Fig 2: Cells from ZS patients show defects in cholesterol enrichment in the ciliary membrane and Shh signal transduction APrimary skin fibroblasts from a normal individual, SLOS patient, ZS patients, X-ALD patient, and NPC patient incubated for 24 h without serum were immunostained with anti-pericentrin (red) and anti-acetylated-tubulin (blue) antibodies. Cholesterol was stained with Filipin III (green). Arrows indicate primary cilia. Scale bar, 5 µm.BThe intensity of Filipin III signal at primary cilia from (a) was remarkably reduced in SLOS and ZS patient cells (**P < 0.01: one-way ANOVA with Tukey's HSD, n = 3: 45–50 cells per experiment). In the boxplot, medians, 25th/75th percentile, and min/max were represented by the central lines, the box limits, and the whiskers/error bars, respectively.CImmunofluorescent staining of primary skin fibroblasts in quiescent G0 phase treated with 50 nM Shh-N for 24 h using anti-Smo (green), anti-acetylated-tubulin (blue), and anti-?-tubulin (red) antibodies. Arrows indicate primary cilia. Scale bar, 2.5 µm.DQuantification of proportion of Smo-positive ciliated cells upon Shh-N stimulation from (C). The populations of Smo-positive ciliated fibroblasts from the SLOS and ZS patients were significantly reduced (mean ± s.d.: ***P < 0.001: one-way ANOVA with Tukey's HSD, n = 3: 100 cells per experiment).EThe Smo intensity at primary cilia in the fibroblasts from (C). The ciliary localization of Smo upon Shh-N stimulation was significantly impaired in fibroblasts from the SLOS and ZS patients (***P < 0.001: one-way ANOVA with Tukey's HSD, n = 3: 90–100 cells per experiment). In the boxplot, medians, 25th/75th percentile, and min/max were represented by the central lines, the box limits, and the whiskers/error bars, respectively.FPrimary fibroblasts from a normal individual, and SLOS and ZS patients were treated with 1.5% methyl-ß-cyclodextrin for 45 min to remove cellular cholesterol and then incubated with or without cholesterol (cholesterol/methyl-ß-cyclodextrin complex) for 1 h. After washing out the exogenous cholesterol, the fibroblasts were stimulated with 50 nM Shh-N for 24 h in the presence of pravastatin and then immunostained with anti-Smo (green), anti-acetylated-tubulin (blue), and anti-pericentrin (red) antibodies. For the alternative cholesterol complementation, LDL (0.06 mg/ml) was co-incubated with Shh-N and pravastatin for 24 h after methyl-ß-cyclodextrin-mediated cholesterol depletion. Scale bar, 2.5 µm.GQuantification of the Smo intensity at primary cilia in the fibroblasts from (F). The ciliary accumulation of Smo upon the Shh-N stimulation of fibroblasts from an SLO patient was restored by the complementation of both exogenous cholesterol/methyl-ß-cyclodextrin complex and LDL, while that of cells from ZS patients was efficiently rescued by not LDL but cholesterol/methyl-ß-cyclodextrin complex (*P < 0.05, **P < 0.01, ***P < 0.001: one-way ANOVA with Tukey's HSD, n = 3: 90–100 cells per experiment). In the boxplot, medians, 25th/75th percentile, and min/max were represented by the central lines, the box limits, and the whiskers/error bars, respectively.