Induction of HIF-1α expression in HUVECs drives angiogenesis. HUVECs were treated with DFO (380 μM) and evaluated for HIF-1α expression by Western blot (A) and confocal (B) analyses. In panel A, the breast carcinoma MDAMB 231 cell line was used as a positive control. In panel B, HIF-1α (in red) colocalizes with nuclear DAPI (pseudo-colored in green; top panels); GLUT-1 staining demonstrated the transcriptional activity of HIF-1α (bottom panels). (C) HUVECs seeded on Matrigel layers in the presence or absence of DFO or hypoxia were analyzed for their capacity to form tubule-like structures. VEGF (10 ng/mL) was used as positive control. The contribution of the VEGF/VEGFR₂ autocrine loop to hypoxia-induced tubule formation was assessed by using the blocking anti-VEGFR₂ mAb. After 18 hours of incubation, images were captured under a Nikon TS 100 microscope (original magnification ×10), 3 independent experiments were performed, and structures were counted with Lucia software on 3 different fields (D).