miR-27b targets Dll4 and Spry2. (A) Putative miR-27b binding sequences in Dll4 and Spry2 3′UTRs. (B) 3′UTR reporter assay. Reporter constructs with luciferase reporter fused to Dll4 and Spry2 3′UTR sequences, wild-type or mutated (MT), were cotransfected into HEK-293T cells together with vectors encoding miR-27b precursor, wild-type or scrambled (SCR). Luciferase activity was measured after 24 hours and normalized to Renilla luciferase control. *Statistically significant differences from SCR and/or MT controls by the 1-tailed Student t test. (C) Human microvascular endothelial cells were treated as indicated and Western blots probed with the Abs for Dll4, Spry1, Spry2, and GAPDH (loading control). (D-E) HUVECs were treated with MO-27b, MO-C, or transfected with lentiviral miRNA inhibitor Zip-27b or Zip-C control. Dll4 and Spry2 were measured by Western blot. GAPDH was used as loading control. (F-G) Whole-mount in situ hybridization with spry2 (F) and dll4 probes (G).