FIGURE 3.

Duox2 and DuoxA2 are required for IFN-γ-mediated extracellular H₂O₂ production blocked by the flavin dehydrogenase inhibitor diphenylene iodonium (DPI) or the calcium chelator BAPTA. A, BxPC-3 cells grown in serum-free RPMI 1640 medium were treated with 50 ng/ml of IFN-γ for 22 h and then incubated with or without different concentrations of DPI (left panel) or BAPTA (right panel) for another 2 h; cells were harvested and extracellular H₂O₂ formation was measured for 1 h in the presence or absence of ionomycin (1 μm). Error bars represent standard deviations from quadruplicate samples. B, Duox2 siRNA abrogates IFN-γ-induced Duox expression and H₂O₂ production. Upper panel, control or Duox2-specific siRNAs were transiently transfected into BxPC-3 cells; 24 h after transfection, cells were incubated in serum-free medium with or without 25 ng/ml of IFN-γ for a subsequent 24 h. Immediately thereafter, H₂O₂ production was measured over 1 h as previously described. The lower panel demonstrates the expression of Duox, Stat1, or β-actin by Western analysis for BxPC-3 cells exposed to IFN-γ or solvent, and transfected with Duox2 or control siRNAs as outlined for the measurement of H₂O₂. C, the left panel demonstrates the effect of control or DuoxA2-specific siRNAs on Duox2 or DuoxA2 expression in BxPC-3 cells treated with IFN-γ (25 ng/ml). Experiments shown in the right panel examine the effect of DuoxA2-specific siRNAs on IFN-γ-induced extracellular H₂O₂ production. H₂O₂ formation was determined for 1 h as previously described.