Figure 3. The effect of ETV1 and dETV1 on ARF promoter activity. (A) The structure of the ARF-luciferase reporter pARF-luc. A fragment of human genome that corresponds to a portion of INK4A exon 1b and the putative ARF promoter was introduced into pGL3Basic plasmid upstream of the firefly luciferase coding region. Translation start sites corresponding to p14ARF and luciferase (show in bold) are maintained in the same open reading. Nucleotide positions are numbered relative to the transcription start site of ARF (“TSS”). The scheme is not drawn to scale. (B) ETV1 increases ARF promoter activity. pARF-luc was co-transfected with a constitutive β-galactosidase reporter pRSV-βgal and either an ETV1-expressing construct or the corresponding empty vector control (pBabePuro). The luciferase activity was normalized for that of β-galactosidase and shown relative to that in vector-transfected cells. (C) ETV1 does not increase the activity of CMV immediate early promoter. CMV-driven luciferase construct pLNCLuc was co-transfected with pRSV-βgal and either an ETV1-expressing construct or the corresponding empty vector control (pBabePuro). (D) ETV1 and dETV1 promote ARF gene expression in Saos-2 cells. p14ARF promoter activity in Saos-2 cells was measured following co-transfection of pARF-luc and pRSV-βgal into Saos-2 cells pre-engineered with the indicated transgenes (see Fig. 2). Luciferase activity was normalized to that of β-galactosidase and is presented relative to the levels in cells expressing empty vector control (pBabePuro). Results are shown as the means and standard deviations of three independent replicas.