Fig. 7

LY6E inhibited the production of mtROS, ROS, oxidized mtDNA and 8-OHdG but not OGG-1 induced by IFN-α and IC stimulation. BMDMs were electroporated with siLY6E or siCtl and then stimulated with or without IFN-α or ICs for 24 h. After adding 5 μM MitoSOX™ to the culture and incubating for 0.5 h, the intensity of MitoSOX fluorescence was measured and used as an indicator of mtROS levels (A). The generation of cellular ROS was measured by DCFDA staining followed by flow cytometry analysis (B). To determine the status of mtDNA oxidation, we used formamidopyrimidine DNA glycosylase (Fpg)-sensitive real-time PCR analysis. Treatment of mtDNA with Fpg removes oxidized purines from DNA and creates single-strand breaks, leading to blockade of PCR amplification at these sites. The different intensities of qPCR amplification between Fpg-treated and Fpg-untreated DNA reflect oxidative base damage and the percentage of intact DNA; recognition and cleavage by Fpg causes a decrease in the percentage and indicates an increase in the number of sequences harboring oxidized base products (C). Accordingly, the mtDNA oxidation status was measured in both the mitochondrial and cytosolic fractions of IFN-α- and IC-treated BMDMs with or without LY6E knockdown (D). The generation of 8-OHdG was determined by flow cytometry (E and F). The expression of Ogg1 mRNA was determined by qPCR (G). Each data point represents one mouse, and the values are fold changes relative to the mean value of the siCtl in RT‒qPCR and Western blotting. The values for the flow cytometry results are presented as the geomeans (MFIs). Statistical analysis was performed with two-way ANOVA with Holm‒Sidak multiple comparisons to compare differences among different treatments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 and ****, P < 0.0001. MFI, mean fluorescence intensity