Figure 6

Role of ROS and cellular glutathione status in macrophage-mediated NF-κB activation in RLE cells. A. Modulation of intracellular glutathione levels to investigate the influence of intracellular redox status on phosphorylation and degradation of IκBα induced by supernatant of quartz-treated macrophages (Q-MS). Macrophages were treated with 40 μg/cm² DQ12 or PBS for 24 h. RLE cells were pretreated with 0.1 mM buthionine sulfoximine (BSO) for 24 h to deplete glutathione or with 1 mM N-acetyl cysteine (NAC) for 2 h to enhance intracellular levels. IκBα and phospho-IκBα protein was visualized using Western blot; blots have been reprobed for β-tubulin as a loading control. B. Investigation of the direct potential of ROS to activate the classical NF-κB pathway by measuring IκBα degradation using Western blotting. RLE cells were treated with 50 μM hydrogen peroxide (H₂O₂) in a medium-free environment for 5-20 min. Additionally, the intracellular glutathione level was modulated using BSO and NAC at concentrations of 0.1 mM and 1 mM. β-tubulin served as loading control.