Figure 4. C14 sterol reductase activity is impaired in LBR-deficient cells.

(A) LBR-deficient primary fibroblasts and controls were cultured in lipid-depleted medium for 5 days, and total lipids were extracted and analyzed by gas chromatography/mass spectrometry. ACG1A_1 carries the homozygous LBR mutation described in this study; CTL_2 is representative of 3 matched wild-type controls (n = 3). Peak 1: 5β-cholestan-3α-ol (internal standard). Peak 2: cholesterol. Peak 3 (red number) was only detected in ACG1A_1 and corresponds to 8,14-cholestadien-3β-ol previously described in Greenberg dysplasia. The data are representative of 3 independent experiments (n = 3). (B) Indicated primary cells were cultured under cholesterol-restrictive growth conditions for 2 days, the medium was supplemented either with 2.5 mM methyl-β-cyclodextrin (MβCD, cholesterol depletion), or with 2.5 mM MβCD:cholesterol-saturated complexes (cholesterol replacement), and imaged by bright-field microscopy. The data are representative of 3 experiments (n = 3). (C) Growth curves of LBR-deficient cells and 3 matched controls (n = 3) in lipid-depleted medium for 9 days; 6.5 μM cholesterol was added to the medium of ACG1A_1 (ACG1A_1+CHOL) and 3 controls (CTL+CHOL). Cell numbers were determined by automated counting; data points represent the mean of triplicates values; for controls the mean of 3 pooled triplicates was determined (n = 9); error bars indicate SD. Statistical differences were assessed by 2-way ANOVA using Bonferroni’s correction for multiple comparisons; ns., not significant. *P ≤ 0.05, ****P < 0.0001.