Figure 2

<p>CNEs Regulate Luciferase Reporter Gene Expression in Transiently Transfected Human Cell Lines. (A) Diagrams of the reporter constructs employed to test the regulatory potential of CNEs from the introns of human <i>GLI3</i>. CNEs were cloned into in a pGL3-Basic vector containing either a minimal <i>GLI3</i> promoter (pGL3-CNE-prom<i>GLI3</i>-300-luc) or a heterologous SV40 promoter (pGL3-CNE promSV40-luc) upstream of a firefly luciferase gene. (B) Luciferase activity of reporter constructs in human H661 cells that express endogenous <i>GLI3</i>. (C) Luciferase activity of reporter constructs in human H441 cells that do not express endogenous <i>GLI3</i>. The pGL3-Basic vector, with no promoter/enhancer insert was used as a negative control. Luciferase activity in cells transiently transfected with the positive control, a construct containing a SV40 promoter upstream of the reporter gene (pGL3-promSV40-luc), was taken as 100% (blue dotted line). A plasmid expressing <i>Renilla</i> luciferase was co-transfected as a standard for transcription efficiency. Average firefly luciferase reporter activities relative to <i>Renilla</i> luciferase activity from three triplicate transfection experiments are depicted as percentage of activity obtained with the positive control vector (B, C). Standard errors of the mean are shown. Black dotted lines indicate the luciferase expression level reached in each cell line with the pGL3-prom<i>GLI3</i>-300-luc vector.