Sonic hedgehog blocks synaptamide-mediated axon growth in cortical neurons. (A) Immediately after isolation, cortical neurons were electroporated with 0.5 µg pCMV6-Flag-Shh or equal amount of pCDNA3 plasmids per 1×107 cells. At 24 h after plating, neurons were treated with 100 nM synaptamide for 48 h. Calculation of average axonal length was based on all neurons, regardless of transfection. Results are plotted as percent change in average axon length from vehicle-treated, pCDNA-transfected sample. (B) DIV1 mouse cortical neurons were supplemented with synaptamide (100 nM) or Shh at 50 ng/ml or 500 ng/ml concentrations. Results are plotted as % change in average axon length from vehicle-treated sample. (C) Representative images of electroporated, SMI-312-stained neurons that were used for quantification. In A,B data represents the average of at least three independent experiments performed in triplicates, error bars are ±s.e.m. At least, 1368 (A) and 905 (B) cells were analyzed across all the replicates per point. Statistical significance was calculated using one-way ANOVA (A, P=8.71×10−7; B, P=7.43×10−8) followed by Tukey–Kramer post hoc tests.