Ninein depletion increases the number of MT minus ends present in the soma and leading process of migratory neurons. (A) A cultured cerebellar migratory neuron, transfected to express GFP-CAMSAP3 for 2 h. Neurons were cotransfected with RFP-pericentrin to label the centrosome (inset, blue asterisk). (B) Bar graph of ninein intensity under control siRNA and ninein siRNA conditions. n = 30 per group; ***, P < 0.001. Black lines represent median values. (C) 3D z-stack projection of neurons transfected with control siRNA (top) or ninein siRNA (bottom) and coimmunostained for CAMSAP2 (red) and ninein (green). More CAMSAP2 speckles appear in the soma and leading process of ninein-depleted neurons compared with controls. Speckles vary in apparent size because of CAMSAP dynamicity (Jiang et al., 2014). (D) Scatterplot of CAMSAP2 speckles versus ninein intensity. Speckle number in the soma (orange squares), leading process (gray triangles), and the total number of speckles (blue diamonds) were all found to have significant negative correlations with ninein intensity (P < 0.01, 0.028, and 0.01, respectively; n = 20). (E) Merged phase and green channel images of migratory neurons under control siRNA + GFP, ninein siRNA + GFP, and ninein siRNA + GFP-hNinein conditions. Arrows identify the leading process of each neuron. (F) Graph of leading process length quantification under control, ninein siRNA, and ninein siRNA + GFP conditions. Process length is significantly increased after ninein depletion. This change is rescued after ectopic expression of hNinein (n = 10 per group; *, P < 0.05). Black lines represent median values. See also Figs. S2 and S3 and Video 1. Data are represented as mean ± SEM. N.S., not significant.