Fig. 5. Activation of the MAP kinase pathway, but not the PI 3-kinase pathway, is required to induce neurite extension of PC12 cells. (A) MEK inhibitor dose-dependent inhibition of neurite outgrowth induced by the activation of the full-length ALK-FH protein. PC12 cells were electroporated with ALK-FH construct (25 µg), cultured overnight in a 1% horse serum medium and treated with the dimerizer (20 nM) for two days in the presence of increasing concentrations of the MEK inhibitor U0126 dissolved in DMSO. As a control, cells were treated without DMSO (No vehicle). DMSO control (0 µM) showed no adverse effects. (B,C) Inhibition of neurite outgrowth by the MEK inhibitor U0126 but not by the PI 3-kinase inhibitor wortmannin. PC12 cells were electroporated with normalized quantities of the indicated constructs, cultured as described above and treated with the dimerizer (20 nM) in the presence or not of U0126 (25 µM) (B) or wortmannin (50 nM) (C) for two days. All the data (A-C) were obtained following immunofluorescence detection with the anti-HA-tag antibody (12CA5), neurite outgrowth of transfected cells being scored as indicated in the Materials and Methods. The experiments were performed in triplicate and values are expressed as the mean ± s.e.m. (%).