PMC

Effects of SOD on tube formation in irradiated HUVECs. HUVECs were irradiated with 20 Gy and then cultured on Matrigel® without (control) or with 5 units/ml of SOD for 16 h. For quantification of tube formation, the number of endotubes was counted in each microscopic field, and data from 4 fields of each well were analyzed. The number of endotubes in control was assumed to have 100% activity. Data represent mean ± SE from 3 experiments. (A) Representative photographs of capillary-like tube structures on Matrigel. (B) Quantification of numbers of endotubes in irradiated HUVECs. *p = 0.0164, **p = 0.0456,***p = 0.0198, ****p = 0.0139, *****p = 0.0399.

Effects of SOD on survival of HUVECs after irradiation. HUVECs were cultured with 50 units/ml of SOD for 1 h and then irradiated with γ-ray (8 Gy/min) in the presence of SOD (squares, SOD + Irradiation). In parallel, cells were irradiated and then SOD was added to cell cultures (triangles, Irradiation + SOD). As control, cells were exposed to irradiation alone (circles, control). Colonies were stained with Giemsa 13 days after irradiation, and those having over 50 cells were counted. Results are presented as the mean of 5 separate experiments. Plating efficiencies of untreated control cells and SOD-treated cells were 0.027 ± 0.005 and 0.027 ± 0.004, respectively.

Effects of SOD on phosphorylation of ERK1/2 in irradiated HUVECs. (A) Effects of irradiation on phosphorylated ERK1/2. HUVECs were irradiated with 20 Gy and cultured for different durations. Cells were sequentially harvested at indicated times and subjected to western blot analysis using anti-ERK1/2 (ERK) or phospholylated ERK1/2 (p-ERK1/2) antibody. Levels of β-actin were used as loading control. (B) and (C) Effects of SOD on phosphorylated ERK1/2. Cells were treated with either 5 or 50 units/ml of SOD. Levels of phospholylated form of ERK1/2 were determined by western blotting. (D) Effects of SOD treatment either before or after irradiation on phosphorylation of ERK1/2 in irradiation. HUVECs were pre-treated with SOD (50 units/ml) for 1 h and then irradiated with 20 Gy in the presence of SOD (SOD + Irradiation), or they were irradiated and then SOD was added to cell cultures 30 min later (Irradiation + SOD). After culturing with SOD for 4 h, both groups of cells were subjected to western blot analysis. For inhibition of the MEK pathway, cells were treated with 50 µM of PD98059 for 30 min and then irradiated and/or treated with SOD.