Dependence of Proximal GC Boxes and Binding Transcription Factors in the Regulation of Basal and Valproic Acid-Induced Expression of t-PA

Figure 3

Induction of t-PA reporter construct by VPA and its dependence on proximal GC boxes. HT-1080 (a) and HUVEC (b) cells were cotransfected with a firefly luciferase construct containing different variants of a 318 bp insert of the t-PA proximal promoter and a SV40 driven pGL4.73 renilla luciferase control vector. Firefly luciferase signals were normalized to renilla luciferase and the figures show relative data and mean values of three (HT-1080) and four (HUVEC) independent experiments. The variants of the t-PA insert were unmutated (intact), CRE mutated, GC box II mutated, and GC box III mutated. After overnight incubation, cells were stimulated with 3 mM VPA for 24 h. Data are presented as relative inductions of respective construct upon stimulation with VPA, where the dotted line in each figure represents untreated controls (=1). Statistical evaluation was performed using paired Student’s -test and comparisons are relative to respective untreated control. The marks indicate the statistical comparison of the VPA-induction in the t-PA construct with intact insert versus the inductions of respective mutated insert. ; and NS = nonsignificant.

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(b)