BioMed Research International / 2017 / Article / Fig 5

Research Article

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

Figure 5

Activated ERK5 is essential for low-concentration H2O2-induced HUVEC angiogenesis. (a) Cell proliferation assessed by CCK-8 assays. Cells were treated with PBS or H2O2 (50 μmol/L) for 30 min. Then, 10 μl CCK-8 was added. The OD value at 450 nm was measured 2.5 h later (mean ± SD of six separate experiments, , and one-way ANOVA followed by SNK post hoc test). (b, c) Cell migration assessed by scratch wound assays. Cells were seeded at confluence and cultured overnight. Following treatment with PBS or H2O2 (50 μmol/L) for 30 min, a scratch was made. The wounded cell monolayers were then incubated with medium containing 2% FBS for 12 h. Photographs were taken at 0 h and 12 h at fixed locations along the scratch, and closure of the wound was quantified (mean ± SD of six separate experiments, , 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 or 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, , and one-way ANOVA followed by SNK post hoc test). (g) VEGF secretion assessed by ELISA. Cells were treated with PBS or H2O2 (50 μmol/L) for 30 min; then the medium was changed to fresh normal medium. After 12 h or 24 h culture, the cell supernatant was collected, and the concentration of VEGF was determined by a human VEGF ELISA kit (mean ± SD of six separate experiments, , and one-way ANOVA followed by SNK post hoc test).
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