Figure 5

Mechanisms of USP18-regulated mtDNA release examined by specific inhibitors. A549 cells (2 × 10⁵/ml) delivered with siCtrl or siUSP18 were treated with indicated concentrations of RUR and Xec for 2 h and then infected with mock or DENV (MOI = 0.5) for 24 h. The levels of DENV RNA were measured (A). A549 cells (2 × 10⁵/ml) delivered with siCtrl or siUSP18 were treated with Mitotempo (100 μM) for 2 h and then infected by DENV (MOI = 0.5) for 24 h. The collected cells were mixed with 10 nM calcein and 400 μM CoCl₂ in calcein-quenching assays, and the fluorescence intensity was determined by flow cytometry to measure DENV-induced opening of mPTP (B). A549 cells (2 × 10⁵/ml) delivered with siCtrl or siUSP18 were infected with mock or DENV (MOI = 0.5) for 24 h. The total cell lysates were incubated with 200 μM EGS at 30 °C for 15 min to stabilize the VDAC oligomer and then examined by Western blot (C). H₂O₂ (1 mM) treatment for 6 h served as a positive control. A549 cells (2 × 10⁵/ml) delivered with siCtrl or siUSP18 were treated or not with various chemical compounds, including CsA (5 μM), VBIT-4 (10 μM), and Mitotempo (100 μM) for 2 h and then infected by DENV (MOI = 1) for 24 h. The DENV RNA and mtDNA levels were determined. A549 cells (5 × 10⁴/ml) were treated or not with 100 μM ddc for 7 days to deplete mtDNA. Cells were passaged every 2–3 days. The mtDNA-depleted cells (2 × 10⁵/ml) delivered with siCtrl or siUSP18 were infected with mock or DENV (MOI = 0.5) for 24 h. After collecting total cell lysates, the expression of USP18 and DENV NS2B was determined by Western blotting (E). More than 3 independent experiments were carried out and analyzed for each condition. Statistical analysis was done using two-way ANOVA with Holm-Sidak’s multiple comparisons (A, B, D, and 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. 12.