Fig. 1

Knockdown of DLK in Neuro-2a cells. Neuro-2a cells were infected with an empty lentiviral vector (pLKO.1) or with lentivirus expressing mouse DLK shRNAs (shDLK#1 and shDLK#2), followed by selection with puromycin to establish stable cell lines. (A) Representative Western blots showing the levels of DLK, phospho-JNK, total JNK, phospho-c-Jun (Ser-63 or Ser-73), c-Jun and actin in control and DLK-depleted Neuro-2a cells incubated without (-) or with (+) 20 µM retinoic acid (RA) for 24 h. Bands of interest were excised from different blots and grouped together. Full-length blots are presented in Supplementary File 1. (B) Quantification of DLK, phospho-JNK, total JNK, phospho-c-jun (Ser-63 or Ser-73) and c-Jun protein levels in control and DLK-depleted cells. All chemiluminescent signals were quantified using a Bio-Rad ChemiDoc imaging system and normalized to the actin level in control cells exposed to RA. The data are presented as the means ± SEMs (error bars) from two or three independent experiments. Each replicate has its own actin control. Statistical significance was determined using a two-tailed unpaired Student’s t test. For simplicity, only the statistical analysis of the shDLK#2-depleted Neuro-2a cells treated with RA is shown. *, p < 0,05; **, p < 0,01, relative to control cells exposed to RA. ns, not significant. (C) Neurite length (mm) per cell-body cluster was measured every 4 h in control and DLK-depleted cells that were exposed to RA for 76 h. Data on graph represent the individual values obtained from five independent experiments at each time point with means connected. Multiple unpaired t tests were used for statistical analysis. **, p < 0,01; ***, p < 0,001; ****, p < 0,0001, relative to control cells. (D) Representative phase contrast images of control (pLKO.1) and shDLK#1- or shDLK#2-depleted Neuro-2a cells cultured with RA for 48 h. Scale bar, 400 μm