Fig. 6. Neurite outgrowth of PC12 cells mediated by ALK-FH-derived protein is accompanied by arrest of DNA synthesis. Cells were electroporated with normalized quantities of the indicated constructs, continually cultured in a normal serum medium (10% horse serum, 5% fetal calf serum) in order to study their normal asynchronous growth and treated with the dimerizer (20 nM) for 12 hours in the presence or not of U0126 (25 µM). At 6 hours after dimerizer application, cells were incubated with BrdU (10 µM) for 6 hours of incorporation. Then immunofluorescence assay was performed using the anti-HA-tag (3F10) and the FITC-conjugated anti-BrdU antibodies; 100 transfected cells were counted and cells with positive BrdU staining were scored as having undergone DNA replication during the time of labeling. The experiment was performed in triplicate and values are expressed as the mean ± s.e.m. (%). Statistical analyses were carried out by Student's t-test (*P<0.05; **P<0.01).