Figure 6

Effect of Sp1 expression on ACVRL1 promoter activity. (A) Dose-response effect of Sp1 on the transcriptional activity of ACVRL1 promoter in Schneider S2 and HEK293T cells. S2 (Sp1-less) and HEK293T cells were cotransfected with the pGL2 empty vector or the ACVRL1 promoter construct -1,035/+210 and with increasing amounts of the Sp1 expression vector (pPac-Sp1 and pCIneo-Sp1, respectively). Luciferase activity was corrected with β-galactosidase activity and expressed as fold induction of the transcriptional activity of pALK1 in the absence of exogenous Sp1. (B) Left, scheme showing the distribution of the different Sp1 consensus binding sites along the ACVRL1 promoter (black ovals) in the different constructs. Right, transient transfection of Schneider S2 cells with 25 ng of pPac-Sp1 and the indicated ACVRL1 promoter constructs. Fold-induction values respect to basal activity are indicated on top of each bar. (C) Effect of Sp1-knock down on ACVRL1 transcriptional activity. HEK293T cells were transfected with Sp1 siRNA. Left, Sp1 mRNA and protein levels were measured by semiquantitative RT-PCR and western blot after 48 hr. Right, 24 hr after the siRNA Sp1 transfection, the different ACVRL1 promoter constructs were transfected. The transcriptional activity of all the fragments was measured and normalized by the β-galactosidase activity. Basal pALK1 activity (100%) and the reduction after Sp1 silencing (grey bars) are shown. In every case, Sp1 suppression resulted at least in a decrease of 50% in ACVRL1 transcriptional activity (***p < 0.005).