Fig. 3. Effects of perturbation of the Wnt signaling pathway on neurite extension by PC12 cells. (A) Morphology of PC12 cells infected with EGFP, caß-catenin-EGFP, caLEF-EGFP or dnLEF-EGFP retroviruses. PC12 cells were infected with retrovirus encoding EGFP, caß-catnin-EGFP, caLEF-EGFP or dnLEF-EGFP; the cells were then incubated with 50 ng/ml NGF for 5 days to cause differentiation, and subsequently fixed. Cells were immunostained with anti-ßIII-tubulin and anti-GFP antibodies and visualized with Alexa 546 and Alexa 488, respectively. DAPI was used for staining nuclei. Bar, 100 µm. (B-E) Neurite length distribution of the PC12 cells expressing EGFP, dnLEF-EGFP, caß-catenin-EGFP or caLEF-EGFP. Neurite lengths of more than 75 cells of EGFP-positive and -negative cells were measured. Each experiment was conducted in triplicate. Error bars represent s.e.m. of three independent results. (F) Normalized neurite length of PC12 cells expressing EGFP, caß-catenin-EGFP, caLEF-EGFP or dnLEF-EGFP. The average neurite length of EGFP-positive transfected cells (n>75) were normalized by the average neurite length of EGFP-negative non-transfected cells (n>75). Each experiment was conducted in triplicate. Error bars represent s.e.m. ^*P<0.05; ^***P<0.005 by the Student's t-test. (G) Effect of the ß-catenin-Lef-1 signaling pathway on MAPK-dependent transcription activity. PC12 cells were co-transfected with the c-fos promoter-luciferase plasmid (0.1 µg) together with caMEK or caLEF plasmids (0.1 µg), as described in Materials and Methods. After 10 hours of culture, cells were harvested and the luciferase activities were measured.