Figure 6

Effect of DDS on PQ-induced PKC activation and superoxide anion generation in primary mouse lung fibroblasts. (A) Primary mouse lung fibroblasts were exposed to 250 µM PQ at 3, 5, 10, 15, and 30 min, and then the phosphorylation of PKCµ at Ser744/748 and Ser916 was determined using Western blot analysis. (B) Primary mouse lung fibroblasts were pretreated with DDS (0.1, 1, 5, or 20 µM) for 3 h or with 5 µM Gö6983 for 30 min. After treatment with 250 µM PQ for 5 min, cells were lysed. Proteins were analyzed by Western blotting using antibodies against phosphorylated PKCµ. β-Actin was used as loading control. (C) Intracellular superoxide anion levels were measured by dihydroethidium (DHE) assay using fluorescence microscopy. After treatment with 250 µM PQ for 30 min, cells were incubated with 5 µM DHE for 15 min at 37℃ in the dark, and fluorescence was observed under an LSM 510 META fluorescence microscope. (D) HPLC analysis of superoxide anion production. HPLC samples were prepared and analyzed as described in Methods. The levels of 2-OH-Et⁺ (peak 1) and Et⁺ (peak 2) were expressed as HPLC peak area per mg protein. The 2-OH-Et⁺ and Et⁺ generation data graphed is representative of three independent experiments. ^#P < 0.05 relative to control; ^*P < 0.05 relative to PQ.