Regulation of targets by EWS in vivo.

A. Protein domain organization of EWS and FLI1. The black vertical arrows indicate common breakpoints in Ewing sarcoma. Numbers correspond to exons and a typical EWS-FLI1 fusion protein is also shown. Note that the RNA-binding domain of EWS is lost in the process of translocation. B. Pie diagram showing the distribution of PAR-CLIP clusters across 3’UTR, 5’UTR, intronic and coding regions of Refseq RNAs. The three diagrams give the cluster distribution of all sequenced EWS PAR-CLIP targets, all targets regulated by EWS and the four targets we validated (FGF9, MDM2, CBFB, CCDC6). C. Relative mRNA levels of targets genes FGF9, MDM2, CBFB, CCDC6 and EWS in HEK293T cells following EWS knockdown assayed by qRT-PCR (mock: only transfection reagent used; scrambled: AllStars Negative Control siRNA; EWS: siRNA targeting EWS). Relative mRNA levels were normalized to beta actin and quantified relative to the mock and scrambled control levels. Results are shown as mean SEM values (*P < 0.05; n = 3 per group). D. Amount of CCDC6 mRNA transcript percentage is measured upon knocking down of EWS as compared to control. The level of transcript was measured by qRT-PCR after knocking down for 24 hours followed by treatment with actinomycin D. The linear regression and slopes were calculated and the data is presented as Mean and SEM on a linear scale. E. Luciferase activity of CCDC6 upon EWS transfection (normalized to empty psiCHECK-2 plasmid). Data is shown as the fold increase in luciferase activity (RLU units) relative to control. Results are shown as the mean SEM values (*P<0.05; n = 3 per group). F. Relative mRNA levels of CCDC6 and EWS in mock, control and EWS knockdown in MHH-ES-1 cells. Knockdown of EWS decreased the expression of CCDC6. The mRNA levels were normalized to beta actin. Data is represented as mean SEM values (*P<0.05; n = 3 per group). G. Western blot showing the downregulation of CCDC6 upon EWS knockdown in MHH-ES-1 cells. Antibodies are indicated.