Figure 1.

PLD activates the HIF-1-VEGF pathway. (a) Vector (Vec)- and PLD-transfected cells (PLD) were lysed to obtain whole cell lysates. PLD1 and PLD2 protein levels were monitored by western blot. (b) PLD activity in vector (Vec)- and PLD-transfected cells (PLD) was assessed as described in Materials and Methods. The data are means±s.e.m. (n=3). *P<0.01 and **P<0.001 vs Control (c) Vector (Vec)- and PLD-transfected cells (PLD) were lysed to obtain nuclear extracts and HIF-1α protein was immunodetected by western blot. (d) Vector (Vec)- and PLD-transfected cells (PLD) were transfected with a HIF-1-dependent luciferase reporter plasmid and the luciferase activities were measure 10 h after the transfection. The data are means±s.e.m. (n=4). *P<0.01 vs Control (e) Cell culture media of vector (Vec)- and PLD-transfected cells (PLD) were replaced with serum-free media 8 h before analysis of VEGF. Secreted VEGF was detected in the cell culture media by an ELISA kit. The data are means±s.e.m. (n=3). *P<0.05 and **P<0.01 vs Control (f) SiRNA specific for HIF-1α (HIF-1α siRNA, HS, 200 nmol l⁻¹) was transfected in PLD-transfected cells. A nontargeting siRNA (NS) was used as a nonspecific control. One day post transfection, HIF-1α in nuclear extracts was analyzed by western blot. (g) One day post transfection with HIF-1α siRNA (HS) or non-targeting siRNA (NS) in PLD-transfected cells, cell culture media were replaced with serum-free media 8 h before analysis of VEGF. PLD-transfected cells were treated with echinomycin (20 nM) for 8 h following replacement of cell culture media. Secreted VEGF was detected in the cell culture media by an ELISA kit. The data are means±s.e.m. (n=3). *P<0.05 and **P<0.01 vs Control, ^#P<0.05 and ^##P<0.01.