The GAX 3′-UTR confers responsiveness to serum and proangiogenic factors in ECs. (A) Construction of the psiCHECK2-GAX-3′-UTR. The 280-bp sequence in the GAX 3′-UTR containing 2 miR-130a consensus sequences was inserted into the vector at the 3′ end of the hRluc (Renilla luciferase) reporter gene. hRluc indicates Renilla reniformis luciferase. (B) Serum and proangiogenic factors suppress luciferase activity in HUVECs transfected with psiCHECK2-GAX-3′-UTR compared with psiCHECK2 empty vector. HUVECs were transduced with either psiCHECK2-GAX-3′-UTR or psiCHECK2 (control) and then incubated in 0.1% FBS, in 0.1% FBS supplemented with 10 ng/mL of either VEGF, bFGF, or TNF-α, or in 2% FBS. Renilla luciferase activities were measured as described in “Dual luciferase reporter assays.” In parallel, identical passaged HUVECs from the same split were transfected with psiCHECK2 empty vector and subjected to the same conditions. Results are expressed as a ratio of the signal from cells transfected with psiCHECK2-GAX-3′-UTR to the signal from cells transfected with psiCHECK2 empty vector. (C) Inhibition of miR-130a reverses the suppression of luciferase activity by serum in a dose-dependent fashion. HUVECs were treated as in B but were cotransfected with increasing concentrations of miR-130a inhibitor. A dose-dependent reversal of the down-regulation of reporter activity attributable to serum was observed. Error bars represent SEM.