Figure 1: The PDGF-induced proliferation of hVSMCs is DNA-PK dependent. (A) Proliferation assay of hVSMCs treated with the DNA-PK inhibitor NU7026 (1 µmol/l) and PDGF-BB (10 ng/ml); BrdU incorporation was quantified. The fold induction is the ratio of stimulated to unstimulated cells. ***p < 0.001 (B) Immunoblot analysis of cyclin D1expression in hVSMCs after PDGF treatment and DNA-PK inhibition by the DNA-PK inhibitor NU7026; vinculin was used as a loading control. (C) mRNA expression of cyclin D1 in hVSMCs incubated for 6, 18, 24, and 48 hours with PDGF-BB (10 ng/ml) in the absence or presence of the DNA-PK inhibitor NU7026 (1 µmol/l). The fold induction is the ratio of stimulated to unstimulated cells, and error bars represent the S.D.s of three experiments (four measurements) *p < 0.05.

Figure 2: PDGF-dependent upregulation of DNA-PKcs. (A and B) Immunoblot analysis of DNA-PKcs expression in hVSMCs after PDGF-BB (10 ng/ml) treatment in a time- (A) and dose-dependent manner (B); ß-actin was used as a loading control. (C) mRNA expression of DNA-PKcs in hVSMCs incubated for 18 hours with PDGF-BB (10 ng/ml); the fold change in induction is the ratio of stimulated to unstimulated cells. **p < 0.01. (D) Luciferase assay of hVSMCs after transfection of the pGL3-Basic luciferase reporter plasmid with and without a cloned fragment of the DNA-PKcs promoter (-3 to -606) treated with PDGF-BB (10 ng/ml). The fold induction is the ratio of stimulated to unstimulated cells, and error bars represent the S.D.s of three experiments (three measurements). **p < 0.01 (e). Immunoblot analysis of p- DNA-PKcs expression in hVSMCs after PDGF-BB treatment (10 ng/ml) in a time-dependent manner; ß-actin was used as a loading control.

Figure 3: PDGF induces double-strand break repair. (A and B) (A) Immunofluorescence microscopy of ?-H2AX foci in nuclei and (B) a bar chart of ?-H2AX foci per nucleus as an indicator of double-strand breaks in hVSMCs treated with PDGF-BB and irradiated for 1, 3, 5 and 7 hours. ***p < 0.001. (C and D) Immunoblot analysis of phosphorylated ?-H2AX expression in hVSMCs incubated with PDGF-BB and treated with irradiation for 1 (C) and 6 (D) hours; tubulin used as a loading control. (E and F) Immunoblot analysis of DNA-PKcs (E) and phosphorylated DNA-PKcs (F) expression in hVSMCs treated with PDGF-BB and irradiated for 6 hours; phosphorylated Chk1 (E) and Chk2 (F) indicate DNA-PK activation; vinculin was used as a loading control.