H₂O₂activation of PDGF-D requires Ets-1. (A) H₂O₂ increases levels of PDGF-D and Ets-1, but not Sp1. SMCs were treated with H₂O₂ (10 nM) for 0, 4, and 8 hours prior to extraction of total RNA and assessment of PDGF-D, Ets-1, and Sp1 levels by RT-PCR. (B) H₂O₂ increases PDGF-D mRNA expression in SMCs and bovine aortic endothelial cells (ECs) at various concentrations (10-1000 nM) and in a time-dependent manner (10 nM) by RT-PCR. Real-time PCR (bottom left panel) confirms H₂O₂ induction of PDGF-D mRNA expression. (C) H₂O₂ stimulates PDGF-D promoter activity. SMCs were transfected with 10 μg wild-type p1168PDGF-Dluc, p1168PDGF-Dluc–bearing mutant Ets-D3 element, or D4 elements. H₂O₂ was added to a final concentration of 10 nM. Luciferase activity was determined after 24 hours. (D) Ets-1 and Sp1 siRNA block Ets-1 activation of PDGF-D expression. SMCs were treated with 0.2 μM Ets-1 siRNA, Sp1 siRNA, and irrelevant (Irr) siRNA overnight, then incubated with 10 nM H₂O₂ for 8 hours prior to extraction of total RNA and RT-PCR. The histograms on the right refer to band intensity in the blots on the left. (E) Assessment of PDGF-D mRNA expression by real-time PCR. SMCs were treated with 0.2 μM Ets-1 siRNA, Sp1 siRNA, and irrelevant siRNA overnight then incubated with 10 nM H₂O₂ for 8 hours prior to extraction of total RNA and real-time PCR. (F) SMCs were exposed to H₂O₂ (100 nM) in the absence or presence of goat polyclonal PDGF-D antibodies or goat IgG and total cell counts were determined after 3 days using a Coulter counter. Alternatively the cells were exposed to 5% serum for control purposes. The data are representative of at least 2 independent experiments. Error bars represent SEM performed in duplicate or triplicate. *P < .05 relative to control using Student t test.