Figure 1: The UPS and NGF maintain the local protein synthesis dependence of Sema3A-induced growth cone collapse.

(a) Growth cones can undergo sequential rounds of Sema3A-induced growth cone collapse. Representative phase images of the same growth cones at t=0 min (before any treatment), t=60 min (after 1 h of Sema3A or vehicle exposure), t=120 min (after washout) and t=180 min (after 1 h of the second Sema3A or vehicle treatment). Scale bar represents 5 μm. The insets show magnified images of the growth cones. (b) Quantification of results in a. The red line represents growth cones exposed to 450 ng ml⁻¹ Sema3A, whereas the black line represents vehicle-treated growth cones. In the last hour of the experiment (right side), the dashed red line indicates growth cones that were treated with vehicle in the first hour and Sema3A in the final hour, whereas the solid red line represents growth cones that had been exposed to Sema3A in the first hour and were again exposed to Sema3A in the final hour of the experiment. Growth cones treated with vehicle during the final hour are indicated with a black line (dashed for vehicle-treated during the first hour, solid for Sema3A-treated during the first hour). n≥15 growth cones per time point from at least three independent experiments. (c) The second Sema3A-induced growth cone collapse is dependent on local protein synthesis. Treatment of growth cones with the protein synthesis inhibitors cycloheximide (CHX, 10 μM) or anisomycin (Aniso, 40 μM) abolished the second Sema3A-induced growth cone collapse. **P<0.01, one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test, n≥38 growth cones per condition from at least three independent experiments. (d) The requirement of local protein synthesis in Sema3A-induced growth cone collapse depends on UPS activity and NGF levels during washout. Addition of proteasome inhibitors (50 μM LLnL or 5 μM MG132) during the washout phase or washout in media containing minimal NGF concentrations (1 ng ml⁻¹) rendered the second collapse protein synthesis independent. *P<0.05, one-way ANOVA with Tukey’s multiple comparison test, n≥34 growth cones per condition from at least three independent experiments. NS, not significant.