Figure 8

Forskolin does not alter expression of DYRK1A. A) Potential cAMP responsive element (CRE) upstream of TSS A. The sequence shown is fully conserved in the mouse, dog, and platypus genomes and comprises a full CRE sequence (with one mismatch to the canonical octamer) and a CRE half-site (boxed). B) Forskolin stimulates phosphorylation of CREB on Ser133. PC12 cells were treated for 30 min with forskolin (10 μM) or DMSO (Ctrl) before nuclear extracts were prepared. Phospho-Ser133 in CREB was detected by Western blot analysis with the help of a phosphospecific antibody. Migration of molecular mass standards is indicated in kDa. C) Northern blot. PC12 cells were treated for 24 h with forskolin (10 μM) or DMSO as the vehicle (Ctrl) before total RNA was isolated and subjected to Northern blot analysis with probes for DYRK1A, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and c-fos. Duplicate lanes contain RNA samples from parallel plates. Migration of the ribosomal RNAs is indicated in the right margin (28S, 18S). D) Luciferase assays. The indicated cell lines (PC12, PC3, Saos2) were transfected with the reporter gene constructs as schematically depicted. Luciferase activity was determined from cells treated with forskolin (10 μM) for 24 h and from untreated cells. Data were normalized to β-galactosidase activity and are presented as fold induction relative to untreated cells. Bars reflect the means +/- SD of 3 independent experiments (Saos2, PC3) or the means of duplicate wells for the experiment with PC12 cells.