Fig. 8

C21ORF2-V58L affects NEK1 expression at the post-transcriptional level. A–L Analysis of NEK1 distribution and expression in iPSC-derived motor neuron (MN) cultures at days in vitro 25 (DIV25, 7 days after plating). C1 and C2, healthy controls; P1, ALS patient with C21ORF2-V58L mutation; P2, ALS patient with C21ORF2-V58L/R60W mutations; iso, isogenic control 1 generated from P1. A Co-immunostaining for NEK1 and CHAT/ISL1 to label MNs. B, C Quantification of NEK1 expression in soma and nucleus compartments based on immunostaining as in A. D Ratio of NEK1 immunostaining in nucleus compared to soma. E RT/q-PCR analysis of NEK1 gene expression. For relative mRNA expression the ΔCt values (ΔCt = Ct(NEK1) – Ct (ACTINB)) for each line are graphed. F, G Western blot analysis using anti-NEK1 and anti- β -actin antibodies. β-actin was used as loading control. F Representative Western blot. G Quantification of NEK1/actin intensity ratio in Western blot experiments as in F. H–L Immunostaining (H, I), RT-q-PCR (J), Western blot analysis (K, L) and corresponding quantifications using MNs derived from the C1, P1 and iso1 lines and performed as described for panels A–G. Results of three independent experiments are plotted as means with SEM **p < 0.01; ***p < 0.001; ****p < 0.0001, (G, L) One-Way ANOVA, Tukey multiple comparison post-hoc test. (B, C, I) Kruskal–Wallis test with Dunn’s multiple comparison post-hoc test. Scale bars: A, 5 µm; I, 20 µm