Fig. 2

ALS-linked NEK1 variants perturb primary ciliogenesis and Shh signaling in patient fibroblasts. A Subcellular distribution of GFP-tagged NEK1 WT in transfected NSC-34 cells under basal culture conditions (left panel) or serum starvation for 48 h (right panel). Fluorescence images of the primary cilia in the transfected NSC-34 cells. Cells were stained with ACIII (red, neuronal cilia marker). Nuclei were stained with DAPI. Scale bar: 10 µm. B Representative fluorescence images of endogenous NEK1 (green) and acetylated α-tubulin (red, ciliary axoneme marker) in control fibroblasts under basal culture conditions. Nuclei were stained with DAPI. Scale bar: 10 µm. C Representative fluorescence images of endogenous NEK1 (green) and acetylated α-tubulin (red, ciliary axoneme marker) for primary cilia formation in control and patient fibroblasts stimulated with serum starvation for 48 h. The bottom panels indicate higher magnification views of the primary ciliary regions. Nuclei were stained with DAPI. Scale bar: 10 µm. D-E Quantification of the ciliary frequency (D) and the ciliary length (E) in C. The >100 cells per condition were quantified per replicate experiment (n = 3). Data represent mean ± SEM. Comparisons were made against the control (**P < 0.01, ****P < 0.0001; one-way ANOVA with post-hoc Tukey’s tests). F Representative fluorescence images of Smo (green) and ARL13B (red, cilia marker) in the control and patient fibroblasts stimulated with serum starvation for 48 h. We examined the Smo translocation to the cilium in response to the Shh ligand-mediated signaling in the fibroblasts treated with DMSO or 200 nM Smo agonist (SAG) for 24 h. The right panels illustrate higher magnification views of the primary ciliary regions. Nuclei were stained with DAPI. Scale bar: 10 µm. G Quantification of Smo⁺ cells frequency in F. The >100 cells per condition were quantified per replicate experiment (n = 3). Data represent mean ± SEM. Comparisons were made against the DMSO-treated fibroblast (*P < 0.05, ****P < 0.0001; one-way ANOVA with post-hoc Tukey’s tests). H Relative changes in the GLI1 mRNA levels in the control and patient fibroblasts treated with DMSO or 200 nM SAG for 24 h. Data represent mean ± SEM (from three independent experiments). Comparisons were made against the DMSO-treated fibroblasts (*P < 0.05, ****P < 0.0001; one-way ANOVA with post-hoc Tukey’s tests)