Detection and quantitation of cholesterol 27-hydroxylase in THP-1 cells exposed to NS398. (a) Dose-dependent decrease in 27-hydroxylase mRNA expression in THP-1 monocytes treated with the COX-2 inhibitor NS398. Cultured THP-1 monocytic cells were untreated or exposed to NS398 for 18 hours. After isolation of total RNA, the RNA was reverse-transcribed and the cDNA amplified by quantitative real-time polymerase chain reaction as described. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) Dose-dependent decrease in 27-hydroxylase protein expression in THP-1 monocytes treated with the COX-2 inhibitor NS398. Cultured THP-1 monocytic cells were untreated or exposed to NS398 for 18 hours. Total cell protein was isolated and 27-hydroxylase detected with specific rabbit polyclonal anti-human 27-hydroxylase antibody. Western blotting was performed with an anti-β-actin antibody to confirm equal protein loading. At 100 mM NS398 concentration, cell death was statistically significant (14.8% ± 6.3%). COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS398, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide. ** p < 0.01.