Knockdown of EFA6A and ARF6 by siRNA results in a decrease in spine formation. A, Cultured neurons were transfected at DIV 13 singly with pSUPER.gfp/neo EFA6A (si-EFA6A), pSUPER.gfp/neo ARF6 (si-ARF6), or pSUPER.gfp/neo alone (si-vec), or doubly with pSUPER constructs plus rescue FLAG-EFA6A or ARF6-HA expression constructs (Rescue), were immunostained at DIV 16 with the antibodies indicated. Scale bar, 5 μm. B–D, Quantitation of the effects of siRNA knockdown of EFA6A and ARF6 on the linear densities of spines, filopodia, and total protrusions. si-vec (protrusion density, 45.51 ± 1.347; spine density, 40.49 ± 1.36; filopodia density, 5.03 ± 0.44; length, 1.91 ± 0.04; width, 1.21 ± 0.02); si-EFA6A (protrusion density, 37.73 ± 1.24; spine density, 25.27 ± 1.03; filopodia density, 12.45 ± 0.84; length, 2.66 ± 0.07; width, 1.07 ± 0.02); si-EFA6A + Rescue (protrusion density, 45.68 ± 1.46; spine density, 42.19 ± 1.49; filopodia density, 3.48 ± 0.48; length, 1.70 ± 0.04; width, 1.19 ± 0.02); si-ARF6 (protrusion density, 38.92 ± 1.16; spine density, 21.92 ± 0.94; filopodia density, 17.0 ± 0.69; length, 3.13 ± 0.06; width, 0.99 ± 0.01); si-ARF6 + Rescue (protrusion density, 46.06 ± 1.83; spine density, 39.84 ± 1.74; filopodia density, 6.23 ± 0.67; length, 2.03 ± 0.04; width, 1.21 ± 0.02). E–H, Quantitation of the effects of siRNA knockdown of EFA6A and ARF6 on the lengths and widths of protrusions. I, Cultured neurons transfected with si-EFA6A, si-ARF6, or si-vec were doubly labeled for EGFP and PSD-95. Scale bar, 5 μm. J, Quantitation of the effects of siRNA knockdown of EFA6A and ARF6 on the localization of PSD-95 in spines. si-vec (47.48 ± 1.99); si-EFA6A (28.42 ± 2.80); si-ARF6 (30.98 ± 3.46). Data were collected from 1200–1800 protrusions on 30–39 dendrites of 22–37 neurons for each group in the three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA).