Autophagy of VEGFR2 Impairs Diabetic Angiogenesis Figure 5. MGO increases markers of autophagy through ONOO2 generation. A–C: The MGO-increased autophagy marker (Beclin 1) could be prevented by blocking ONOO2 formation. BAEC were incubated respectively with mTempol (1 mM for 1 h), L-NAME (1 mM for 1 h), and UA (100 mM for 1 h) prior to MGO (25 mM, 1 h) incubation. D: ONOO2 per se induced both autophagy markers. BAEC were incubated with ONOO2 (100 mM) for up to 8 h. Cells were collected for Western blotting with a rabbit derived Beclin-1 (or LC3B) antibody and a mouse derived b-actin antibody. All blots shown are representative of three independent experiments. *P,0.05 vs control (n = 3). NS: not significant vs control. doi:10.1371/journal.pone.0046720.g005 found more in cytosol than in the cell membrane. MGO incubation reduced the staining in cytosol and membrane. However, pre-incubation of either mTempol or CQ restored the staining of VEGFR2 protein levels (Fig. 7B/C). In any cases, the nuclear VEGFR2 staining is not evident (Fig. 7A/B), although a functional nuclear localization of VEGR2 is recently character- ized with reporter assays [59]. Incubation of MGO with Mouse Aortas ex vivo Reduces Angiogenesis, which can be Abolished by Autophagy Suppression To replicate the findings from cell culture study in a more physiologically relevant setting, we first performed ex vivo experiments in which the aortic rings from normglycemic mice (C57BL/6J) were challenged with MGO, with or without autophagy suppression and subjected to angiogenesis assessment by aortic ring assays. As presented in Fig. 8, MGO vs vehicle PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e46720