Exosomal miR-135b targets FIH-1 in hypoxic HUVECs. (A) miR-135b binding sites in the FIH-1 3′-UTR were cloned into pMIR luciferase reporter vector (left, sensor vector). Identical construct mutation was generated (right, mutated vector). RPMI8226^1%O2/miR-135b exosomes (exosomes derived from RPMI8226^1%O2 transfected with miR-135b mimics), RPMI8226^1%O2/control exosomes (derived from RPMI8226^1%O2 transfected with scramble control miR), or RPMI8226HR^1%O2-exosomes were treated with HUVEC/Luc/β-gal (HUVECs cotransfected with pMIR-FIH1-3′-UTR vector and β-gal control vector). Luciferase activity of the sensor vector displayed a significant decrease by treatment with RPMI8226^1%O2/miR-135b exosome (**P < .01, Student t test, n = 3) and RPMI8226-HR^1%O2-exosome (*P < .05, Student t test, n = 3) compared with the control (HUVECs/Luc/β-gal without exosomes). These experiments were performed in triplicate, and the results are shown as mean ± SD. (B) FIH-1 protein expression levels measured by immunoblot after treatment with RPMI8226^20%O2-exosome, RPMI8226^1%O2-exosome, or RPMI8226-HR^1%O2-exosome of normoxic HUVECs (solid bars) and hypoxic HUVECs (open bars). The intensity of each band was quantified and normalized to FIH-1 expression signals by β-actin (**P < .01 vs control, Student t test, n = 3). (C) Effect of exosomes derived from HR-MM cells (RPMI8226-HR) on HIF-1–dependent transactivation of luciferase activity in normoxic HUVECs (solid bars) and hypoxic HUVECs (open bars) transfected with luciferase reporter genes linked to hypoxia response element (HRE). Luciferase activity was measured using the Dual-Glo Luciferase System (Promega). All assays were performed in triplicate. Means ± SD are shown (**P < .01 vs control; HUVECs^1%O2 without exosomes, Student t test, n = 3). exo, exosome.