Type I IFNs upregulate IFITM3 and block dengue infection of human MKs. (A-F) Human, CD34⁺-derived MKs (culture day 12) were stimulated with IFNα or IFNβ at various concentrations or vehicle control (Veh) for 18 hours. IFITM3 mRNA expression (A) or protein expression (B) in IFNα-stimulated MKs was determined by using quantitative RT-PCR and immunoblot. (C) Immunoblots shown in panel B were quantified by densitometry. (D) IFITM3 mRNA expression in IFNβ-stimulated MKs was determined by using quantitative RT-PCR. (E-F) IFITM3 protein expression in IFNβ-stimulated MKs was measured by immunoblot and quantified by densitometry. (G-H) CD34⁺ MKs (culture day 11) were left alone (NT) or preconditioned with IFNα (100 U/mL) or Veh (phosphate-buffered saline) for 18 hours. CD34⁺ MKs were then infected with DENV2 at an MOI of 0.01 (G) or an MOI of 0.1 (H) for 18 hours. Dengue infection was determined by measuring the titers of virus released by MKs in LLC-MK2 cells. Data are from >4 to 6 independent experiments per group.