Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome (2020)
Figure 2

Figure 2 CRISPR/Cas9-mediated disruption of the PEX genes phenocopies the dysfunction of cholesterol trafficking to ciliary membranes

(A) Western blot analysis showing the depletion of PEX1 and PEX14 and the normal processing of Shh protein in the PEX1−/− and PEX14−/− hTERT-RPE1 cell clones. GAPDH served as a loading control.

(B) Immunostaining with anti-PEX1 (red) or PEX14 (red), anti-PMP70 (green), anti-ninein (blue), and anti-acetylated tubulin (white) in wild-type, PEX1−/−, and PEX14−/− hTERT-RPE1 cells in quiescent G0 phase. Arrowheads indicate primary cilia. Scale bar, 10 μm.

(C) Quantification of the number of PMP70-positive peroxisomes per cell from (B). Peroxisome formation in PEX1−/− and PEX14−/− hTERT-RPE1 cells was significantly impaired compared with that of the parental cells (mean ± s.d.: ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 20-25 cells per experiment).

(D) Quantification of proportion of ciliated cells from (B). Ciliogenesis in PEX1−/− and PEX14−/− hTERT-RPE1 cells was not significantly altered compared with that of the parental cells (mean ± s.d.: one-way ANOVA with Tukey's HSD, n=3: 190-200 cells per experiment).

(E) Quiescent G0-phase wild-type, PEX1−/−, and PEX14−/− hTERT-RPE1 cells transfected with AcGFP1-tagged D4 as a cholesterol probe were immunostained with anti-pericentrin (white) and anti-acetylated tubulin (blue) antibodies. Cholesterol was stained with Filipin III (red). Arrows and arrowheads indicate primary cilia and cytosolic accumulations of AcGFP1-tagged D4, respectively. Scale bar, 5 μm.

(F) Quantification of the Filipin III intensity at primary cilia from (E). PEX1−/− and PEX14−/− hTERT-RPE1 cells had significant reductions in the ciliary signal of Filipin III (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 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, representatively.

(G) Quantification of the AcGFP1-tagged D4 intensity at primary cilia from (E). The ciliary signal of AcGFP1-tagged D4 in PEX1−/− and PEX14−/− hTERT-RPE1 cells was significantly diminished (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 25-30 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, representatively.

(H) Quiescent G0-phase wild-type, PEX1−/−, and PEX14−/− hTERT-RPE1 cells were treated with or without 1.5% methyl-β-cyclodextrin for 45 min, and then incubated with or without cholesterol (cholesterol/methyl-β-cyclodextrin complex) for 1 h. After removing exogenous cholesterol, they 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-γ-tubulin (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-β-cyclodextrin-mediated cholesterol depletion. Scale bar, 2.5 μm.

(I) Quantification of the Smo intensity at primary cilia in wild-type, PEX1−/−, and PEX14−/− hTERT-RPE1 cells from (H). PEX1−/− and PEX14−/− hTERT-RPE1 cells exhibited the dampened Shh-N ligand-induced ciliary accumulation of Smo. The complementation of exogenous cholesterol (cholesterol/methyl-β-cyclodextrin complex) restored the ciliary accumulation of Smo in both PEX-knockout cells, while the LDL complementation did not rescue the ciliary phenotypes efficiently (*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, representatively.