Primary cerebellar granule neurons were prepared from P6 Long-Evans rat pups as described (Konishi et al. 2002). One day after culture preparation, neurons were treated with cytosine arabinofuranoside (AraC) at a final concentration of 10 mM to prevent glial proliferation. For morphology assays, granule neurons were transfected 8 h after plating using amodified calciumphosphate method (Konishi et al. 2002, 2004; Gaudillie`re et al. 2004). High-efficiency transfection of granule neurons for biochemical analyses was achieved using a nucleofection method with the Amaxa electroporation device. To visualize endogenous MAP2 and Tau1, granule neurons were transfected with the Amaxa electroporation device and plated at a density of 100–200 cells per square millimeter. For time-lapse analyses, neurons were plated on etched coverslips (Bellco) and transfected 8 h later. To rule out the possibility that the effects of RNAi or protein expression on neuronal morphogenesis were due to any effects of these manipulations on cell survival, the anti-apoptotic protein Bcl-xl was coexpressed in our experiments. The expression of Bcl-xl had little or no effect on neuronal polarity, and on axonal or dendrite development (Supplemental Fig. 12) (Gaudillie`re et al. 2004; Konishi et al. 2004). In addition, FOXO knockdown impaired neuronal polarity in the presence or absence of Bcl-xl (Supplemental Fig. 12). Polarity gene expression analyses in neurons Granule neurons were transfected at high efficiency by nucleofection with the control U6 or U6/foxo RNAi plasmid. Two days later, RNA was extracted using Trizol according to the manufacturer's instructions, and cDNA was prepared using oligodT primers and SuperScript III reverse transcriptase (Invitrogen) according to manufacturer's protocol. Quantitative PCR was performed with the LightCycler 480 SYBR Green 1Master Kit on a LightCycler 480 thermocycler (Roche). For all quantitative PCR experiments, gene expression was normalized to GAPDH levels. Specific amplification of target genes was confirmed by agarose gel electrophoresis and calculation of melting temperature of the amplified product. Primer sequences for RT–PCR analyses and details of the method are available on request.