5′-deletion study of the VE-statin/egfl7 promoter region.
A. H5V endothelial (black bars) and L929 fibroblast (right, white bars) were transfected with the pGL3basic luciferase reporter vector (Ctrl) or pGL3basic in which −12969/+38 VE-statin/egfl7 promoter region or 5′ deletions of it were inserted. These reporters (80 fmoles) were transfected together with 54 fmoles pCH110 normalization vector. After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Numbers associated with the black bars represent the calculated endothelial/fibroblast ratio of activity for the corresponding construct. Left: scaled schematic representation of the constructs, names are given according to the cloned 5′ and 3′ end positions relative to the exon-1b transcription start, Luc; luciferase. The experiment is representative of a set of at least three experiments performed in similar conditions. Constructions were designed so that the luciferase gene was placed within exon-1b because initial experiments showed that placing it where the coding sequence starts in exon-3 resulted in no detectable activity (not shown). ** p<0.01, * p<0.05. B. H5V endothelial cells (black bars) or 3T3 fibroblasts (white bars) were transfected with 80 fmoles of pGL3promoter (Ctrl, Promega), −8409/−7563SV40Luc, −7563/−8409-SV40Luc, or −7770/−7563-SV40Luc and with 54 fmoles of pCH110 normalization vector. After 48h of culture, cells were lyzed and the luciferase value of each sample was measured and normalized with its β-galactosidase value. Bars represent normalized activity as fold over Ctrl mean value set to 1. Results are displayed as in Figure 3A. The −8409/−7563 fragment is active in endothelial cells regardless of orientation, the most active sequence in this fragment corresponds to region A (−7770/−7563) which shows an endothelial/fibroblast ratio similar to the reporter containing the whole −8409/−7688 region.
