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. 2008 Jun 4;3(6):e2353. doi: 10.1371/journal.pone.0002353

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This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

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A. Schematic representation of ENT4 gene. The dark grey box indicated by +1 represents the first non-coding exon and the transcription start site (+1). The second dark grey box marked by ATG represents exon 2 containing the starting ATG. The light grey box represents the GA-repeat sequence. The black box represents the GC-rich sequence. B. Schematics showing the luciferase reporter constructs (P1–P4) containing different fragments of the ENT4 promoter. P3 promoter construct contains the deletion of GA-repeat sequences (−915 to +272). The numbers are relative to the transcription start (+1). C. Direct transcriptional activation of ENT4 promoters by EWS/WT1. U2OS cells were co-transfected with either pcDNA3-EWS/WT1 (− or +KTS; 0.5 µg) or with pcDNA3 along with the promoter-reporter constructs (P1, P2, P3 or P4; 0.5 µg) and Renilla luciferase (0.1 µg) using FuGENE 6. Luciferase activity was measured at 48 hrs post-transfection and expressed as relative luciferase activity compared to the empty vector. Data represent the mean±S.D. from three independent experiments. D. Schematics showing the three putative EWS/WT1(−KTS) binding sites (black boxes) within the GC-rich region of the P2 promoter and the mutated derivatives, M1, M2, M3 and M2/M3 constructs (see Materials and Methods for detail). E. Identification of the EWS/WT1(−KTS) responsive element M2. Luciferase-reporter assay was performed as in (C) except with the promoter-reporter constructs shown in (D). Data represent the mean±S.D. from three independent experiments.