(A-F) Primary cultured neurons were transfected with FAM-labelled negative control siRNA (siCTL) or FAM-labelled NELL2 siRNA (siNELL2). The cells were fixed at 2 days after transfection and stained with anti-Tau1 antibody, and their axon development was analyzed: representative microphotographs (A), NELL2 mRNA level determined by real-time PCR analysis (B), axon length (C), average neurite length (D), average neurite number (E), and progression of development stages (F). n = 46 (siCTL) and 48 (siNELL2) cells. (G) Representative microphotographs showing neurons transfected with FAM-labelled negative control siRNA (siCTL) or FAM-labelled NELL2 siRNA (siNELL2). Scale bar = 20 µm (A and G). (H and I) The cultured neurons transfected with siRNA were analyzed to determine the average number of axon branches (H) and length of average axon branches at different branch order (I). All data are presented as mean ± SEM. n = 75 (siCTL) and 82 (siNELL2) cells. *P < 0.05; **P < 0.01; ***P < 0.001. AU, arbitrary units. P values for unpaired comparisons were analyzed by two-tailed Student’s t-test. Two-way repeated-measures ANOVA was performed to detect significant interaction between groups.