Fig 5.

Identification and characterization of IFIT1/ISG56 as an miR-203 target. (A) Illustration of the 3′ UTR of IFIT1/ISG56, with boxes indicating the positions of two partially overlapping miR-203 seed matches. (B) A549 cells were transfected with 25 nM nontargeting miRNA mimic (Control) or nontargeting control miRNA inhibitor (Inhib) or miR-203-specific mimic or miR-203 inhibitor, as indicated. Cells were either untreated (−) or treated (+) with 1,000 units/ml IFN-α. RNA was purified from cells 10 h later and analyzed by RT-qPCR for the induction of IFIT1/ISG56 mRNAs. In parallel, levels of miR-203 were measured by TaqMan assay. Statistical significance was determined by a two-tailed t test (*, P < 0.05; **, P < 0.01). (C) A549 cells were transfected with miRNA mimics and inhibitors and then treated with IFN-α as described for panel B Whole-cell lysates were prepared 10 h later and analyzed by immunoblotting with antiserum specific for IFIT1/ISG56, MDA5, or GAPDH protein.