Expressing, Derivative Assay

Regulation of Habp2, Serpine1, F2, F12, Casp7 , and Nrap mRNA expression by TGF-β. Primary hepatocytes were stimulated with TGF-β (10 ng/ml) for 24 or 48 h. Relative mRNA levels were normalized to Gusb and represent mean ± S.D. of three independent experiments performed in duplicate. Student's t test: *, p
Figure Legend Snippet: Regulation of Habp2, Serpine1, F2, F12, Casp7 , and Nrap mRNA expression by TGF-β. Primary hepatocytes were stimulated with TGF-β (10 ng/ml) for 24 or 48 h. Relative mRNA levels were normalized to Gusb and represent mean ± S.D. of three independent experiments performed in duplicate. Student's t test: *, p

Techniques Used: Expressing

c-fos interacts with the minimal Habp2 promoter. A , DNA sequence representing the minimally active and TGF-β-responsive promoter region (−1/−177) and other non-responsive regions (−313/−427 and −177/−313) were biotinylated and incubated with nuclear extracts from AML12 cells. After pull-down with streptavidin magnetic beads the bound fraction was subjected to Western blotting analysis with an anti-c-fos antibody or anti-c-jun antibody. Band density was quantified, and results represent mean ± S.E. ( n = 3). B / C , ChIP was performed on nuclear extracts of AML12 cells with or without TGF-β stimulation for 24 h. Pull down was performed with an anti-c-fos ( B ) or an anti-SMAD 2/3 ( C ) antibody. Intact extract was used as an input control. PCR was performed to amplify the minimally active and TGF-β-responsive promoter region (−1/−177) and other non-responsive region (−313/−427). Values represent mean ± S.E. ( n = 3 independent experiments). Statistically significant results ( p
Figure Legend Snippet: c-fos interacts with the minimal Habp2 promoter. A , DNA sequence representing the minimally active and TGF-β-responsive promoter region (−1/−177) and other non-responsive regions (−313/−427 and −177/−313) were biotinylated and incubated with nuclear extracts from AML12 cells. After pull-down with streptavidin magnetic beads the bound fraction was subjected to Western blotting analysis with an anti-c-fos antibody or anti-c-jun antibody. Band density was quantified, and results represent mean ± S.E. ( n = 3). B / C , ChIP was performed on nuclear extracts of AML12 cells with or without TGF-β stimulation for 24 h. Pull down was performed with an anti-c-fos ( B ) or an anti-SMAD 2/3 ( C ) antibody. Intact extract was used as an input control. PCR was performed to amplify the minimally active and TGF-β-responsive promoter region (−1/−177) and other non-responsive region (−313/−427). Values represent mean ± S.E. ( n = 3 independent experiments). Statistically significant results ( p

Techniques Used: Sequencing, Incubation, Magnetic Beads, Western Blot, Chromatin Immunoprecipitation, Polymerase Chain Reaction

31) Product Images from "Tissue Tropism of SV40 Transformation of Human Cells"

Article Title: Tissue Tropism of SV40 Transformation of Human Cells

Journal: Genes & Cancer

doi: 10.1177/1947601910395580

Role of Notch-1 in SV40-mediated transformation of HM and Ast. ( A ) Notch-1 silencing in HM infected with wtSV40 and 1ESV40. Western blotting was performed 48 hours after transfection with Notch-1 siRNA (si-N1) or with “scramble” control
Figure Legend Snippet: Role of Notch-1 in SV40-mediated transformation of HM and Ast. ( A ) Notch-1 silencing in HM infected with wtSV40 and 1ESV40. Western blotting was performed 48 hours after transfection with Notch-1 siRNA (si-N1) or with “scramble” control

Techniques Used: Transformation Assay, AST Assay, Infection, Western Blot, Transfection

32) Product Images from "Hormonal regulation of cardiac KCNE2 gene expression"

Article Title: Hormonal regulation of cardiac KCNE2 gene expression

Journal: Molecular and cellular endocrinology

doi: 10.1016/j.mce.2008.06.003

The specific estrogen antagonist ICI 182,780 inhibits estrogen-induced upregulation of KCNE2 transcription in different cell lines. ( A ). Scheme of the −1461/+UTR construct used in this study. The construct was transfected in MCF7 cells ( B ) or
Figure Legend Snippet: The specific estrogen antagonist ICI 182,780 inhibits estrogen-induced upregulation of KCNE2 transcription in different cell lines. ( A ). Scheme of the −1461/+UTR construct used in this study. The construct was transfected in MCF7 cells ( B ) or

Techniques Used: Construct, Transfection

KCNE2 basal and estrogen responsive promoter activity. ( A ), Scheme of deletion constructs from the TSS used to measure basal (unstimulated) activity. All constructs had KCNE2 5′-UTR fused with the luciferase reporter gene. ( B ), Similar basal promoter
Figure Legend Snippet: KCNE2 basal and estrogen responsive promoter activity. ( A ), Scheme of deletion constructs from the TSS used to measure basal (unstimulated) activity. All constructs had KCNE2 5′-UTR fused with the luciferase reporter gene. ( B ), Similar basal promoter

Techniques Used: Activity Assay, Construct, Luciferase

33) Product Images from "Hormonal regulation of cardiac KCNE2 gene expression"

Article Title: Hormonal regulation of cardiac KCNE2 gene expression

Journal: Molecular and cellular endocrinology

doi: 10.1016/j.mce.2008.06.003

Outline of KCNE2 gene structure. ( A) , red letters). ( B ), Product of retained intronic sequence in
Figure Legend Snippet: Outline of KCNE2 gene structure. ( A) , red letters). ( B ), Product of retained intronic sequence in

Techniques Used: Sequencing

Homology plot of the mouse and human KCNE2 genes upstream sequences (−10000 of the stop codon). Analysis was performed using Vista browser and LAGAN algorithm. Gray regions indicate conserved sequences with at least 70% homology in 100 bp segments,
Figure Legend Snippet: Homology plot of the mouse and human KCNE2 genes upstream sequences (−10000 of the stop codon). Analysis was performed using Vista browser and LAGAN algorithm. Gray regions indicate conserved sequences with at least 70% homology in 100 bp segments,

Techniques Used:

Direct interaction of ERα with the ERE of the KCNE2 gene. ( A ), Duplex oligos used in EMSA. Boxes delineate the KCNE2 ERE in wild type, mutated and the perfect consensus ERE of the commercial oligo (Santa Cruz Biotechnology). In the KCNE2 mutated
Figure Legend Snippet: Direct interaction of ERα with the ERE of the KCNE2 gene. ( A ), Duplex oligos used in EMSA. Boxes delineate the KCNE2 ERE in wild type, mutated and the perfect consensus ERE of the commercial oligo (Santa Cruz Biotechnology). In the KCNE2 mutated

Techniques Used:

Determination of KCNE2 gene TSS by 5′-RACE and primer extension analysis. (A)
Figure Legend Snippet: Determination of KCNE2 gene TSS by 5′-RACE and primer extension analysis. (A)

Techniques Used:

The specific estrogen antagonist ICI 182,780 inhibits estrogen-induced upregulation of KCNE2 transcription in different cell lines. ( A ). Scheme of the −1461/+UTR construct used in this study. The construct was transfected in MCF7 cells ( B ) or
Figure Legend Snippet: The specific estrogen antagonist ICI 182,780 inhibits estrogen-induced upregulation of KCNE2 transcription in different cell lines. ( A ). Scheme of the −1461/+UTR construct used in this study. The construct was transfected in MCF7 cells ( B ) or

Techniques Used: Construct, Transfection

KCNE2 basal and estrogen responsive promoter activity. ( A ), Scheme of deletion constructs from the TSS used to measure basal (unstimulated) activity. All constructs had KCNE2 5′-UTR fused with the luciferase reporter gene. ( B ), Similar basal promoter
Figure Legend Snippet: KCNE2 basal and estrogen responsive promoter activity. ( A ), Scheme of deletion constructs from the TSS used to measure basal (unstimulated) activity. All constructs had KCNE2 5′-UTR fused with the luciferase reporter gene. ( B ), Similar basal promoter

Techniques Used: Activity Assay, Construct, Luciferase

E2 induced KCNE2 promoter activation
Figure Legend Snippet: E2 induced KCNE2 promoter activation

Techniques Used: Activation Assay

Cardiac KCNE2 transcripts are upregulated by estrogen but not by DHT. ( A ), Cardiac KCNE2 fluorescence vs. cycle number plots in NP at diestrus-2 (●) and LP (○). ( B), Bar graph showing the mean relative transcript expression normalized
Figure Legend Snippet: Cardiac KCNE2 transcripts are upregulated by estrogen but not by DHT. ( A ), Cardiac KCNE2 fluorescence vs. cycle number plots in NP at diestrus-2 (●) and LP (○). ( B), Bar graph showing the mean relative transcript expression normalized

Techniques Used: Fluorescence, Expressing

34) Product Images from "Matrix Metalloproteinase-9 Genotype as a Potential Genetic Marker for Abdominal Aortic Aneurysm"

Article Title: Matrix Metalloproteinase-9 Genotype as a Potential Genetic Marker for Abdominal Aortic Aneurysm

Journal: Circulation. Cardiovascular genetics

doi: 10.1161/CIRCGENETICS.112.963082

Functional consequences of MMP-9 Exon SNPs. A, A cartoon of the MMP-9 protein denoting the various functional domains and the relative locations of the SNPs investigated resulting in amino acid changes. C indicates the cysteine switch, Zn the zinc binding site, and the various functional domains are indicated by the different shades. B , Nonreducing PAGE of the culture supernatants collected 24 hours after transfection of HEK293 cells with the denoted constructs. The membrane after transfer was blotted with anti–MMP-9 antibody (1:1000 in TBST containing 1% dry milk) and shows the upper and lower-MMP-9 immunoreactive bands corresponding to the dimeric and monomeric MMP-9 protein. C, A bar plot summary of densitometric analysis of monomeric (open bars) and dimeric (hatched bars) protein amount normalized by the respective values for the wild-type. D ). E, Absolute (open bars) and specific (hatched bars) enzymatic activity normalized to that of the wt-MMP-9 transfected experiment (first columns). The enzymatic activity quantified is the total activity including both the monomeric and dimeric MMP-9 activity. Bar plots are mean±SEM from 5 independent experiments. * P
Figure Legend Snippet: Functional consequences of MMP-9 Exon SNPs. A, A cartoon of the MMP-9 protein denoting the various functional domains and the relative locations of the SNPs investigated resulting in amino acid changes. C indicates the cysteine switch, Zn the zinc binding site, and the various functional domains are indicated by the different shades. B , Nonreducing PAGE of the culture supernatants collected 24 hours after transfection of HEK293 cells with the denoted constructs. The membrane after transfer was blotted with anti–MMP-9 antibody (1:1000 in TBST containing 1% dry milk) and shows the upper and lower-MMP-9 immunoreactive bands corresponding to the dimeric and monomeric MMP-9 protein. C, A bar plot summary of densitometric analysis of monomeric (open bars) and dimeric (hatched bars) protein amount normalized by the respective values for the wild-type. D ). E, Absolute (open bars) and specific (hatched bars) enzymatic activity normalized to that of the wt-MMP-9 transfected experiment (first columns). The enzymatic activity quantified is the total activity including both the monomeric and dimeric MMP-9 activity. Bar plots are mean±SEM from 5 independent experiments. * P

Techniques Used: Functional Assay, Binding Assay, Polyacrylamide Gel Electrophoresis, Transfection, Construct, Activity Assay

Basal promoter activity of MMP-9 promoter SNPs. A, A cartoon of the MMP-9 genomic sequence upstream of the start methionine depicting the relative location of the 5 promoter SNPs and the (CA) n microsatellite polymorphism. Locations of the DNA consensus binding motifs for some of the common transcription factors are also noted. B, Relative promoter activity normalized to the wt promoter–reporter construct. HEK293 cells transfected with the noted promoter–reporter constructs in a 24-well dish (0.4 μg promoter–reporter plasmid + 0.013 μg Renilla plasmid/well) were harvested 24 hours later and replated into 3-replicate wells of a 96-well plate at an approximate cell density of 10 4 cells/well in DMEM supplemented with 1% FBS. The firefly luciferase promoter–reporter activity normalized by the Renilla luciferase activity to compensate for potential transfection efficiency and cell density differences were assessed 24 hours later. Bars represent mean±SEM from 6 independent experiments each with triplicate readings. * P
Figure Legend Snippet: Basal promoter activity of MMP-9 promoter SNPs. A, A cartoon of the MMP-9 genomic sequence upstream of the start methionine depicting the relative location of the 5 promoter SNPs and the (CA) n microsatellite polymorphism. Locations of the DNA consensus binding motifs for some of the common transcription factors are also noted. B, Relative promoter activity normalized to the wt promoter–reporter construct. HEK293 cells transfected with the noted promoter–reporter constructs in a 24-well dish (0.4 μg promoter–reporter plasmid + 0.013 μg Renilla plasmid/well) were harvested 24 hours later and replated into 3-replicate wells of a 96-well plate at an approximate cell density of 10 4 cells/well in DMEM supplemented with 1% FBS. The firefly luciferase promoter–reporter activity normalized by the Renilla luciferase activity to compensate for potential transfection efficiency and cell density differences were assessed 24 hours later. Bars represent mean±SEM from 6 independent experiments each with triplicate readings. * P

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

35) Product Images from "Transcriptional Dominance of Pax7 in Adult Myogenesis is Due to High-Affinity Recognition of Homeodomain Motifs"

Article Title: Transcriptional Dominance of Pax7 in Adult Myogenesis is Due to High-Affinity Recognition of Homeodomain Motifs

Journal: Developmental Cell

doi: 10.1016/j.devcel.2012.03.014

Myf5 −111kb (ECR111) Is a Common Evolutionarily Conserved Target of Pax3/7
Figure Legend Snippet: Myf5 −111kb (ECR111) Is a Common Evolutionarily Conserved Target of Pax3/7

Techniques Used:

36) Product Images from "Circadian Clock Control of Nox4 and Reactive Oxygen Species in the Vasculature"

Article Title: Circadian Clock Control of Nox4 and Reactive Oxygen Species in the Vasculature

Journal: PLoS ONE

doi: 10.1371/journal.pone.0078626

Nox4 promoter is regulated by the circadian clock. Human Nox4 promoter transactivation was assessed by a dual luciferase assay in transfected COS cells expressing the Nox4 promoter Gaussian luciferase in the presence and absence CLOCK, Bmal1, NPAS2, Bmal1+NPAS2 andBmal1+Clock. Cotransfection with Bmal1 and NPAS2 or Bmal1 and Clock significantly induced Nox4 promoter activity (*p
Figure Legend Snippet: Nox4 promoter is regulated by the circadian clock. Human Nox4 promoter transactivation was assessed by a dual luciferase assay in transfected COS cells expressing the Nox4 promoter Gaussian luciferase in the presence and absence CLOCK, Bmal1, NPAS2, Bmal1+NPAS2 andBmal1+Clock. Cotransfection with Bmal1 and NPAS2 or Bmal1 and Clock significantly induced Nox4 promoter activity (*p

Techniques Used: Luciferase, Transfection, Expressing, Cotransfection, Activity Assay

37) Product Images from "Elevated CO2 selectively inhibits interleukin-6 and tumor necrosis factor expression and decreases phagocytosis in the macrophage"

Article Title: Elevated CO2 selectively inhibits interleukin-6 and tumor necrosis factor expression and decreases phagocytosis in the macrophage

Journal: The FASEB Journal

doi: 10.1096/fj.09-136895

Hypercapnia inhibits IL-6 promoter-driven luciferase activity but does not alter IL-6 mRNA degradation. A ) RAW 264.7 cells were transfected with pGL4.10-IL-6/luc and pGL4.74-hRLuc/TK (1 μg and 50 ng/10 6 cells/well, respectively) and 24 h later stimulated with LPS (1 or 100 ng/ml) or medium alone in 5 or 20% CO 2 . After 6 h, cells were harvested and assayed for firefly luciferase and Renilla luciferase activities. Normalized firefly luciferase activity for each condition is represented as fold-change relative to unstimulated normocapnic cells ( n =3). * P
Figure Legend Snippet: Hypercapnia inhibits IL-6 promoter-driven luciferase activity but does not alter IL-6 mRNA degradation. A ) RAW 264.7 cells were transfected with pGL4.10-IL-6/luc and pGL4.74-hRLuc/TK (1 μg and 50 ng/10 6 cells/well, respectively) and 24 h later stimulated with LPS (1 or 100 ng/ml) or medium alone in 5 or 20% CO 2 . After 6 h, cells were harvested and assayed for firefly luciferase and Renilla luciferase activities. Normalized firefly luciferase activity for each condition is represented as fold-change relative to unstimulated normocapnic cells ( n =3). * P

Techniques Used: Luciferase, Activity Assay, Transfection

38) Product Images from "Expression of Human Frataxin Is Regulated by Transcription Factors SRF and TFAP2"

Article Title: Expression of Human Frataxin Is Regulated by Transcription Factors SRF and TFAP2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0012286

Transcription factors SRF and TFAP2 bind to the FXN gene promoter region in vivo . (A) Sequence of 5′-end of the human FXN gene. Sequences are color-coded: intronic sequences in green, exon 1 and exon 2 in black. Putative transcription factor binding sites identified by Genomatix analysis are annotated in bold black for SRF, in bold blue for TFAP2, in bold red for SP1, and in bold pink for EGR3. Start codon (AUG) in exon 1 is in bold black. (B) Schematic diagram of human FXN promoter region showing the location of the putative regulatory sequences. Black-filled square: SRF binding site; blue-filled square: TFAP2 binding site; red-filled square: SP1 binding site; pink-filled square: EGR3 binding site. Primers used in the ChIP assays are designated as: P189, P195, P204, P205, P206, P364, and P365. (C) Binding of SRF and TFAP2 to the FXN promoter in vivo was quantified by chromatin immunoprecipitation (ChIP) and quantitative real-time PCR (qRT-PCR). The upper panel is the quantification of FXN promoter chromatin, immunoprecipitated using antibodies raised against SRF, TFAP2, and SP1. Three pairs of primers flanking the putative binding sites of these transcription factors were used for the qPCR analysis. The lower panel shows the end-products of the qPCR reactions separated by agarose gel electrophoresis. Primers specific for human FXN intron 4 were included as an internal negative control (data not shown).
Figure Legend Snippet: Transcription factors SRF and TFAP2 bind to the FXN gene promoter region in vivo . (A) Sequence of 5′-end of the human FXN gene. Sequences are color-coded: intronic sequences in green, exon 1 and exon 2 in black. Putative transcription factor binding sites identified by Genomatix analysis are annotated in bold black for SRF, in bold blue for TFAP2, in bold red for SP1, and in bold pink for EGR3. Start codon (AUG) in exon 1 is in bold black. (B) Schematic diagram of human FXN promoter region showing the location of the putative regulatory sequences. Black-filled square: SRF binding site; blue-filled square: TFAP2 binding site; red-filled square: SP1 binding site; pink-filled square: EGR3 binding site. Primers used in the ChIP assays are designated as: P189, P195, P204, P205, P206, P364, and P365. (C) Binding of SRF and TFAP2 to the FXN promoter in vivo was quantified by chromatin immunoprecipitation (ChIP) and quantitative real-time PCR (qRT-PCR). The upper panel is the quantification of FXN promoter chromatin, immunoprecipitated using antibodies raised against SRF, TFAP2, and SP1. Three pairs of primers flanking the putative binding sites of these transcription factors were used for the qPCR analysis. The lower panel shows the end-products of the qPCR reactions separated by agarose gel electrophoresis. Primers specific for human FXN intron 4 were included as an internal negative control (data not shown).

Techniques Used: In Vivo, Sequencing, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Immunoprecipitation, Agarose Gel Electrophoresis, Negative Control

The SRF and TFAP2 binding sites in the FXN promoter are important for frataxin expression. (A) Luciferase analysis of a novel intronic regulatory region of the FXN gene. The upper panel portrays the upstream region of the FXN gene including the exon 1 and 2 and intron 1. Bottom panel: luciferase activity was measured in cells transfected with luciferase constructs containing truncated FXN promoter fragments containing the SRF and TFAP2 binding sites. Filled square: SRF binding site; open square: TFAP2 binding site; dotted square: EGR3 binding site. The four luciferase constructs are designated as I, II, III, IV (see MATERIALS AND METHODS ). (B) Mutation of the SRF and TFAP2 binding sites in the FXN promoter dramatically decreased luciferase activity driven from FXN promoter fragment IV. Mutation of the predicted EGR3 transcription factor binding site in intronic sequence of the FXN gene showed cell line-specific effects on transcriptional activity. Three separate experiments were carried out. For each experiment, duplicate transfections were performed. Error bars represent the standard deviation.
Figure Legend Snippet: The SRF and TFAP2 binding sites in the FXN promoter are important for frataxin expression. (A) Luciferase analysis of a novel intronic regulatory region of the FXN gene. The upper panel portrays the upstream region of the FXN gene including the exon 1 and 2 and intron 1. Bottom panel: luciferase activity was measured in cells transfected with luciferase constructs containing truncated FXN promoter fragments containing the SRF and TFAP2 binding sites. Filled square: SRF binding site; open square: TFAP2 binding site; dotted square: EGR3 binding site. The four luciferase constructs are designated as I, II, III, IV (see MATERIALS AND METHODS ). (B) Mutation of the SRF and TFAP2 binding sites in the FXN promoter dramatically decreased luciferase activity driven from FXN promoter fragment IV. Mutation of the predicted EGR3 transcription factor binding site in intronic sequence of the FXN gene showed cell line-specific effects on transcriptional activity. Three separate experiments were carried out. For each experiment, duplicate transfections were performed. Error bars represent the standard deviation.

Techniques Used: Binding Assay, Expressing, Luciferase, Activity Assay, Transfection, Construct, Mutagenesis, Sequencing, Standard Deviation

39) Product Images from "PU.1 is linking the glycolytic enzyme HK3 in neutrophil differentiation and survival of APL cells"

Article Title: PU.1 is linking the glycolytic enzyme HK3 in neutrophil differentiation and survival of APL cells

Journal: Blood

doi: 10.1182/blood-2011-09-378117

PU.1 and PML-RARA are direct regulators of the HK3 promoter. (A) Schematic representation of a 6.6-kb human HK3 promoter fragment. Lanes A, B, and C are 3 putative PU.1 binding sites (circles) found in the HK3 promoter as determined by MatInspector. (B)
Figure Legend Snippet: PU.1 and PML-RARA are direct regulators of the HK3 promoter. (A) Schematic representation of a 6.6-kb human HK3 promoter fragment. Lanes A, B, and C are 3 putative PU.1 binding sites (circles) found in the HK3 promoter as determined by MatInspector. (B)

Techniques Used: Binding Assay

Transcriptional regulation of HK3 by PU.1 in NB4 and HT93 APL cell lines. NB4 and HT93 cells were stably transduced with nontargeting shRNA (SHC002) or shRNAs targeting PU.1 (shPU.1_256, shPU.1_928) and differentiated with 1μM ATRA for 6 days.
Figure Legend Snippet: Transcriptional regulation of HK3 by PU.1 in NB4 and HT93 APL cell lines. NB4 and HT93 cells were stably transduced with nontargeting shRNA (SHC002) or shRNAs targeting PU.1 (shPU.1_256, shPU.1_928) and differentiated with 1μM ATRA for 6 days.

Techniques Used: Stable Transfection, Transduction, shRNA

Inhibition of HK3 decreases cell viability of ATRA-differentiated APL cells and renders APL cells more sensitive to anthracyclin therapy. (A) NB4 control (SHCOO2) or HK3 knockdown (shHK3_560, shHK3_1420, shHK3_1748) APL cells were grown in the presence
Figure Legend Snippet: Inhibition of HK3 decreases cell viability of ATRA-differentiated APL cells and renders APL cells more sensitive to anthracyclin therapy. (A) NB4 control (SHCOO2) or HK3 knockdown (shHK3_560, shHK3_1420, shHK3_1748) APL cells were grown in the presence

Techniques Used: Inhibition

Inhibition of HK3 by shRNA impairs neutrophil differentiation of APL cells. NB4 cells were stably transduced with nontargeting shRNA (SHC002) or shRNAs targeting HK3 (shHK3_560, shHK3_1420, shHK3_1748) and differentiated with 1μM ATRA for 6 days.
Figure Legend Snippet: Inhibition of HK3 by shRNA impairs neutrophil differentiation of APL cells. NB4 cells were stably transduced with nontargeting shRNA (SHC002) or shRNAs targeting HK3 (shHK3_560, shHK3_1420, shHK3_1748) and differentiated with 1μM ATRA for 6 days.

Techniques Used: Inhibition, shRNA, Stable Transfection, Transduction

Repression of HK3 mRNA in primary AML patient samples
Figure Legend Snippet: Repression of HK3 mRNA in primary AML patient samples

Techniques Used:

Significantly reduced HK3 and PU.1 mRNA levels in primary AML patient samples and induction of HK3 expression during neutrophil differentiation of NB4 APL cells. (A) Primary AML blasts were isolated using a Ficoll gradient, total RNA was extracted, and
Figure Legend Snippet: Significantly reduced HK3 and PU.1 mRNA levels in primary AML patient samples and induction of HK3 expression during neutrophil differentiation of NB4 APL cells. (A) Primary AML blasts were isolated using a Ficoll gradient, total RNA was extracted, and

Techniques Used: Expressing, Isolation

40) 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

Related Articles

Transfection:

Article Title: Muscleblind-like 1 (Mbnl1) Promotes Insulin Receptor Exon 11 Inclusion via Binding to a Downstream Evolutionarily Conserved Intronic Enhancer *
Article Snippet: .. Transient transfections of cells with plasmid DNA were performed with TransFast reagent (Promega, Madison, WI) according to the manufacturer's protocol. .. For cotransfection experiments, cells were transfected with 500 ng of minigene plasmid DNA and 1 μg of an expression vector for splicing factors of interest.

Article Title: The NS5A-binding heat shock proteins HSC70 and HSP70 play distinct roles in the hepatitis C viral life cycle
Article Snippet: .. Seventy-two hours post transfection, Renilla and Firefly luciferase activity were determined using Dual Luciferase Assay System (Promega, E1910). .. Error bars reflect the standard deviation.

Article Title: Human SNP Links Differential Outcomes in Inflammatory and Infectious Disease to a FOXO3-Regulated Pathway
Article Snippet: .. Cells were seeded at 2 × 105 cells/ml and cultured overnight without antibiotics before transient transfection with a firefly pGL4.14 hLuc vector containing the TGFβ1 promoter (666 ng/ml, Promega), a pCMV renilla luciferase control vector (6.6 ng/ml), and one of two FOXO3A siRNAs, or a scrambled control (66 nM/ml, Origene) using lipofectamine 2000 (Invitrogen). ..

Luciferase:

Article Title: MiR-195 inhibits proliferation and growth and induces apoptosis of endometrial stromal cells by targeting FKN
Article Snippet: .. Luciferase activity was measured 48 h post-transfection using the Dual-Luciferase Reporter Assay System, according to the manufacturer’s instructions (Promega). .. For each sample, Renilla luciferase activity was normalized by the total protein content.

Article Title: Structure and ubiquitination-dependent activation of Tank-Binding Kinase 1
Article Snippet: .. At 48 h post-transfection, luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) or RNA was prepared for QRT-PCR, as described in . .. Lysates were prepared 60 h following transient transfection of HEK293T cells with TBK1 constructs using standard RIPA buffer.

Article Title: Improvement of the CRISPR-Cpf1 system with ribozyme-processed crRNA
Article Snippet: .. Two days post-transfection, luciferase expression was measured with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. .. The ratio of Firefly to Renilla was calculated as the relative Luc activity.

Article Title: The NS5A-binding heat shock proteins HSC70 and HSP70 play distinct roles in the hepatitis C viral life cycle
Article Snippet: .. Seventy-two hours post transfection, Renilla and Firefly luciferase activity were determined using Dual Luciferase Assay System (Promega, E1910). .. Error bars reflect the standard deviation.

Article Title: Human SNP Links Differential Outcomes in Inflammatory and Infectious Disease to a FOXO3-Regulated Pathway
Article Snippet: .. Cells were seeded at 2 × 105 cells/ml and cultured overnight without antibiotics before transient transfection with a firefly pGL4.14 hLuc vector containing the TGFβ1 promoter (666 ng/ml, Promega), a pCMV renilla luciferase control vector (6.6 ng/ml), and one of two FOXO3A siRNAs, or a scrambled control (66 nM/ml, Origene) using lipofectamine 2000 (Invitrogen). ..

Article Title: Yin Yang 1 Intronic Binding Sequences and Splicing Elicit Intron-Mediated Enhancement of Ubiquitin C Gene Expression
Article Snippet: .. Luciferase Assay Cell extracts were subjected to luciferase assay 48 h post-transfection using the Luciferase assay reagent (Promega), essentially as reported in . .. Luciferase activity was determined on a FLUOstar OPTIMA multifunction microplate reader (BMG-LABTECH GmbH).

Reporter Assay:

Article Title: MiR-195 inhibits proliferation and growth and induces apoptosis of endometrial stromal cells by targeting FKN
Article Snippet: .. Luciferase activity was measured 48 h post-transfection using the Dual-Luciferase Reporter Assay System, according to the manufacturer’s instructions (Promega). .. For each sample, Renilla luciferase activity was normalized by the total protein content.

Article Title: Improvement of the CRISPR-Cpf1 system with ribozyme-processed crRNA
Article Snippet: .. Two days post-transfection, luciferase expression was measured with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. .. The ratio of Firefly to Renilla was calculated as the relative Luc activity.

DNA Purification:

Article Title: Novel 45-Kilodalton Leptospiral Protein That Is Processed to a 31-Kilodalton Growth-Phase-Regulated Peripheral Membrane Protein
Article Snippet: .. Genomic DNA was extracted from L. kirschneri with a Wizard genomic DNA purification kit (Promega). ..

Quantitative RT-PCR:

Article Title: Structure and ubiquitination-dependent activation of Tank-Binding Kinase 1
Article Snippet: .. At 48 h post-transfection, luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) or RNA was prepared for QRT-PCR, as described in . .. Lysates were prepared 60 h following transient transfection of HEK293T cells with TBK1 constructs using standard RIPA buffer.

Activity Assay:

Article Title: MiR-195 inhibits proliferation and growth and induces apoptosis of endometrial stromal cells by targeting FKN
Article Snippet: .. Luciferase activity was measured 48 h post-transfection using the Dual-Luciferase Reporter Assay System, according to the manufacturer’s instructions (Promega). .. For each sample, Renilla luciferase activity was normalized by the total protein content.

Article Title: Structure and ubiquitination-dependent activation of Tank-Binding Kinase 1
Article Snippet: .. At 48 h post-transfection, luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) or RNA was prepared for QRT-PCR, as described in . .. Lysates were prepared 60 h following transient transfection of HEK293T cells with TBK1 constructs using standard RIPA buffer.

Article Title: The NS5A-binding heat shock proteins HSC70 and HSP70 play distinct roles in the hepatitis C viral life cycle
Article Snippet: .. Seventy-two hours post transfection, Renilla and Firefly luciferase activity were determined using Dual Luciferase Assay System (Promega, E1910). .. Error bars reflect the standard deviation.

Cell Culture:

Article Title: Human SNP Links Differential Outcomes in Inflammatory and Infectious Disease to a FOXO3-Regulated Pathway
Article Snippet: .. Cells were seeded at 2 × 105 cells/ml and cultured overnight without antibiotics before transient transfection with a firefly pGL4.14 hLuc vector containing the TGFβ1 promoter (666 ng/ml, Promega), a pCMV renilla luciferase control vector (6.6 ng/ml), and one of two FOXO3A siRNAs, or a scrambled control (66 nM/ml, Origene) using lipofectamine 2000 (Invitrogen). ..

Expressing:

Article Title: Improvement of the CRISPR-Cpf1 system with ribozyme-processed crRNA
Article Snippet: .. Two days post-transfection, luciferase expression was measured with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. .. The ratio of Firefly to Renilla was calculated as the relative Luc activity.

Plasmid Preparation:

Article Title: Muscleblind-like 1 (Mbnl1) Promotes Insulin Receptor Exon 11 Inclusion via Binding to a Downstream Evolutionarily Conserved Intronic Enhancer *
Article Snippet: .. Transient transfections of cells with plasmid DNA were performed with TransFast reagent (Promega, Madison, WI) according to the manufacturer's protocol. .. For cotransfection experiments, cells were transfected with 500 ng of minigene plasmid DNA and 1 μg of an expression vector for splicing factors of interest.

Article Title: Human SNP Links Differential Outcomes in Inflammatory and Infectious Disease to a FOXO3-Regulated Pathway
Article Snippet: .. Cells were seeded at 2 × 105 cells/ml and cultured overnight without antibiotics before transient transfection with a firefly pGL4.14 hLuc vector containing the TGFβ1 promoter (666 ng/ml, Promega), a pCMV renilla luciferase control vector (6.6 ng/ml), and one of two FOXO3A siRNAs, or a scrambled control (66 nM/ml, Origene) using lipofectamine 2000 (Invitrogen). ..

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    Promega pgl4 10
    A 3′ ss can mimic a promoter in the pGL4 system by inducing production of spliced readthrough transcripts. ( A ) Expression of luciferase from <t>pGL4.10</t> and pGL4.17 with and without the globin 3′ ss inserted into the MCS. Error bars, SD. ( B ) 5′ RACE products from pGL4.17 containing the globin insert.
    Pgl4 10, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pgl4 10 basic plasmid
    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, <t>PGL4.10-TGF-β1</t> 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
    Pgl4 10 Basic Plasmid, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Promega pgl4 10 promoterless luciferase vector
    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 <t>promoterless</t> luciferase construct, <t>pGL4.10.</t> 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
    Pgl4 10 Promoterless Luciferase Vector, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A 3′ ss can mimic a promoter in the pGL4 system by inducing production of spliced readthrough transcripts. ( A ) Expression of luciferase from pGL4.10 and pGL4.17 with and without the globin 3′ ss inserted into the MCS. Error bars, SD. ( B ) 5′ RACE products from pGL4.17 containing the globin insert.

    Journal: Nucleic Acids Research

    Article Title: Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function

    doi: 10.1093/nar/gks451

    Figure Lengend Snippet: A 3′ ss can mimic a promoter in the pGL4 system by inducing production of spliced readthrough transcripts. ( A ) Expression of luciferase from pGL4.10 and pGL4.17 with and without the globin 3′ ss inserted into the MCS. Error bars, SD. ( B ) 5′ RACE products from pGL4.17 containing the globin insert.

    Article Snippet: The SacI–NcoI fragment of pGL3-Enhancer containing the 3′ ss was transferred to pGL4.17 and pGL4.10 to generate the β-globin 3′ ss-containing variants of these plasmids.

    Techniques: Expressing, Luciferase

    Dual luciferase reporter assays examining promoter and enhancer activities for DNA fragments containing HS site . The pGL4.10 vector, which contains the promoterless synthetic firefly luc2 , was used in the promoter assay (left). The pGL4.23 vector, which contains the synthetic firefly luc2 driven by a minimal promoter, was used in the enhancer assay (right). The histograms show a summary of the ratio of luciferase activity (adjusted by dividing the firefly luciferase with the control Renilla luciferase) for each insert (from the HS sites shown in lower panel) relative to the luciferase activity for pGL4.10 or pGL4.23. Each fragment was tested in triplicate and experiments were carried out three times independently. Error bars represent the standard deviations of three trials. While fragments upstream of the XIST promoter containing HS sites showed background luciferase activity, fragment -65 displayed five fold and seven fold increases in promoter activity and ten fold and six fold increases in enhancer activity in the XIST and antisense orientation, respectively.

    Journal: BMC Molecular Biology

    Article Title: Identification of regulatory elements flanking human XIST reveals species differences

    doi: 10.1186/1471-2199-11-20

    Figure Lengend Snippet: Dual luciferase reporter assays examining promoter and enhancer activities for DNA fragments containing HS site . The pGL4.10 vector, which contains the promoterless synthetic firefly luc2 , was used in the promoter assay (left). The pGL4.23 vector, which contains the synthetic firefly luc2 driven by a minimal promoter, was used in the enhancer assay (right). The histograms show a summary of the ratio of luciferase activity (adjusted by dividing the firefly luciferase with the control Renilla luciferase) for each insert (from the HS sites shown in lower panel) relative to the luciferase activity for pGL4.10 or pGL4.23. Each fragment was tested in triplicate and experiments were carried out three times independently. Error bars represent the standard deviations of three trials. While fragments upstream of the XIST promoter containing HS sites showed background luciferase activity, fragment -65 displayed five fold and seven fold increases in promoter activity and ten fold and six fold increases in enhancer activity in the XIST and antisense orientation, respectively.

    Article Snippet: PCR fragments were first cloned into pGEM® -T Easy vector (Promega) via TA cloning prior to insertion into pGL4.10 and pGL4.23 reporter vectors (Promega) upstream of the firefly luciferase gene.

    Techniques: Luciferase, Plasmid Preparation, Promoter Assay, Activity Assay

    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

    Journal: Oncology Letters

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

    doi: 10.3892/ol.2017.6291

    Figure Lengend 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

    Article Snippet: TGF-β1 promoter was subcloned into the pGL4.10 basic plasmid (Promega Corporation, Madison, WI, USA) at the KpnI and HindIII restriction enzyme sites to drive luciferase expression.

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

    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

    Journal: eLife

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

    doi: 10.7554/eLife.17304

    Figure Lengend 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

    Article Snippet: Forward (5’-GAGCTC GCTAGC TAAACGTGTTCACATTTTGTTGCC- 3’) and reverse (5’-TGCC AAGCTT CAGTCCCCGGAGACCCTC-3’) primers containing NheI and HindIII restriction sites respectively, indicated by underlined regions, were used to amplify the promoter region and allow cloning into the Promega pGL4.10 promoterless luciferase vector.

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