cdna encoding erg  (OriGene)

Journal: Nature Communications

Article Title: The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature

doi: 10.1038/s41467-019-12897-w

Figure Lengend Snippet: ERG regulates thrombomodulin expression and activity in vitro. a qPCR analysis of thrombomodulin ( TM ) mRNA expression in control (siCtrl) and ERG-deficient (siERG) HUVEC after 12, 24 and 48 h treatment ( n = 3 independent experiments). Data were normalized to GAPDH . b Representative immunoblot and quantification of TM following siCtrl or siERG treatment on HUVEC for 12, 24 and 48 h ( n = 4 independent experiments). Data were normalized to GAPDH. c Representative image and quantification of TM expression (green) in HUVEC transfected with siCtrl or siERG siRNA for 48 h by immunofluorescence; nuclei are identified by DAPI (blue) and cells are co- stained for ERG (magenta). Scale bar 40 µm. Quantification represents the mean pixel intensity for TM signal (arbitrary unit, A.U.) per cell. d , e Representative immunoblot and quantification of ERG and TM expression in control (siCtrl) and ERG-deficient (siERG) d HDMEC (microvascular EC) or e HDBEC (microvascular EC) after 48 h siRNA treatment ( n = 3). f qPCR analysis of TM mRNA expression in HUVEC transfected with control pcDNA or ERG cDNA expression plasmid (ERG) ( n = 3). Data were normalised to GAPDH . g siCtrl or siERG-treated HUVEC for 48 h were incubated with protein C (300 nM), CaCl 2 (5 mM), thrombin (10 nM). After 10, 20, 30 and 60 min of incubation at 37 °C, anti-thrombin III (100 nM) and heparin (15 U per ml) were added to neutralise thrombin, and protein C activity was measured using chromogenic substrate S-2366 (0.5 mM) ( n = 3). Data are expressed as relative activated protein C (APC) concentration normalised to siCtrl-treated condition following 60 min of incubation. All graphical data are mean ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t -test. Source data are provided as a Source Data file

Article Snippet: Human ERG cDNA (NCBI Accession NM_182918) was cloned into the mammalian vector pcDNA3.1 (Invitrogen).

Techniques: Expressing, Activity Assay, In Vitro, Western Blot, Transfection, Immunofluorescence, Staining, Plasmid Preparation, Incubation, Concentration Assay

Journal: Nature Communications

Article Title: The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature

doi: 10.1038/s41467-019-12897-w

Figure Lengend Snippet: ERG binds to and transactivates the thrombomodulin promoter in vitro. a Putative ERG binding sites (black bars) are located within the TM promoter upstream and downstream of the transcription start site (black arrow); ERG ChIP-sequencing data show a significant ERG peak on TM proximal promoter. ENCODE sequence conservation between 100 vertebrates is shown across this gene. ENCODE ChIP-seq data profiles for H3K4Me3 (tri-methylation of lysine (K) 4 on histone 3), H3K27Ac (acetylation of lysine (K) 27 on histone 3) and RNA polymerase II (RNA pol II) in HUVEC indicate open chromatin and active transcription. Location of qPCR primers covering regions R1 and R2 (black bar) are indicated. b ChIP-qPCR using primers to region R1 and R2 on ERG-bound chromatin from siCtrl or siERG-treated HUVEC. Primers for a region within 5′UTR region of TM gene (TM neg con) were used as negative control. Data are shown as fold change over IgG ( n = 3 independent experiments). Graphical data are mean ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t -test. c TM promoter luciferase reporter assay. ERG cDNA expression plasmid (ERG) or empty expression plasmid (pcDNA) were co-transfected with TM promoter-luciferase constructs (TM wild type (WT), TM mutant 1 or TM mutant 2) or a pGL4 empty vector in HUVEC, and luciferase activity was measured. Values represent the fold change in relative luciferase activity over the empty pGL4 vector alone ( n = 4 independent experiments). Graphical data are mean ± s.e.m., ** P < 0.01, *** P < 0.001, One-way ANOVA; ## P < 0.01 compared to pcDNA-TM WT condition, Student’s t -test. d ChIP-qPCR using primers to region R1 and R2 on H3K27Ac-bound chromatin from HUVEC treated with DMSO or p300 inhibitor (10 µM) for 1 h. Data are shown as fold change over IgG ( n = 3 independent experiments). e – f ChIP-qPCR using primers to region R1 and R2 on e p300- ( n = 3 independent experiments) or f H3K27Ac-bound ( n = 4 independent experiments) chromatin from siCtrl or siERG-treated HUVEC. Data are shown as fold change over IgG. All graphical data for ChIP experiments are mean ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t -test. Source data are provided as a Source Data file

Article Snippet: Human ERG cDNA (NCBI Accession NM_182918) was cloned into the mammalian vector pcDNA3.1 (Invitrogen).

Techniques: In Vitro, Binding Assay, ChIP-sequencing, Sequencing, Methylation, Negative Control, Luciferase, Reporter Assay, Expressing, Plasmid Preparation, Transfection, Construct, Mutagenesis, Activity Assay

Journal: Nature Communications

Article Title: The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature

doi: 10.1038/s41467-019-12897-w

Figure Lengend Snippet: ERG cooperates with KLF2 to drive thrombomodulin expression. a – d TM promoter luciferase reporter assays. Values represent the fold change in relative luciferase activity over the empty pGL4 vector alone. b pcDNA or ERG expression plasmid (ERG) were co-transfected with a TM wild type (WT) promoter-luciferase construct (TM WT) in siCtrl or siKLF2-treated HUVEC, and luciferase activity was measured ( n = 3 independent experiments). b pcDNA or KLF2 cDNA expression plasmid (KLF2) were co-transfected with TM WT promoter construct in siCtrl or siERG-treated HUVEC ( n = 3 independent experiments). Graphical data presented in a and b are mean ± s.e.m., * P < 0.05, ** P < 0.01, Student’s t -test. c pcDNA, KLF2 or both KLF2 and ERG plasmids were co-transfected with TM WT promoter construct ( n = 4 independent experiments). Graphical data are mean ± s.e.m., *** P < 0.001, **** P < 0.0001, One-way ANOVA, compared to pcDNA-TM WT condition. d pcDNA or KLF2 plasmid were co-transfected with TM promoter constructs (TM WT, TM mutant 1 or TM mutant 2) ( n = 4 independent experiments). Graphical data are mean ± s.e.m., *** P < 0.001, **** P < 0.0001, One-way ANOVA. ## P < 0.01, compared to pcDNA-TM WT, Student’s t -test. e , f ChIP-qPCR using primers to region R1 and R2 on e ERG- or f KLF2-bound chromatin from siCtrl, siERG or siKLF2-treated HUVEC ( n = 3 independent experiments). Primers for a region within 5’UTR region of TM gene (TM neg con) were used as negative control. Data are shown as fold change over IgG. Graphical data are mean ± s.e.m., NS: Not Significant, * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t -test. g Representative image of proximity ligation assay (PLA) for nuclear ERG-KLF2 interaction was performed on confluent HUVEC using anti-ERG and anti-KLF2 antibodies; nuclei are identified by DAPI (blue). Scale bar, 20 μm. h Model: In homeostatic EC, ERG is required for p300 recruitment at the TM promoter region, leading to acetylation of H3K27 which opens the chromatin and allows KLF2 binding to the TM promoter. ERG binds to and cooperates with KLF2 to drive TM expression. In the absence of ERG, the chromatin is not accessible; KLF2 is not binding to the TM promoter and is not able to transactivate the TM promoter. Source data are provided as a Source Data file

Article Snippet: Human ERG cDNA (NCBI Accession NM_182918) was cloned into the mammalian vector pcDNA3.1 (Invitrogen).

Techniques: Expressing, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Construct, Mutagenesis, Negative Control, Proximity Ligation Assay, Binding Assay

Journal: Nature Communications

Article Title: The endothelial transcription factor ERG mediates Angiopoietin-1-dependent control of Notch signalling and vascular stability

doi: 10.1038/ncomms16002

Figure Lengend Snippet: ( a ) mRNA levels of Dll4 in HUVEC treated with siCtrl or siERG for 24 and 48 h ( n =6). ( b ) Representative WB and quantification of Dll4 protein expression in siCtrl and siERG-treated HUVEC for 24 and 48 h ( n =3). ( c ) mRNA levels of Jag1 in siCtrl and siERG-treated HUVEC for 24 and 48 h ( n =6). ( d ) Representative WB and quantification of Jag1 expression in siCtrl and siERG-treated HUVEC for 24 and 48 h ( n =6). ( e ) Putative ERG binding sites (grey bars) are located within the Dll4 promoter downstream of the transcription start site (TSS) (arrow); ENCODE sequence conservation between 100 vertebrates is shown across this region. ENCODE ChIP-seq data profiles for H3K4me3, H3K27Ac and RNA polymerase II (RNA pol) in HUVEC indicate open chromatin and active transcription. Location of qPCR amplicon covering region R1 is indicated. ( f ) ChIP-qPCR using primers to region R1 on ERG-bound chromatin from siCtrl or siERG-treated HUVEC. Primers for a region within exon11 of the Dll4 gene were used as negative control. Data are shown as fold change over IgG ( n =3). ( g ) Dll4 promoter luciferase reporter assay. ERG cDNA expression plasmid (pcDNA-ERG) or empty expression plasmid (pcDNA) were co-transfected with a Dll4 promoter-luciferase construct (pGl4-Dll4, covering region R1) in HUVEC, and luciferase activity was measured. Values represent the fold change in relative luciferase activity over the empty pGL4 vector alone ( n =4). ( h ) Putative ERG binding sites (grey bars) located within the Jag1 genomic locus. TSS is indicated (arrow); ENCODE sequence conservation between 100 vertebrates and ChIP-seq data profiles for H3K4me3, H3K27Ac and RNA polymerase II in HUVEC are shown across this region. Location of qPCR amplicons covering R1, R2 and R3 are indicated. ( i ) ChIP-qPCR using primers covering Jag1 promoter regions R1, R2, R3 and Ctrl region on ERG-bound chromatin from siCtrl or siERG HUVEC ( n =4). ( j ) Control or Jag1 promoter luciferase construct (pGl3-Jag1, covering regions R1 and R2) activity after siERG treatment. Results are expressed as luciferase activity relative to siCtrl-treated cells ( n =3). All graphical data are mean±s.e.m., * P <0.05, ** P <0.01, *** P <0.001, Student’s t -test.

Article Snippet: Human ERG cDNA (NCBI Accession NM_182918) was cloned into the mammalian expression vector pcDNA3.1 (Invitrogen).

Techniques: Expressing, Binding Assay, Sequencing, ChIP-sequencing, Amplification, Negative Control, Luciferase, Reporter Assay, Plasmid Preparation, Transfection, Construct, Activity Assay

Journal: Nature Communications

Article Title: The endothelial transcription factor ERG mediates Angiopoietin-1-dependent control of Notch signalling and vascular stability

doi: 10.1038/ncomms16002

Figure Lengend Snippet: ( a ) Dll4 mRNA expression in siCtrl and siERG-transfected HUVEC treated in the presence or absence of the γ-secretase inhibitor DAPT ( n =4). ( b ) Dll4 mRNA expression in HUVEC transfected with ERG cDNA expression plasmid (pcDNA-ERG) or an empty expression plasmid (pcDNA) and treated in the presence or absence of the γ-secretase inhibitor DAPT ( n =3). ( c ) ERG mRNA expression in siCtrl and siERG-transfected HUVEC treated in the presence or absence of DAPT ( n =4). ( d ) mRNA expression of ERG and the Notch target gene Hey1 in HUVEC stimulated with control BSA or Dll4 ( n =4). Representative images and quantification of ERG (red) staining of P6 retinal vessels in the ( e ) vascular plexus and ( f ) angiogenic front from control ( Rbpj fl/fl ) and Rbpj i Δ EC mice. Retinas are co-stained for isolectin B4 (IB4, green). Quantification represents the ratio between the sum of pixel intensity and isolectin B4 area ( n =4 fields per mouse, n =4 mice per genotype). Scale bar, 70 μm. Arteries (A) and veins (V) are indicated. All graphical data are mean±s.e.m., * P <0.05, ** P <0.01, *** P <0.001, Student's t -test.

Article Snippet: Human ERG cDNA (NCBI Accession NM_182918) was cloned into the mammalian expression vector pcDNA3.1 (Invitrogen).

Techniques: Expressing, Transfection, Plasmid Preparation, Staining