Identification of miR-17 targets. (A) mRNA expression of the putative targets after overexpression of Pre-17 in HUVECs for 24 hours; n = 3. *P ≤ .05. (B-C) Expression of Jak1 in HUVECs overexpressing the indicated miR precursors. (B) A representative Western blot. (C) Quantification; n = 4. (D) Effect of Jak1 silencing using 40nM siRNA on spheroid sprouting. An siRNA directed against firefly luciferase or cells treated with the transfection reagent was used as control; n = 5 or 6. Down-regulation of Jak1 protein 48 hours after siRNA transfection is shown in the representative Western blot. (E) Jak1 siRNA reduces the proangiogenic effect of miR-17 inhibition. HUVECs were transfected with Jak1 siRNA (40nM), miR-17 inhibitor (50nM), and the respective controls as indicated. Spheroids were generated and sprouting was quantified; n = 3. *P < .05. (F) Firefly luciferase activity normalized to Renilla luciferase activity measured in homogenates of HEK cells transfected with the wild-type (wt) or mutated luciferase constructs and Pre-17 or control Pre-miR (mutations were applied to underlined nucleotides). Measurements were done 48 hours after transfection; n = 4.