FIGURE 1.
NADPH oxidase inhibitors decrease SOD1G93A-induced ROS generation in mouse glial cells. A, mouse glial cells were infected with Ad.Empty or Ad.SOD1G93A virus for 28 h prior to changing to serum-deprived (0.2% serum) medium for 8 h, and then incubated with DHE (1 μm) for 30 min at 37 °C. Cells were then assessed by FACScan flow cytometry. Top panel, two representative graphs for samples with Ad.Empty and Ad.SOD1G93A; bottom panel, quantification of flow cytometry data as either the percentage of total cell population (left y axis) or relative DHE intensity (right y axis). B, mouse glial cells were infected with Ad.SOD1G93A or Ad.Empty for 28 h prior to changing to serum-deprived (0.2% serum) medium for 8 h. Diphenylene iodonium (DPI, 10 μm) or apocynin (Apo, 100 μm) were added 60 min before washing with PBS. Cells were then incubated with DHE (1 μm) for 30 min at 37 °C before fluorescence microscopy (top panel) or FACScan flow cytometry (bottom panel). In the fluorescent images, the mitochondria are stained green and the nucleus is stained blue. C, mouse glial cells were infected with Ad.G93A or Ad.Empty for 36 h prior to cell harvesting. A 1-μg portion of each 100,000 endomembrane pellet was used to evaluate NADPH-dependent production of O2˙̄ by lucigenin-based chemiluminescence. D, mouse glial cells were infected with the indicated adenoviral vectors for 28 h prior to changing to serum-deprived (0.2% serum) medium for 8 h. Cells were then incubated with DHE (1 μm) for 30 min at 37 °C before FACScan flow cytometry. Data in all graphs represent the mean ± S.E. with n = 3; marked comparisons (* or #) demonstrate significant differences as determined by one-way analysis of variance followed by Student's t test (p < 0.05).