Figure 6. TGIF1 has no effect either on radiation-induced Smad pathway activation or on radiation-induced PAI-1 overexpression in endothelial cells.

(A) HUVECs were transfected with (CAGA)9-Luc reporter plasmid for 24 h and were serum starved for 18 hours. Cells were irradiated at 10 Gy in presence or not of 10 ng/mL TGF-β1. Relative luciferase activity was measured 24 h after irradiation (B) HUVECs were co-transfected for 24 h with CAGA9-Lux and myc-Smad3 with or without myc-TGIF1. Relative luciferase activity was measured 24 h after irradiation and/or treatment with 10 ng/mL of TGF-β1. (C) HUVECs were transfected with wt-PAI-1 Luc reporter or CAGA box-mutated PAI-1 Luc reporter (Δ123-PAI-1 Luc) and myc-Smad3 with or without myc-TGIF1. Luciferase activity was assayed 24 hours after irradiation. Relative luciferase activity is the mean ± SEM of at least 3 independent experiments realized in triplicates (D) HUVECs were transfected for 24 h with myc-Smad3 with or without myc-TGIF1. Cells were irradiated at 10 Gy and PAI-1 expression was measured 24 h after irradiation. E-G) HUVECs were transfected with non–targeting siRNA (siRNA control) or siRNA TGIF1 for 24 h and irradiated at 10 Gy. Silencing efficiency was confirmed by western-blot (E). mRNA level (F) and protein levels of PAI-1 and Phospho-Smad3 were performed by western-blots (G). Results are the mean +/- SEM of two independent experiments realized in triplicates and representative blots from three independent experiments are shown. *p<0.05