Neurite outgrowth assay

Individual Tuj1-positive neurons used for Sholl analyses⁵⁸ were randomly selected and imaged using a Nikon Eclipse TE300 with a 40× objective. The neurites were traced using the ImageJ (NIH) plugin NeuronJ⁵⁸, and Sholl analysis was performed using the Sholl tool of Fiji⁵⁹, quantifying the number of intersections at 10-μm intervals from the cell body. The Sholl analysis was performed on a minimum of n= 50 neurons. Statistical analysis was performed by comparing the number of intersections of KO clones with the parental WT line for each 10-μm interval using Prism 7 (Graph Pad, La Jolla, CA, USA). Significance was assessed by a standard Student’s t-test, with a p value of p<0.05 considered as significant.