FIGURE 4.

Down-regulation of Kidins220/ARMS in hippocampal neurons induces the formation of multiple axon-like processes. A, PC12 cells were transfected with control shRNA (GFP-shC) or Kidins220/ARMS-specific shRNA (GFP-sh1 and -sh2) vectors. After 96 h, cells were lysed, and Kidin220/ARMS knockdown was examined by immunoblot analysis. Neuronal specific enolase (NSE) levels were used as control. B, hippocampal neurons were transfected with GFP-shC (a) or GFP-sh2 (b) and immunostained for Kidins220/ARMS (red). GFP-sh2 neurons showed decreased Kidins220/ARMS labeling. Merged images are also shown (a and b). Scale bar, 20 μm. C, hippocampal neurons transfected with GFP-shC (a) or GFP-sh2 (b) and fixed at stage 3 were immunostained with the axonal marker SMI-31 (red). Only merged images are shown. Early down-regulation of Kidins220/ARMS increased the number of SMI-31-positive processes per neuron. Scale bar, 20 μm. D, quantification of the percentage of hippocampal neurons bearing several axon-like processes after knocking down Kidins220/ARMS (sh2) compared with control (shC). The data shown are the means ± S.E. of three independent experiments (shC n = 148 cells, sh2 n = 128 cells), and statistical significance was evaluated by Student's unpaired t test (**, p < 0.01). E, frequency histogram of the percentage of hippocampal neurons with distinct axonal lengths in control or in Kidins220/ARMS knockdown cells (shC n = 100 cells, sh2 n = 100 cells).