Fig. 8

NEK1-LOF abnormality in the ALS fibroblasts rescued by pharmacological inhibition of HDAC6. A Western blot analysis of the lysates from DMSO or tubastatin A (Tub-A)-treated control and NEK1-LOF (p.E853Rfs*9 and p.M1?) patient fibroblasts using anti-acetylated α-tubulin (Ac-tub) and anti-α-tubulin (tub) antibodies. α-tubulin (tub) was used as a loading control. B Quantification of the normalized acetylated α-tubulin (Ac-tub) protein expression. Ac-tub intensities were normalized to that of the α-tubulin (tub). Data represent mean ± SEM (from three independent experiments). Comparisons were made against the DMSO-treated control (**P < 0.01, ***P < 0.001, ****P < 0.0001; one-way ANOVA with post-hoc Tukey’s test). C Representative fluorescence images of the tubulin acetylation and mitochondrial distribution in control and NEK1-LOF (p.E853Rfs*9 and p.M1?) patient fibroblasts by Tub-A treatment. Cells were treated with DMSO or 1 μM Tub-A for 24 h and then stained with anti-acetylated α-tubulin antibody (red), TOM20 (green, mitochondrial marker), and DAPI (blue). Scale bar: 10 µm. D Quantification of the mitochondrial length in C. The >50 cells per condition were quantified per replicate experiment (n = 3). Data represent mean ± SEM. Comparisons were made against the DMSO-treated control (****P < 0.0001; one-way ANOVA with post-hoc Tukey’s test). E Representative fluorescence images of the primary cilia formation in the control and NEK1-LOF (p.E853Rfs*9 and p.M1?) patient-derived fibroblasts pretreated with DMSO or 1 μM Tub-A. Cells were stained with acetylated α-tubulin (green, ciliary axoneme marker) and ARL13B (green, ciliary membrane marker) after serum starvation for 48 h. The right panels show the higher-magnification views of the primary ciliary regions. Scale bar: 10 µm. F Quantification of the ciliary length in E. The >100 cells per condition were quantified per replicate experiment (n = 3). Data represent mean ± SEM. Comparisons were made against the DMSO-treated control (****P < 0.0001; one-way ANOVA with post-hoc Tukey’s test)