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CEP76 deficiency in the family 7 proband fibroblasts impairs both primary cilium formation and structural integrity. (A) Representative fluorescence images, costained with an axoneme marker (ARL13B) and centrosome/basal body marker (γ-tubulin) (red), CEP76 (green), and nuclei (Hoechst; blue). (B) Quantification of CEP76 intensity (a.u.) at centrosomes/basal bodies of control (n = 130 nuclei) and proband (n = 90 nuclei). (C) Representative fluorescence images, costained with an axoneme marker (ARL13B; green), centrosome/basal body marker (γ-tubulin; red), and nuclei (Hoechst; blue). (D) Percentage ciliation in control (n = 612 nuclei, 10 tiles) and proband (n = 965 nuclei, 8 tiles) fibroblasts, with each tile measuring 300.9 by 301.29 μm. (E to G) Quantification of cilium structure using CiliaQ. Cilium length (E), volume (F), and shape complexity (G). Cilia with <0.07-μm size were excluded to minimize segmentation artifacts. Each data point represents one primary cilium with data combined from three independent technical replicates (control, n = 487; proband, n = 426). (B and D to G) Box plots: center line, median; hinges, first/third quartiles; whiskers, 1.5× interquartile range; outliers shown individually in violin plots. In (A) and (C), subpanels are shown at the same magnification; insets at the top right of each image display zoomed views of representative cilia. Statistical comparisons: (B and D) Student’s t test; (E to G) nonparametric Mann-Whitney test and (E) ANOVA, where indicated. Each assay was conducted in three technical replicates with consistent results. Significance level: ****P < 0.0001.
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