miR-K12-11 represses DUSP1 expression. (A and B) HUVEC were transfected with constructs encoding miR-K12-11 (pc-mi11), xCT (pc-xCT), or an empty control vector (pc). Twenty-four hours later, immunoblotting was performed to identify DUSP1, MAPK, xCT, and 14-3-3β protein expression (A), and qRT-PCR was performed to identify DUSP1 transcript expression (B). Error bars represent the standard errors of the means for three independent experiments. **, P < 0.01. (C) Cells were cotransfected with the pGL3-DUSP1 3′ UTR and either a control vector or pc-mi11, and luciferase expression was quantified as described in Materials and Methods. (D) HUVEC were cotransfected with either a control vector or pc-mi11, along with the luciferase reporter construct for miR-K12-11 (mi11-sensor). Aliquots were also incubated with 300 pmol 2′ OMe RNA antagomirs targeting either miR-K12-11 or miR-K12-12 (the latter was used as a negative control). Forty-eight hours later, luciferase expression was quantified as described in Materials and Methods. (E) Immunoblotting was performed 48 h after the transfection of cells with either a control vector or pc-mi11 in the presence of 300 pmol of either a control antagomir or a miR-K12-11-specific antagomir. (F) HUVEC were incubated with purified KSHV; then miR-K12-11 expression was quantified at the time points indicated using qRT-PCR. Expression at each time point was quantified relative to that in cells incubated with KSHV for 10 min. (G) HUVEC were transfected with luciferase reporter constructs for miR-K12-11 for 24 h and were then incubated with purified KSHV or medium (mock). Luciferase expression was quantified as described in Materials and Methods for cells collected at the indicated time points. Error bars represent the standard errors of the means for three independent experiments.