Effect of Vangl2 knockdown on neuronal morphology. (A, B) Knockdown (KD) effect of shRNA-Vangl2 on transiently expressed HA-Vangl2 (A) or HA-Vangl1 (B) using the calcium phosphate method in COS-7 cells. The lysate of the cells (20 μl of the lysate each) was analyzed by western blotting using anti-HA and -tubulin antibodies. (C) The shRNA-control and -Vangl2 vectors were transfected by nucleofection into neurons at 0 DIV. Endogenous Vangl2 at 8 DIV neurons (10 μg of protein each) was analyzed by western blotting using anti-Vangl2 and -actin antibodies. (D) The intensities of protein bands for Vangl2 and actin (n =3 for each band) were analyzed. *p <0.05, Students t-test. (E, F) Cultured hippocampal neurons transfected with control or Vangl2 KD vector were visualized by immunohistochemistry using the anti-GFP antibody. (G) The spine density measured at 21 DIV was significantly decreased in Vangl2 KD neurons. Data are means ± SEM (n =10). ***p <0.001, Students t-test. (H, I) The dendritic complexity of neurons transfected with the indicated vectors was measured by Sholl analysis, which shows the number of dendrites crossing circles (vertical axis) at various radial distances from the cell soma (horizontal axis). Intersections at various radial distances (10100Δm) and total intersections were significantly suppressed by the Vangl2 KD vector. Data are means ± SEM (n =20). ***p <0.001, Students t-test.