Figure 5

Sp1 and Sp3 interact with the proximal PDPN promoter in vitro and in vivo. A) Supershifts were performed by incubating nuclear proteins either with anti-Sp1 antibody or with anti-Sp3 antibody, both antibodies together, or non immune IgG before addition of probe. Anti-Sp1 antibody supershifted complex I (ss-Sp1), while anti-Sp3 antibody specifically blocked the formation of protein-DNA complex II. B) Mutation of Sp binding sites (mut) abolished the binding complexes I and II versus the wild-type probe (wt) with nuclear extracts from MG63 cells. The mutated oligonucleotides used in this study are listed in Table 1. C) Deletion mutants of the four Sp-elements in the PDPN promoter. The sequences above the Wild-Type construct exhibit the core sequences of the Sp binding sites. Above each mutation construct, the respective mutated nucleotides are drawn in bold italics. In construct Sp.4-1mut, all four Sp elements are mutated. D) The wild type reporter construct and five mutation constructs were transiently transfected into MG63 and Saos-2 cells. Firefly luciferase values were divided by those of the internal control Renilla luciferase to represent the absolute promoter activity. Stars indicate the statistical difference of the promoter activities between the unmutated and mutated transfection experiments. *, p < 0.05; **, p < 0.01.