FIG. 4.

dRP acts intracellularly following its internalization by the transporter GLUT1. (A) LC-MS detection of intracellular dRP. HUVECs were treated with 200 μM dRP, before three washes in PBS and ultrasonication. Example chromatogram (upper panel) and data quantification (lower panel) are shown. Statistical significance was tested by t-test (*p < 0.05, compared with vehicle, n = 4). (B) STF-31 and fasentin (10 μM) inhibited dRP-dependent, but not VEGF-dependent, tube formation. Example pictures (upper panel) and data quantification (lower panel) are shown. Bar graphs represent quantification of tube number per optical field compared by one-way ANOVA with Bonferroni post hoc test (*p < 0.05 compared with vehicle, n = 5). Bar: 300 μm. (C) siRNA-dependent silencing of GLUT1 inhibits dRP-induced ROS generation. Following GLUT1 silencing displayed in top panels, ROS was measured as described over a period of 2 h. Time courses were analyzed by two-way ANOVA (n = 4) with Bonferroni post hoc test (*p < 0.05, compared with vehicle/scrambled siRNA; **p < 0.05 compared with dRP/scrambled siRNA). (D) siRNA-dependent silencing of GLUT1 inhibits dRP-induced tube formation. Example pictures for dRP response by HUVECs treated with scrambled siRNA or GLUT1 siRNA are shown in top panels. Bar graphs represent quantification of tube number per optical field compared by one-way ANOVA with Bonferroni post hoc test (*p < 0.05 compared with scrambled siRNA/vehicle, **p < 0.05 compared with scrambled siRNA/dRP, ns, nonsignificant, n = 4). Bar: 300 μm. GLUT1, glucose transporter 1; PBS, phosphate-buffered saline; VEGF, vascular endothelial growth factor. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars