FIGURE 6.

ROS generation was deregulated in the absence of autophagy and negatively regulated IFN-γ-induced STAT1 activation. A, confocal fluorescence microscopic observation of the co-localization (Merge, yellow) of EGFP-LC3 punctate formation (green) and MitoSOX Red staining (red) in WT MEFs with or without IFN-γ (10 ng/ml) treatment for 3 h. 3D, three-dimensional. Scale bars, 20 μm. B, Western blotting, PCR, and flow cytometry, with COX IV antibodies, COX I primer, and MitoTracker Green staining, respectively, were used to detect mitochondria expression in WT and Atg5^−/− MEFs. The ratios of mitochondrial DNA COX I to the total genomic DNA GAPDH were calculated based on the intensities of their PCR product in agarose gel. MFI, mean fluorescence intensity. C, CM-H₂DCFDA staining and a microplate reader were used to measure ROS generation. Data, obtained from triplicate cultures, are means ± S.D. (error bars). *, p < 0.05. D, Western blotting was used to determine IFN-γ (10 ng/ml)-induced phosphorylation of STAT1α/β (Tyr⁷⁰¹) in WT MEFs 0.25 h after they had been pretreated with (+) and without (−) caffeic acid phenethyl ester (CAPE, 25 μm) or H₂O₂ (10 mm) for 0.5 h. β-Actin was the internal control. Data are representative of three individual experiments. E, with (+) and without (−) H₂O₂ pretreatment for 0.5 h, generation of nitrite in IFN-γ (10 ng/ml)-treated WT MEFs was detected using Griess reagent at 48 h. Data, obtained from triplicate cultures, are means ± S.D. (error bars). *, p < 0.05.