Figure 3. O₃ amino acids selectively inhibited the NF-B system. (A) HeLa cells were incubated with O₃-phosphate-buffered saline (1 hr), followed by TNF stimulation (1 ng/mL, 15 min). The ozonation state is indicated in %. We performed an electrophoretic mobility shift assay to determine NF-B activity as well as Sp-1 binding. (B) Cells were pre-incubated (1 hr) with non-ozonized or ozonized amino acids (medium concentration) dissolved in phosphate-buffered saline (100% ozonation state), followed by stimulation with TNF (1 ng/mL, 15 min). The activation of NF-B was determined by an electrophoretic mobility shift assay. (C) Densitometric analysis of the NF-B activity normalized to Sp-1 is shown (n = 3, mean ± SD). NF-B activity of cells pre-incubated with non-ozonized amino acids in phosphate-buffered saline and stimulated with TNF was defined as 100% (dashed line). (D) HeLa cells were pre-incubated with non-ozonized or ozonized glucose (medium concentration), followed by TNF stimulation. The NF-B activity compared with Sp-1 binding was determined by an electrophoretic mobility shift assay.