Fig. 4

ALS-linked NEK1 variants impair intracellular Ca²⁺ homeostasis and regulate cilia in a Ca²⁺-dependent manner. A Representative images of the cytosolic Ca²⁺ in the control and patient fibroblasts stimulated with serum starvation for 48 h. Fluo3-AM, a calcium indicator with green fluorescence, was visualized using confocal microscopy. Scale bar: 10 µm. B Fluorescence intensities of Fluo 3-AM images (A) of cytosolic Ca²⁺ were quantified using ImageJ with low-power field images. Data represent mean ± SEM (from three independent experiments). Comparisons were made against the control (**P < 0.01, ***P < 0.001; one-way ANOVA with post-hoc Tukey’s test). C Quantitative analysis of the intracellular Ca²⁺ concentration in the control and patient fibroblasts stimulated with serum starvation for 48 h. Data represent mean ± SEM (from three independent experiments). Comparisons were made against the control (*P < 0.05, **P < 0.01; one-way ANOVA with post-hoc Tukey’s test). D Representative fluorescence images of the primary cilia in the starved control and patient fibroblasts treated with DMSO or 10 µM of the Ca²⁺ chelator BAPTA for 60 min. The cilia and basal bodies were visualized with antibodies against ARL13B (red) and γ-tubulin (green), respectively. The nuclei were stained with DAPI (blue). The right panels exhibit higher magnification views of the cilia and basal body. Scale bar: 10 µm. E-F Quantification of the ciliary frequency (E) and ciliary length (F) in D. The >100 cells per condition were quantified per replicate experiment (n = 3). Data represent mean ± SEM. Comparisons were made against the DMSO-treated fibroblasts (**P < 0.01, ****P < 0.0001; one-way ANOVA with post hoc Tukey’s tests). G Representative fluorescence images of activated AurA (p-AurA) in the starved control and patient fibroblasts treated with DMSO or 10 µM of the Ca²⁺ chelator BAPTA for 60 min. Cells were stained with p-AurA (phosphorylated T288) (red), acetylated α-tubulin (green, ciliary axoneme marker), and DAPI (blue). The lower panels demonstrate higher magnification views of the cilia. Scale bar: 10 µm. H Quantification of p-AurA (phosphorylated T288, red) intensity at the ciliary base described in G. The >100 cells per condition were quantified per replicate experiment (n = 3). Data represent mean ± SEM. Comparisons were made against the control (ns, not significant; one-way ANOVA with post-hoc Tukey’s test). I Relative mRNA levels of the cell cycle regulators (CDK4, Cyclin D1, and E2F-1) from G1 to S phase in control and patient fibroblasts pretreated with 10 µM BAPTA for 60 min and stimulated with serum starvation for 48 h. Data represent mean ± SEM (from three independent experiments). Comparisons were made against the control (ns, not significant; one-way ANOVA with post-hoc Tukey’s test)