Figure 2A. Representative stills from time-lapse recordings of DIV6 DRG neurons overexpressing DLK-GFP. Arrowheads point to individual moving DLK-GFP positive structures. B. Kymographs made from the complete time-lapse recording shown in A. The left shows the original kymograph, and the right shows an illustration of the manually traced structure displacements for better visualization. Data information: Scale bar represents 10 μm in (B) 5 μm in (A)C. Representative images of DIV3 DRG neurons nucleofected with a non-targeting siRNA (NT siRNA) or a siRNA against DLK (DLK siRNA), and stained for endogenous DLK and β3-tubulin (TUBB3). On the right, quantification of the DLK mean intensity in neurons expressing control NT or DLK siRNAs (n = 25 neurons/condition; Unpaired t test). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 10 μm in (C) 2 μm in zooms in (C).D-F. Representative kymographs (D), quantification of GFP-positive particle number (E) and percentage of anterograde, retrograde, reverse or non-moving GFP-positive particles (F) from DLK-GFP time-lapse recordings in DIV6 DRG neurons, in control situation (+ NGF) or after 3h of NGF deprivation (-NGF) (n = 45-46 neurons/ condition; Mann-Whitney U test in E and Chi-square test in F). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents , 5 μm in (D)G. Representative images of DIV3 cultured embryonic DRG neurons maintained in the presence (+NGF) or absence (-NGF) of NGF for 3h and stained for endogenous DLK. On the right, quantification of the number of DLK- positive puncta in control situation or after 3h of NGF deprivation (n = 18-19 neurons/condition; Mann-Whitney U test). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 5 μm in (G)H. Representative kymograph and stills from a time lapse recording of 6DIV neurons overexpressing a GFP-tagged N-terminal region of DLK (NtermDLK-GFP). Arrowheads point to individual moving NtermDLK-GFP positive structures. Data information: Scale bar represents 5 μm in (H)I-J. Representative Western blots of p-cJun and GAPDH (I), and quantification of relative c-Jun phosphorylation levels (J) from DIV6 cultured embryonic DRG neurons overexpressing GFP or NtermDLK-GFP, and subjected to NGF withdrawal for 3h (n = 7-8 biological replicates; series of unpaired t tests /Mann-Whitney U tests followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.L. Representative images of HeLa cells expressing the different HA-tagged wild-type (WT) and palmitoyl-mutant (CS) DLK constructs (DLK-WT-ER, DLK-CS-ER, DLK-WT-CAAX, DLK-CS-CAAX, DLK-WT-MTS, DLK-CS-MTS), and stained with markers of the endoplasmic reticulum (KDEL), plasma membrane (CellMask) and mitochondria (cytochrome C). Data information: Scale bar represents 5 μm in (L)M-N. Representative Western blots of DLK, p-cJun and GAPDH (M) and quantification of relative c-Jun phosphorylation levels (N) from HeLa cells transfected with HA alone (HA) and the different HA-tagged wild-type (WT) and palmitoyl-mutant (CS) DLK constructs (DLK-WT-ER, DLK-CS-ER, DLK-WT-CAAX, DLK-CS-CAAX, DLK-WT-MTS, DLK-CS-MTS) (n = 7-8 biological replicates; series of unpaired t tests /Mann-Whitney U tests with DLK-WT or DLK-CS followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.