Figure 1. Effect of forskolin on the proliferation and differentiation of isolated SCs.

Cultures of adult nerve-derived rat SCs were treated for 3 days with the indicated doses of forskolin (A-C), provided alone or together with neuregulin (10 nM). Cells were analyzed for the expression of SC differentiation markers by means of immunofluorescence microscopy (A, B, right panel) and western blot (B, D). Sibling cultures were also analyzed for the incorporation of [³H]-thymidine (C, left panel). The cell permeable analog of cAMP CPT-cAMP (250 µM) was used as a positive control for the induction of differentiation (A, O1 expression) and the dual activation of PKA and EPAC (B). PKA activity was assessed by the immuno-detection of phosphorylated PKA-specific substrates (A, P-PKAs) and the in vitro phosphorylation of the Kemptide substrate (B, P-Kemptide). EPAC activation was determined by pull-down assays of Rap1-GTP (B). PKA and EPAC activity assays used SCs stimulated with forskolin (2 µM) or CPT-cAMP (250 µM) for 30 min. The antibodies used are indicated in the figure. Nuclei were labeled with DAPI (blue). Results in all figures are representative of at least 3 experiments performed independently. In these and all subsequent graphs, bar heights are means of triplicate determinations; error bars represent S.D, and * represents statistical significance for p < 0.05. Scale bars correspond to 50 µm in all figures unless otherwise indicated.