Unlike the side effects of ACAT inhibitors on the formation of foam cells in rodent macrophages via accumulation of free cholesterol, ACAT inhibition is shown in many studies to repress the accumulation of total cholesterol in human macrophages by lowering the uptake of acLDL and facilitating FC efflux. Materials and Methods Materials Oleic acid anilide, a known ACAT chemical, was produced by one of the authors as described. Oleoyl CoA and cholesterol were purchased Dasatinib ic50 from Amersham Biosciences. The radioactivity of related products and services, and oleoyl CoA, cholesterol was measured using a liquid scintillation counter. LDL was isolated from the plasma by sequential ultracentrifugation. AcLDL was prepared by repeated addition of acetic anhydride to LDL, and sterilized by filtration via a membrane with a pore size of 0. 45 fim. The completeness of acetylation was assessed from electrophoretic mobility on agarose fits in. AcLDL was used within four weeks and saved at 4oC. Cell countries Human acute monocytic Cellular differentiation leukemia THP HepG2 cells and 1 cells were developed in RPMI 1640 medium containing DMEM and 10% FBS containing 10% FBS, respectively. THP 1 cells in suspension were plated in RPMI 1640 medium with ten percent FBS and 50 ng/ml of PMA for 3 days to become completely differentiated macrophages before used in experiments. In the vast majority of experiments, macrophages were enriched with cholesterol by addition of acLDL in RPMI 1640 medium containing ten percent lipoproteindeficient serum for 48 h. Full cell cholesterol esterification analysis THP I macrophages were pretreated immediately with or without OAA in total RPMI 1640 medium, followed by incubation in serum free RPMI 1640 medium containing 0. 2 fiCi/ml of oleoyl ubiquitin conjugation CoA BSA complex and 100 fig/ml of acLDL with or without OAA for 18 h. The oleoyl CoA BSA complex was prepared as described. The cells were washed with PBS and extracted with hexane/ isopropyl alcohol. The extracts containing esterified services and products were isolated by thin layer chromatography. Full cell cholesterol esterification activity was assessed by determining the radioactivity of the cholesteryl oleate developed. Parallel artificial membrane permeation assay The permeability of OAA was measured by the parallel artificial membrane permeation assay, that will be based around the usage of 96 properly membrane filter based plate, in 5% DMSO/PBS, pH 7. 4. cholesterol efflux assay The cholesterol efflux assay was done essentially as described with slight modification. Shortly, 1 mg of acLDL was incubated with 10 fiCi of cholesterol for 30 min at 37oC, and then 10 ml of RPMI 1640 medium was added. The THP 1 macrophages were incubated in this medium for 48 h with or without OAA, washed three times with PBS, and then incubated in RPMI 1640 medium containing 0. The next day fatty acid free BSA overnight.