Figure 5

Over-expression of EWS-FLI1 junction region (aa 251- aa 280) inhibits tumorigenicity, EWS-FLI1 target gene expression and EMT marker genes in Ewing’s sarcoma cells. A: Confirmation of junction region (aa 251–280) protein expression in transfected EWS502 cell line by immunoblotting with anti Flag antibody. Lane 1: pCDNA/FLAG expressing EWS502 cells, Lane 2: pCDNA/FLAG/Junction (aa 251–280) expressing EWS502 cells. Figure 5 B: Real time RT-PCR analysis of Junction region (aa 251–280) in pCDNA/FLAG and pCDNA/FLAG/Junction (aa 251–280) stably expressing EWS502 cells. The real time RT-PCR analysis was performed using the same primer set used to clone the junction region and SYBR green chemistry. Figure 5 C: Soft agar assay showing significant reduction in colony number in EWS502 transfected with junction deletion construct, compared to vector control and wild type EWS502. Figure 5 D: Photographs of live pCDNA/FLAG and pCDNA/FLAG/Junction (aa 251–280) stably expressing EWS502 cells in culture obtained at 10X magnification.