(A) IFN-ε induces type I IFN-stimulated genes (ISGs). MDMs were treated with IFN-ε or IFN-α, and gene expression of ISGs was measured by real-time RT-PCR. Induction of each gene (as fold change relative to control) was calculated using the ΔΔCT method, as described in the Methods. The dashed line indicates the level of gene expression of untreated control cells. (B) IFN-ε activates STAT1. STAT1 activation was determined by Western blot using Tyr701 phopho-STAT1 antibody. The blot was stripped and then probed with anti-STAT1 antibody for the loading control. (C) Induction of HIV restriction factors by IFN-ε. MDMs were treated with IFN-ε for 24 hours, and the expression of HIV restriction factors was determined by real-time RT-PCR. Each bar represents the fold change between untreated control and IFN-ε–treated MDMs from different donors. The dashed line indicates the level of gene expression of untreated control cells. (D) Effect of APOBEC3A knockdown on induction of APOBEC3A by IFN-ε. MDMs were transfected with 10 nM control siRNA or siRNA targeting APOBEC3A (A3A) for 24 hours before incubation with IFN-ε for an additional 24 hours. Expression of A3A was determined by quantitative real-time RT-PCR. *P < 0.5, control siRNA vs. A3A siRNA by independent-samples t test. (E) Effect of IFN-ε on HIV infection of APOBEC3A knockdown MDMs. MDMs were incubated with the transfection reagent alone (no siRNA) or the transfection reagent with control siRNA or A3A siRNA for 24 hours. Cells were then treated with IFN-ε for an additional 24 hours followed by HIV-luc (JR-FL) infection. *P < 0.05, ^#P > 0.05, IFN-ε–treated vs. untreated controls PMA-differentiated PMA-differentiated by independent-samples t test. Data are mean ± SD of triplicate samples and represent 3 independent experiments.