Figure 1. DKK1 and DKK2 reciprocally expressed during endothelial morphogenesis distinctively regulate angiogenesis in vitro.
(A) mRNA and protein levels of DKK1 and DKK2 at the same time points (0.5, 8, and 12 hours) during morphogenesis in Matrigel were measured by RT-PCR (left) and Western blotting (right). (B) Human DKK1 or DKK2 promoter activities were measured by luciferase reporter assay. HUVECs cultured on gelatin-coated plates were transfected and transferred to gelatin-coated plates (gelatin) or Matrigel-coated plates (morphogenesis) 6 hours later. Luciferase reporter activity was measured 16 hours later. Data represent mean ± SD. (C) HUVECs stably expressing eGFP plus control (CTL) shRNA, eGFP plus DKK1 shRNA, or DKK1 plus control shRNA were established with lentivirus. Stable transfectants were plated on Matrigel-coated plates at a density of 1.5 × 105 cells/well and incubated for 18 hours. Capillary-like networks, which completely differentiated into tube-like structure, were quantified with Image-Pro Plus software. (D) Proliferative indices of HUVECs transfected with eGFP or DKK1 were assessed by [3H]-thymidine incorporation assay. (E) HUVECs stably expressing eGFP, DKK2, control shRNA, or DKK2 shRNA. Morphogenesis of the transfectants on Matrigel was analyzed as described in C. (F) Proliferative indices of HUVECs transfected with eGFP or DKK2 were accessed as described in D. Data represent mean ± SD. **P < 0.01; ***P < 0.001.