Fig. 2: Oncogenic transformation capacity of EWS/FLI affected by short regions surrounding the FLI DBD.

A Protein schematic of 3xFLAG-tagged (3 F) EWS/FLI cDNA constructs with deleted FLI domain regions. EF represents a full-length type IV EWS/FLI, EF DBD represents EWS fused directly to the 85-amino acid DNA-binding domain of FLI, and EF DBD+ represents EWS fused to a 102-amino acid region of FLI that contains the 85 amino-acid DNA-binding domain with 7 additional amino-acids on the amino-terminal side and 10 additional amino-acids on the carboxyl-terminal side. B Dual luciferase reporter assay results for the indicated constructs tested on control and 20xGGAA μSat-containing plasmids (as described in Fig. 1). Data are presented as mean ± SEM (N = 6 biological replicates with 3 technical replicates each). Asterisks indicate that the activity of EF DBD and EF DBD+ are each statistically higher than EF (p-value < 0.001). C Representative qRT-PCR results of endogenous EWS/FLI in A673 cells harboring the indicated constructs (iLuc is a control shRNA targeting luciferase and iEF is a shRNA targeting the 3′UTR of endogenous EWS/FLI; N = 1 biological replicate with 3 technical replicates for each sample). EWS/FLI mRNA values are normalized to RPL30 mRNA control values. Asterisks indicate samples are statistically different as compared to control (iLuc + Empty Vector) cells (p-value < 0.001). D Western blot analysis of exogenous EWS/FLI protein expression in the A673 knock-down/rescue cells. Protein constructs were detected using α-FLAG antibody and α-Tubulin was used as a loading control. E Representative soft agar assay results of A673 Ewing sarcoma cells containing the indicated constructs. F Soft agar assay colony formation quantification. Data presented as mean ± SEM (N = 9 biological replicates with 2 technical replicates each). Asterisks indicate p-value < 0.001 as compared to iEF + Empty Vector cells.