FIG 6.
DDX58 is targeted by miR-221 and influences bacterial replication. THP-1 cells were transfected either with miR-221 mimic, miR-579 mimic, or the miRNA mimic pool (miR-125b, miR-221, and miR-579) at a final concentration of 30 nM. As a control, a scrambled precursor (scr) was transfected. At 24 h posttransfection, THP-1 cells were activated with PMA for another 24 h and then infected with L. pneumophila at an MOI of 0.5. (A) DDX58 expression was determined via qPCR and is displayed as the log2 fold change. Luciferase reporter assay analyses were carried out in HEK-293T cells. The plasmid harbored either the wild-type (DDX58WT) or the mutated (DDX58mut) version of the 3′ UTR of DDX58. (B) Ratios of Renilla and firefly luciferase luminescence were normalized to the vector without insert. (C) Intracellular MX1 upon miRNA pool transfection was quantified by flow cytometric analysis, and the relative numbers of MX1-positive cells were calculated. (D) BDMs were transfected with a small interfering RNA-pool targeting DDX58 (siDDX58) or with a scrambled siRNA as control (scr) and infected at an MOI of 0.5 or left untreated. The downregulation of DDX58 expression with siRNA was verified by qPCR and is displayed as the log2 fold change. (E) The CFU of Legionella were determined 24, 48, and 72 h postinfection after a knockdown of DDX58. Boxes show the upper and lower quartiles, with medians (when n = 3 independent biological replicates) and whiskers indicating minimal and maximal values (when n = 4 to 6 independent biological replicates). A two-way ANOVA with Sidak’s correction was performed. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (compared to scramble). #, P ≤ 0.05 compared to the wild type (B); ##, P ≤ 0.01 (for global treatment effects) (E).