Figure 4

Detection and quantitation of cholesterol 27-hydroxylase in THP-1 cells exposed to SC560. (a) 27-Hydroxylase mRNA expression in THP-1 monocytes is decreased by the specific COX-1 inhibitor SC560. Cultured THP-1 monocytic cells were untreated or exposed to increasing doses of SC560 for 24 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) Decrease in 27-hydroxylase protein expression in THP-1 monocytes treated with the COX-1 inhibitor SC560. Cultured THP-1 human monocytes were untreated or exposed to SC560 for 24 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. COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SC560, 5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazol. ** p < 0.01.