FIGURE 2.

IFN-γ enhances intracellular ROS and extracellular H₂O₂ production. A, upper panel, interferon-γ induces Duox2-mediated ROS production measured by confocal microscopy. Starved BxPC-3 cells were treated with or without IFN-γ for 24 h. Live staining was performed as described under “Experimental Procedures.” Higher ROS levels were observed in BxPC-3 cells that were pre-treated with IFN-γ followed by stimulation with ionomycin than in those cells treated with IFN-γ or ionomycin alone. Representative confocal images of ROS production (green) or phase-contrast fields (immediately below) are shown. The lower panel provides the quantitation of fluorescent intensities under the different treatment conditions for three separate experiments. B, IFN-γ induces intracellular ROS production as detected by analytical cytometry. Serum was withdrawn from BxPC-3 cells overnight; cells were then treated with or without IFN-γ for 24 h; intracellular ROS production was confirmed using the redox-sensitive dye CM-H₂DCFDA. A right-shift in fluorescence intensity indicates increasing amounts of ROS. C, production of extracellular H₂O₂ after exposure of BxPC-3 cells to IFN-γ. BxPC-3 cells were grown using serum-free RPMI 1640 medium in the presence or absence of IFN-γ for 24 h; after collection, 2 × 10⁴ BxPC-3 cells were mixed with the Amplex Red reagents with or without 1 μm ionomycin for the indicated times. H₂O₂ levels were calculated using a standard curve of 0–2 μm H₂O₂. H₂O₂ production for cells exposed to IFN-γ has been compared with cells treated with solvent alone at each time point. *, p < 0.05; **, p < 0.01; ***, p < 0.001.