Figure 2. Effect of O₃ medium on IB proteolysis, NF-B-dependent gene expression, and B-dependent transcription. (A) IB proteolysis was prevented in O₃-medium-cultivated cells. BHY (left panel) and HeLa cells (right panel) were pre-incubated with serum-free O₃ medium (BHY, 15 min; HeLa, 1 hr) (ozonation state in %) before TNF was added (BHY, 20 ng/mL, 45 min; HeLa, 1 ng/mL, 15 min). IB and actin were determined by Western blot analysis. (B) and (C) NF-B target gene expression was inhibited by pre-incubation with O₃ medium. (B) BHY and HGF-1 cells were pre-incubated with serum-free O₃ medium (BHY, 15 min; HGF-1, 45 min), followed by TNF (BHY, 20 ng/mL, 45 min; HGF-1, 5 ng/mL, 45 min). The supernatant was then replaced by regular medium, and interleukin-8 was measured after 5 hrs by an enzyme-linked immunosorbent assay (BHY, n = 2; HGF-1, n = 3; mean ± SD). (C) BHY cells were pre-treated with O₃ medium (15 min). The supernatant was replaced by regular medium, TNF (20 ng/mL, 45 min) was added, and interleukin-1β was measured after 12 hrs (immunoassay). The interleukin-1β level after TNF stimulation following pre-treatment with non-ozonized medium was defined as 100% (dashed line) (n = 3, mean ± SD). (D) B-dependent transcription was inhibited by pre-incubation with O₃ medium. HeLa cells were transfected with pGL2-3B-Luc and the Renilla plasmid. After 24 hrs, cells were treated with serum-free O₃ medium (1 hr), followed by TNF (1 ng/mL, 15 min). The supernatant was replaced by regular medium, and relative luciferase activity was measured after 5 hrs (n = 3, mean ± SD).