Figure 3. Content analysis of sterol levels and sterol esterification.

(A, B) Sterol and sterol esterification levels of MEFs with different PS genotypes (A), and H4 cells with and without pharmacological inhibition of γ-secretase with DAPT (B), determined by the Amplex Red Cholesterol Assay. Significantly reduced levels of cholesterol esterification were observed in cells lacking functional PS expression or upon inhibition of γ-secretase activity. Total sterol levels were not significantly. Data shown are average and individual values ± SEM, n = 12 for MEF and n = 8 for H4 cells. (A) In experiments with MEF cells of different PS genotypes (A), significance was analyzed by one-way ANOVA and Holms–Sidak multiple comparison test. (B) For comparison of cells with and without DAPT treatment (B), significance was analyzed by unpaired t test with Welch’s correction. (C) Content analysis of a panel of sterols in MEFs with different PS genotypes, determined by GC-FID and GC-MS. Analyzed sterols where cholesterol precursors (lathosterol and desmosterol), as well as cholesterol and esterified cholesterol. Significantly increased levels of lathosterol and desmosterol can be observed in MEFs lacking functional PS expression, together with significantly decreased cholesterol levels. Data shown are average and individual values ± SEM, n = 9 for lathosterol, desmosterol, and cholesterol levels, and n = 7 for esterification ratio. Significance was analyzed by one-way ANOVA and Holms–Sidak multiple comparison test. (D) Secreted cholesterol and cholesterol esterification in culture media were analyzed by GC-FID. Significantly increased levels of secreted cholesterol can be observed in cells lacking expression of functional PS. Data shown are average and individual values ± SEM n = 4. Significance was analyzed by one-way ANOVA and Holms–Sidak multiple comparison test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.