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Figure 6 | Particle and Fibre Toxicology

Figure 6

From: Regulation of the arachidonic acid mobilization in macrophages by combustion-derived particles

Figure 6

NAC inhibits fly ash-induced formation of ROS, mobilization of AA, phosphorylation of ERK1/2, JNK, c-Jun and COX-2 expression. (A) Adherent H2DCF-loaded RAW264.7 cells were pre-incubated with 5 mM NAC and further incubated with 10, 25, 50, 100, 200 and 300 μg/ml fly ash particles (6.3, 12.6, 31.3, 62.5, 125, 188 μg/cm2) for 2.5 hours. Values are expressed as a percentage of the untreated controls. For analysis of ERK1/2 (B), c-Jun (F) phosphorylation, and COX-2 expression (D), non-labelled cells were pre-incubated with NAC at 1 and/or 5 mM and further treated with 50 μg/ml MAF02 particles (15.6 μg/m2) for 2.5 hours. Whole cell lysates were analyzed by Western blotting. OD-band intensities of COX-2 and phospho-proteins, analyzed by Odyssey® software, were normalized to the respective loading control protein and expressed in relation to the maximum band intensity of the MAF02-treated sample (100%). To detect mobilization of AA and its metabolites (C, E), RAW264.7 macrophages labelled with [14C]arachidonic acid were pre-incubated with the antioxidant NAC at 1 or 5 mM and subsequently exposed with 50 μg/ml MAF02 particles (13.2 μg/m2) for 2.5 hours. After lipid extraction, the free arachidonic acid and its metabolites were separated by TLC, visualized by autoradiography and analyzed by OptiQuant® software. Data on AA liberation are expressed as percentage of control cells (100% values). Results are presented as the mean ± s.e.m. of three independent experiments (* p < 0.05, **p < 0.01, ***p < 0.001 compared to 50 μg/ml MAF02-treated cells).

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