Figure 4.

Baltimore PM induces reactive oxygen species (ROS) generation in HBEpCs. (A) HBEpCs grown on glass bottom-dishes to approximately 90% confluence were loaded with 10 μM DCFDA for 30 minutes. Cells were rinsed in basal medium without growth factors and exposed to Baltimore PM (100 μg/ml) for 15, 30, and 60 minutes. At the end of exposure, ROS formation was visualized under fluorescence microscope and quantified using image analysis. (B) HBEpCs grown on 35-mm dishes to approximately 90% confluence were challenged with 10 or 100 μg/ml of Baltimore PM for 60 minutes. Media were collected and H₂O₂ levels were measured using the Amplex Red assay. Values are mean ± SD of three experiments. *Significantly different from cells exposed to vehicle alone (P < 0.05). (C) HBEpCs grown on glass-bottom dishes to approximately 90% confluence, challenged with Baltimore PM (100 μg/ml) for 15, 30, and 60 minutes. At the end of each time point, cells were washed with EBM phenol red free media and were loaded with MitoSOX (1 μM) for 10 minutes. Cells were washed three times with EBM phenol red free media and intracellular MitoSOX Red-emitted fluorescence was visualized by immunofluorescence microscopy. (D) Intracellular MitoSOX Red-emitted fluorescence of C quantified by image analysis using MetaVue software. Values are mean ± SD of three independent experiments. *Significantly different from cells exposed to vehicle (P < 0.05).