Effects of inflammation and Golgi glycosylation enzyme inhibitors on intracellular cholesterol accumulation in THP-1 macrophages.

<p>THP-1 macrophages were incubated in serum free medium for 24 h at 37°C. The medium was then replaced by fresh serum-free medium in the absence (control) (I) or presence of 40 ng/ml IL-6 (II) or 50 ng/ml TNF-α (III) or 25 µg/ml LDL (IV) or 40 ng/ml IL-6 plus 25 µg/ml LDL(V) or 50 ng/ml TNF-α plus 25 µg/ml LDL (VI) or glycosylation inhibitors (VII), or 40 ng/ml IL-6 plus inhibitors (VIII), or 50 ng/ml TNF-α plus inhibitors (IX) for 24 h at 37°C. Cell viability was determined using the MTT assay as described in Methods. Cell viability is expressed as the percentage of absorbance from treated cells compared to untreated cells. Graphs represent the average values (M±SD) of three independent experiments with triplicate biological measurements for each experiment (A). The cells were examined for lipid inclusions by Oil Red O (ORO) staining. The results are typical of those observed in four separate experiments (×400). Semi-quantitative analysis of ORO positive staining was performed by the Image-J software. Results represent mean± SD from 4 separate fields (B). Intracellular free cholesterol and total cholesterol were assayed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075650#s2" target="_blank">Materials and Methods</a> section. Values are means ± SD of duplicate wells from 4 experiments (C and D). ^*P<0.05 vs control; ^**P<0.01 vs control; ^***P<0.01 vs IL-6; ^#P<0.05 vs LDL; ^##P<0.01 vs LDL; ^###P<0.01 vs TNF-α.