Figure 3

USP18 regulated mtDNA release, oxidation and fragmentation. A549 cells (2 × 10⁵/ml) treated with siCtrl or siUSP18 via lipofectamine 3000 were infected with mock or DENV (MOI = 0.5) for 24 h. Both total and cytosolic DNA were extracted from collected cells according to the Materials and Methods and quantified using qPCR with specific primers to measure mtDNA levels. The total mtDNA content was normalized to nuclear DNA TERT, and the relative abundance of mtDNA in cytosolic fraction was normalized with exogenously added plasmid encoding the FLAG gene (PCR3.1-flag) as described in the Materials and Methods (A). BMDCs were prepared from ifnar1-KO mice as described in the Materials and Methods and cells (1 × 10⁶/ml) were treated similarly to (A) and mtDNA release was measured (B). A549 cells (2 × 10⁵/ml) treated with siCtrl or siUSP18 via lipofectamine 3000 were infected by DENV (MOI = 0.5) for 24 h and the expression of 8OHdG was measured by intracellular immunostaining with anti-8OHdG Abs (1:500) and then analyzed by flow cytometry (C, upper and left lower). In addition, the 8OHdG levels in the cytosolic fractions were determined by ELISA (C, right lower). A549 cells were treated similarly to (C), collected and the expression of OGG1 in total cell lysates was determined by Western blotting (D). A549 cells (2 × 10⁵/ml) were infected by DENV (MOI = 0.5) for 24 h and DNA was prepared from total, mitochondrial, or cytosolic fractions of treated cells as described in the Materials and Methods and run in agarose gels and analyzed by staining with Midori Green Advance Safe DNA/RNA staining kit (E, left). The signals of 6069 bp and 637 bp fragments of mtDNA and TERT in individual fractions were amplified by PCR using designated primers (Table 1), analyzed in gels, and followed by ethidium bromide staining (E, right). Statistical analysis was done using two-way ANOVA with Holm-Sidak’s multiple comparisons (A–E) to compare differences among different treatments. *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001. Original gels are presented in Supplementary Fig. 11.