Supplementary Figure 6: Functional CRE-luciferase assays in HeLa cells transiently expressing the six PDE3A mutants.

(a) HeLa cells were transfected with an empty vector (sc300-w/o; orange line), a full-length wild-type PDE3A expression construct (red line) and the six full-length PDE3A mutant expression plasmids (gray and black lines). HeLa cells were cotransfected with a cAMP-responsive element (CRE) regulating luciferase transcriptional activity under the influence of increasing forskolin concentrations to further elucidate the functional consequences of the PDE3A mutations (P < 0.002). A Renilla luciferase vector was used for standardization. The data describe the relative increase in luciferase activity normalized to the DMSO control of cells transfected with empty vector. The results are the means of three independent experiments (mean ± s.d.; n = 3; Wilcoxon-Mann-Whitney test, P < 0.002). The more hydrolyzed cAMP there was, the less luciferase expression was detected. In the presence of increasing forskolin concentrations enhancing cAMP levels, the PDE3A mutants showed a significant reduction in CRE-mediated luciferase activity as a result of the higher cAMP hydrolysis compared to wild-type PDE3A. (b) cGMP stimulation with increasing l-arginine concentrations showed that cGMP competitively inhibited cAMP hydrolysis with a significant difference between the mutants and wild-type PDE3A (P < 0.002). The more cGMP that was present, the less cAMP hydrolysis occurred and the greater the luciferase activity was.