Impairing the actin flow–L1 linkage by RNAi inhibits L1-dependent axon outgrowth. Hippocampal neurons were transfected with shRNA against shootin1 or with control shRNA, and cultured on L1-Fc– or polylysine-coated coverslips for 48 h. Some were also cotransfected with shRNA against L1. Neuronal morphology was visualized by EGFP fluorescence. (A) Vectors expressing shRNAs coexpress EGFP. (B) Length of axons or the longest neurites. (C) The percentage reduction in axons by shootin1 RNAi (mean ± SEM; *, P < 0.05, ***, P < 0.01; n = 3 independent cultures; 512 neurons were examined). Bar, 50 μm.