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Fig. 7. Activation of the cytosolic form of the ALK intracellular domain induces DNA synthesis through the PI 3-kinase/AKT pathway. (A) Induced activation of the cytoIA-FH but not of the myrIA-FH protein leads to DNA synthesis in PC12 cells. Cells were electroporated with normalized quantities of the indicated constructs and were submitted to the same protocol as in Fig. 6 except that they were cultured overnight in a 1% horse serum medium before dimerizer treatment. ***P<0.005 (Student's t-test). (B) PI 3-kinase inhibitor inhibition of DNA synthesis induced by the activation of the cytoIA-FH protein. PC12 cells transiently expressing the cytoIA-FH protein were submitted to the same protocol as in A. They were treated with the dimerizer (20 nM) for 12 hours in the presence of the indicated increasing concentrations of PI 3-kinase inhibitors wortmannin or LY294002 dissolved in DMSO. As a control, cells were treated without DMSO (No vehicle). DMSO controls (0 nM or µM) showed no adverse effects. The experiments were performed in triplicate and values are expressed as the mean ± s.e.m. (%). (C) Induced DNA synthesis of PC12 cells expressing the cytoIA-FH protein is dependent on PI 3-kinase but not on ERK 1/2. Cells transiently expressing the cytoIA-FH protein were submitted to the same protocol as in A. U0126 (25 µM) and/or wortmannin (50 nM) were added to the medium 60 and 30 minutes, respectively, before dimerizer treatment. **P<0.01; ***P<0.005 (Student's t-test). (D) Time course of AKT phosphorylation following dimerizer treatment. PC12 cells transfected with the indicated constructs were cultured overnight in 0% horse serum medium and then incubated with the dimerizer (20 nM) for the indicated periods. Cells were lysed in RIPA buffer as described in the Materials and Methods and the lysates (10 µg) were submitted to western blot analysis using the anti-AKT phosphoserine-473 antibody and then reprobed with the anti-AKT antibody. (E) MAP kinase pathway inhibition revealed a potential activation effect of myrIA-FH protein on DNA synthesis through the PI 3-kinase pathway. Cells transiently expressing the myrIA-FH protein were submitted to the same protocol as in C. **P<0.01 (Student's t-test).
