Figure 1. Activation of NF-B was inhibited in the presence of O₃ medium. (A) BHY cells were pre-incubated with serum-free O₃ medium (15 min; the ozonation state is indicated in %) before TNF was added (20 ng/mL, 45 min), and an electrophoretic mobility shift assay was performed (NF-B and Sp-1 binding). The asterisk marks a control reaction in which a 100-fold concentration of unlabeled consensus oligonucleotide was added. (B) Cells were treated with O₃ medium (HGF-1, 45 min; HeLa, 1 hr), followed by stimulation with TNF (HGF-1, 5 ng/mL for 45 min; HeLa, 1 ng/mL for 15 min). NF-B activity normalized to Sp-1 is shown (densitometric analysis). NF-B activity after TNF stimulation following pre-incubation with non-ozonized medium was defined as 100% (dashed line) (n = 3, mean ± SD). (C) Periodontal ligament tissue separated into equal-sized portions was incubated (1 hr, 37°C) with serum-free non-ozonized medium (indicated by "–") or O₃ medium (100% ozonation state, indicated by "+"), followed by two-hour incubation in fresh medium without ozone. The samples were shock-frozen and homogenized, and nuclear extracts were prepared. Left: Electrophoretic mobility shift assays were performed (NF-B and Sp-1 binding). The asterisk marks a control reaction in which a 100-fold concentration of unlabeled consensus oligonucleotide was added. Right: Densitometrically measured NF-B activity was normalized to Sp-1 (n = 3, mean ± SD).