FIGURE 3.

Gpr158 knock down leads to reduced neuronal outgrowth of hippocampal primary neurons. (A) Experimental set-up of in vitro Gpr158 knockdown (KD). Viral transduction with independent shRNAs occurred from DIV7 to DIV14, and morphological analysis occurred at DIV14. (B) Representative images for control (scRNA) and Gpr158 KD (shRNA1–5) neurons (red: MAP2, blue: nuclei). (C–E) Morphological analysis after Gpr158 KD at DIV14 (n = 7–8 wells; see Online Resource 6) shows an overall effect of the shRNA treatment for all parameters [neurite length (C), number of bifurcations (D) and number of extremities (E)], as tested by Kruskal–Wallis (P < 0.001). Whereas shRNA3 affected all 4 parameters, the other shRNAs had a more limited effect for 1–3 of these parameters. Apart from a significant downregulation in number of protrusions, shRNA5 gave a similar strong effect as shRNA3, indicative of a Gpr158-related effect. Data are presented as mean ± SEM with individual data points indicated. Asterisks indicate significant differences compared with control (scRNA) assessed by Student’s t-test (Supplementary Table 3), ^∗P ≤ 0.050; ^∗∗∗P ≤ 0.001.