Journal: Nucleic Acids Research

Article Title: Targeting the DNA-binding activity of the human ERG transcription factor using new heterocyclic dithiophene diamidines

doi: 10.1093/nar/gks971

Figure Lengend Snippet: EPDBi for screening the ERG/EBS-WT binding inhibitors. EPDBi was performed on streptavidin-coated plates in which biotinylated EBS-WT oligonucleotides were fixed. ERG proteins were added together with the indicated DB compounds at 5 µM. Control corresponds to 100% of ERG/DNA complex. EBS-WT DNA-binding specificity was validated by addition of 50-fold excess of EBS-WT (50× S) or non-specific oligonucleotide (50× NS) in the binding buffer. Results are means ± s.e.m. from two experiments both performed in triplicate.

Article Snippet: After two washes for 5 min with 200 µl of washing solution (TBS-Tween 0.5%) at room temperature, ERG proteins expressed from reticulocyte lysate system (Promega, Madison, WI, USA) (2 µl of lysate previously diluted to one-fifth in sterile water) were incubated for 30 min at 4°C with/without 5 µM of the indicated DB compounds in binding buffer (10 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 75 mM NaCl, 6% glycerol, 10 µg BSA).

Techniques: Binding Assay

Journal: Nucleic Acids Research

Article Title: Targeting the DNA-binding activity of the human ERG transcription factor using new heterocyclic dithiophene diamidines

doi: 10.1093/nar/gks971

Figure Lengend Snippet: Validation of the ERG/EBS complex inhibition by the evaluated diamidine compounds. EMSA was performed with the EBS-WT radiolabelled oligonucleotide incubated with ERG protein expressed in reticulocyte lysate systems or empty lysate made with the equivalent empty vector, in the presence of thiophene ( A ), selenophene or furan ( B–D ) derivatives. Bound ERG/EBS-WT complexes ( b , indicated by a black arrow) were competed with increasing concentration of DB compounds (µM) and separated to free EBS-WT oligonucleotide ( f , indicated by black arrow) by electrophoresis migration. DNA binding specificity was determined by addition of 50-fold excess of the specific unlabelled EBS-WT (50× S) or a non-specific unlabelled oligonucleotide EBSm (50× NS).

Article Snippet: After two washes for 5 min with 200 µl of washing solution (TBS-Tween 0.5%) at room temperature, ERG proteins expressed from reticulocyte lysate system (Promega, Madison, WI, USA) (2 µl of lysate previously diluted to one-fifth in sterile water) were incubated for 30 min at 4°C with/without 5 µM of the indicated DB compounds in binding buffer (10 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 75 mM NaCl, 6% glycerol, 10 µg BSA).

Techniques: Inhibition, Incubation, Plasmid Preparation, Concentration Assay, Electrophoresis, Migration, Binding Assay

Journal: Nucleic Acids Research

Article Title: Targeting the DNA-binding activity of the human ERG transcription factor using new heterocyclic dithiophene diamidines

doi: 10.1093/nar/gks971

Figure Lengend Snippet: Sequence selectivity of ERG and DB1255 DNA binding. ( A ) Sequence selectivity of the DB1255/DNA interaction. DNase I footprint assays were performed with a DNA fragment containing three EBS sequences mutated at the indicated positions (positions −1 to +7, DNase I footprint gels and densitometric analyses for the position −1 and 0 and position +3 to +7 are presented in Figure S5A ). For each mutation incubation with DB1255 at the indicated concentrations (µM) revealed protected sites of DB1255 interaction. The track labelled “G” is as in Figure S1 . ( B) Densitometric analyses derived from the gels and EBS-WT (WT) sequence or EBS-mutated sequence are indicated by black boxes or a grey boxes, respectively. ( C ) The logos were drawn using enoLOGOS , where the size of the letter is directly proportional to the binding affinity at 0.8 µM of the compound resulting for densitometric analysis (data shown in Figure S5B ). ( D) Sequence selectivity of the ERG binding to the EBS-WT sequence. ERG binding sequence selectivity are observed by EMSA using radiolabelled EBS oligonucleotide WT (EBS WT) or mutated (EBS mXN) at the indicated position (position X of base N) incubated with ERG protein expressed in reticulocyte lysate system or empty lysate. EBS sequences with mutated positions from −5 to +7 were evaluated but only the EMSA with mutated positions +3 to +7 are shown here (EMSAs of mutated position −5 to +2 are shown in Figure S6A ). ( E) Representation of the percentage of ERG/DNA complex derived from the gel quantification as means ± s.e.m. relatively to EBS-WT. ( F) The logos were drawn as above (full data presented in Figure S6B ).

Article Snippet: After two washes for 5 min with 200 µl of washing solution (TBS-Tween 0.5%) at room temperature, ERG proteins expressed from reticulocyte lysate system (Promega, Madison, WI, USA) (2 µl of lysate previously diluted to one-fifth in sterile water) were incubated for 30 min at 4°C with/without 5 µM of the indicated DB compounds in binding buffer (10 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 75 mM NaCl, 6% glycerol, 10 µg BSA).

Techniques: Sequencing, Binding Assay, Mutagenesis, Incubation, Derivative Assay