Figure 6

Overexpression of USP18 increased DENV replication in USP18-KO cells. A549 cells were treated with puromycin-selectable lenti-CRISPR vector to deliver control sgRNAs or USP18 sgRNAs to generate USP18-knockout (KO) clone as described in the Materials and Methods. The amino acid sequences of the resulting products were shown (A). Expression of USP18, Tom20 and α-tubulin in cytosolic and mitochondrial fractions in wild-type and usp18-KO cells was determined by Western blotting (B). usp18-KO A549 cells (2 × 10⁵/ml) were transfected with different indicated doses of usp18-flag plasmid by lipofectamine 3000 (3 μl/ml) to overexpress USP18 and then infected or not with DENV (MOI = 0.5) for 24 h. Expression of USP18 and NS2B was determined and analyzed (C). In the wild-type and usp18-KO A549 cells (2 × 10⁵/ml) with or without overexpressing USP18, both total and cytosolic fractions were prepared, and mtDNA levels were measured as described in above figures (D). The wild-type and USP18-KO A549 cells were transfected with USP18 or control plasmid by lipofectamine 3000. The treated cells were infected by mock or DENV (MOI = 1) for 24 h and the DENV RNA was measured (E). The wild-type or USP18-KO A549 cells (2 × 10⁵/ml) were transfected with USP18-flag plasmid (2 μg) or control empty vector and then infected by mock or DENV (MOI = 0.5) for 24 h and the expression of USP18 and OGG1 in the whole cell lysates was determined by Western blotting (F). Statistical analysis was done using one-way ANOVA Bonferroni (C) and two-way ANOVA with Holm-Sidak’s multiple comparisons (D–F) to compare differences among different treatments. Original gels are presented in Supplementary Fig. 13.