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. 2014 Aug 5;9(8):e104378. doi: 10.1371/journal.pone.0104378

Figure 5. EWS/FLI-mediated gene expression is highly variable across various GGAA-microsatellite length polymorphisms.

Figure 5

(A) Polymorphic NR0B1 GGAA-microsatellites from white European and African subjects were cloned into luciferase reporter vectors and co-transfected with EWS/FLI into 293 EBNA cells. A bimodal pattern of gene expression was observed, with greatest expression in constructs with 24 GGAA motifs and a lesser peak in constructs with 58 GGAA motifs. (B) A similar bimodal trend was observed using synthetic GGAA constructs identically cloned into the same luciferase reporter construct. (C) In patient derived Ewing sarcoma cell lines, RT-PCR quantified NR0B1 mRNA expression was also maximal in cell lines containing an NR0B1 microsatellite containing 24–26 GGAA motifs. (D) In primary Ewing sarcoma tumors, normalized NR0B1 transcript levels were lowest in tumors with NR0B1 GGAA-microsatellites containing 17–18 GGAA motifs, which was significant less than tumors with microsatellites containing 23–26 GGAA motifs (p = 0.04).