The effect of EWS/ETS expression on the activity of the DKK2 promoter.

<p>A, Schematic representation of the 5′upstream region of the DKK2 gene. The nucleotide numbering represents the distance from the translation start site (+1). <i>Kpn</i>I (−1741), <i>Nhe</i>I (−1241), and <i>Sac</i>I (−521) sites were used to construct a series of deletion mutants of the DKK2 promoter (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004634#s2" target="_blank">Materials and Methods</a>). Consensus ets binding sites are boxed (EBS-1, EBS-2 and EBS-3). B, Western blot analysis for EWS/ETS expression. HEK293 cells were transiently transfected with pcDNA3-flagDEST or pcDNA3-flagEWS/ETS and then analyzed by anti-flag Western blotting. C, The effect of EWS/ETS expression on the activity of the DKK2 promoter. HEK293 cells were co-transfected with reporter plasmids and the expression vector. Luciferase activity was measured after 48 hours. Data are relative values with the SD for triplicate wells. Data are normalized to the value for the empty vector (pcDNA3-flagDEST) which is arbitrarily set to 1. The transfection efficiency was normalized relative to the β–galactosidase activity (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004634#s2" target="_blank">Materials and Methods</a>). D, Schematic representation of a series of deletion mutants of the DKK2 5′ upstream region. KpnI (−1741), NheI (−1241) and SacI (−521) sites were used to construct a series of deletion mutants of the DKK2 promoter (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004634#s2" target="_blank">Materials and Methods</a>). E, F, G, The transactivation of deletion mutants of the DKK2 promoter by EWS/ETS expression. HEK293 cells were co-transfected with reporter plasmids and the expression vector. Data are normalized to the value for the empty vector (pcDNA3-flagDEST) which is arbitrarily set to 1. Luciferase activity was measured after 48 hours. Data are relative values with the SD for triplicate wells. The transfection efficiency was normalized relative to the β–galactosidase activity (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004634#s2" target="_blank">Materials and Methods</a>).