Fig 3. CTEN is transcriptionally regulated by ΔNp63α.

RWPE-1 cells were transfected by control siRNA (si-ctrl) or ΔNp63 siRNA (si-ΔNp63). qPCR (A) and western (B) analyses of ΔNp63 and CTEN expression were performed 48 hr after transfection. α-tubulin in western analysis was used as a loading control. (C) Schematic representation of the CTEN promoter region in mouse and human. Three regions of high homology were identified and are shown as R1 (white boxes), R2 (black boxes) and R3 (grey boxes). (D) The serial deletions at the 5’-end of the CTEN promoter are schematically shown on the left. Different lengths of the CTEN promoter were subcloned into the pGL3-Basic reporter vector (designated pGL), and the numbers represent the position relative to the transcription initiation site. Non-coding exon 1 is shown in ellipse. Each chimeric reporter construct was cotransfected with a ΔNp63α expressing plasmid (■) or with an empty vector (□) into HEK293 cells. In addition, the pRL-TK vector, which contains the Renilla luciferase reporter gene, was also cotransfected as an internal control. Dual luciferase assays were performed 48 hr after transfection and the individual relative light units (RLU) from the firefly luciferase (CTEN promoter activity) were normalized to that from the Renilla luciferase. The final value is expressed as the mean relative fold change compared to RLU from pGL3-Basic for each fragment. Data are expressed as the mean±standard deviation of four different experiments analyzed in triplicate. Numbers next to the error bars indicate the fold induction of activity in ΔNp63α-expressed cells compared to that in the empty vector-transfected cells. (*: P <0.05, **: P <0.001)