Biochemical disruption of EWS-FLI1 and p53 binding to DNA.

<p>Dose response for disruption of recombinant EWS-FLI1 (red lines) and p53 (black lines) binding to DNA in the presence of indicated compounds was measured using AlphaScreen proximity assays. Data plotted as mean +/− SD of triplicate samples and are representative of 2 independent experiments.</p

AlphaScreen Results.

<p>AlphaScreen Results.</p

Effect of actinomycin D on gene expression.

<p>Effects of actinomycin D (<b>A</b>), epirubicin (<b>B</b>) doxorubicin (<b>C</b>), ebselen (<b>D</b>) and Erk inhibitor (<b>E</b>) on NR0B1-Luc (solid line) and UbC-Renilla (dotted line) reporter activity. Data plotted as mean +/− SEM of triplicates. <b>F</b>: Quantitative RT-PCR was used to determine the abundance of NR0B1, TP53 and RPS26 mRNA after overnight treatment of A673 cells with the indicated concentrations of actinomycin D. Results normalized to DMSO control. Data plotted as mean +/− SD of quadruplicates, and are representative of 3 independent experiments.</p

Effects of actinomycin D on the binding of EWS-FLI1 to the NR0B1 promoter.

<p>EWS-FLI1 was immunoprecipiatated using a FLI1 antibody, and quantitative PCR used to determine binding to NR0B1, RPS26, and p53. Data expressed as fold-enrichment over normal IgG control ChIP. Data plotted as mean +/− SD of duplicates are representative of 3 independent experiments.</p

Growth effects of actinomycin D on Ewing sarcoma and other cancer cell lines.

<p><b>Cells were treated with DMSO or actinomycin D for 44 hours.</b> Viable cell number was determined with a MTT assay. Data plotted as mean +/− SD of sextuplets and are representative of 3 independent experiments.</p