BioMed Research International / 2017 / Article / Fig 2

Research Article

Extracellular Signal-Regulated Kinase 5 is Required for Low-Concentration H2O2-Induced Angiogenesis of Human Umbilical Vein Endothelial Cells

Figure 2

Low concentrations of H2O2 promote HUVEC proliferation, migration, and tubule formation. (a) Cell proliferation assessed by CCK-8 assays. HUVECs were treated with PBS, H2O2 (50 μmol/L), or catalase (5 U/ml) and H2O2 (50 μmol/L) for 30 min. Then, 10 μl CCK-8 was added. The OD at 450 nm was measured 2.5 h later (mean ± SD of six separate experiments, versus control, and one-way ANOVA followed by SNK post hoc test). (b, c) Cell migration assessed by scratch wound assays. HUVECs were seeded at confluence and cultured overnight. Following treatment with PBS, H2O2 (50 μmol/L), or catalase (5 U/ml) and H2O2 (50 μmol/L) for 30 min, a scratch was made. The wounded HUVECs monolayers were then incubated with medium containing 2% FBS for 24 h. Photographs were taken at 0 h and 24 h at fixed locations along the scratch, and the closure of the wound area was quantified (mean ± SD of six separate experiments, versus control, and one-way ANOVA followed by SNK post hoc test). (d, e, f) In vitro tubule formation. Tubule formation was assayed on Matrigel. Cells were pretreated with PBS, H2O2 (50 μmol/L), or catalase (5 U/ml) and H2O2 (50 μmol/L) for 30 min and then seeded onto Matrigel-coated wells in normal medium and cultured for 4 to 8 h. Tubule formation was analyzed by measuring branch length and counting tubule number (mean ± SD of six separate experiments, versus control, and one-way ANOVA followed by SNK post hoc test).
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