Figure 4. NGF-induced changes in axonal trafficking require local synthesis of Lis1 or p150^Glued.

DRG neurons were cultured in microfluidic chambers. On DIV 3, the NGF concentration in the axonal chamber was changed to 5 ng ml⁻¹, and axons were selectively transfected with a non-targeting control siRNA or siRNAs targeting Pafah1b1 or Dctn1. (a–c) After 24 h, fresh medium was added to the axonal chamber containing 5 ng ml⁻¹ NGF, no NGF or 100 ng ml⁻¹ NGF together with LysoTracker Green for 15 min. Live-imaging time-lapse series of axonal fields were acquired, with images being taken every 13 s for 4 min. Kymographs of representative 100-μm-long axonal segments are shown. Scale bar, 10 μm. LysoTracker-positive particles with diameters ≥1 μm were scored as anterograde, retrograde, bidirectional or stationary. Means±s.e.m. of 12–18 optical fields per conditions (n=3–6 biological replicates). **P≥0.01; ***P≥0.001. One-way ANOVA with Bonferroni's multiple comparisons test. (d) On DIV 4, axons were treated with 100 ng ml⁻¹ QD-NGF for 15 min and live imaged as above. QD-labelled particles <1-μm diameter were scored as anterograde, retrograde, bidirectional or stationary. Means±s.e.m. of nine optical fields per conditions (n=3 biological replicates). **P≥0.01; ***P≥0.001. Kruskal–Wallis test with Dunn's multiple comparison test. NS, not significant.