Figure 7. Esterification of sitosterol and cholesterol in intact THP-1 macrophages.

The experiments were conducted as described in the Material and methods section. THP-1 macrophage cells were grown in medium A (DMEM, 10% FBS and 10 nM PMA); treated without or with 50 μg/ml AcLDL for 12 h. (A) [³H]Sitosterol or [³H]cholesterol was added to the growth medium for 24 h. (B) [³H]Sitosterol or [³H]cholesterol in LDL was added to the growth medium for 24 h. Cells were harvested and lipids were extracted and separated. Sterol and sterol ester products were counted by scintillation counter. The assays were run in duplicate wells. Error bars represent mean and variation between duplicates. Results shown represent one of the two experiments with similar results. (C) Immunoblot of ACAT1 enzyme in THP-1 macrophages grown in the presence or absence of 50 μg/ml AcLDL for 36 h. Cellular proteins from extracts of cells grown under each growth condition were loaded at doses of 20, 50 or 80 μg (1×, 2.5× or 4×) as indicated. The immunoblot was performed according to procedures described in [11], using the polyclonal anti-ACAT1 antibodies as the primary antibody. Data shown are representative of two separate experiments.