VEGF-induced HRMVEC DNA synthesis, migration, and tube formation require PLD1 activation. (A) Quiescent HRMVECs were treated with and without 40 ng/mL VEGF for the indicated time periods, and cell extracts were prepared and analyzed PLD1 phosphorylation by Western blotting using its phosphospecific antibodies or measured its activity using a kit. The blot was reprobed sequentially with anti-PLD1 and anti–β-tubulin antibodies for normalization. (B) Quiescent HRMVECs in which PLD1 is inhibited by 1-butanol (0.25%) or down-regulated by its siRNA (100nM) were treated with and without VEGF (40 ng/mL) for 24 hours, and DNA synthesis was measured by [³H]-thymidine incorporation. (C-D) All the conditions were the same as in panel B except that cells were treated with and without VEGF (40 ng/mL) for 8 hours and either cell migration (C) or tube formation (D) was measured. (B-D) Bar graphs represent the quantitative analysis of 3 independent experiments. Values are mean ± SD. *P < .01 versus vehicle control or Scr siRNA control. **P < .01 versus VEGF or Scr siRNA plus VEGF.