Journal: PloS one

Article Title: Angiogenesis impairment in diabetes: role of methylglyoxal-induced receptor for advanced glycation endproducts, autophagy and vascular endothelial growth factor receptor 2.

doi: 10.1371/journal.pone.0046720

Figure Lengend Snippet: Figure 2. Knockdown of Glo1 enhances VEGFR2 reduction induced by MGO, whereas Glo1 overexpression prevents the reduction. A–B: MGO did not affect cell viability or apoptotic markers. BAEC were incubated with MGO at the indicated concentrations (for 24 h) or time course (up to 24 h) followed either by viability measurement (MTT assay) or Western blot staining for markers of apoptosis with respective antibodies against Bcl-2, Bax and Caspase 3. C: Knockdown of Glo1 by siRNA partly mimicked the MGO effect and enhanced MGO-induced VEGFR2 reduction. Transfection of control or Glo1 siRNA was performed based on protocols provided by Santa Cruz Biotechnology (Santa Cruz, CA). D: MGO increased levels of MGO-protein adduct which were further enhanced by siRNA knockdown of Glo1. E: Overexpression of Glo1 blocked MGO-induced VEGFR2 reduction. Infection of either control or Glo1 containing plasmid DNA was performed according to the protocol provided by OriGene (Rockville, MD). F: Overexpression of Glo1 blocked MGO-increased MGO-protein adducts. G: Knockdown of RAGE by siRNA prevented MGO-induced VEGFR2 reduction. Transfection of control or RAGE siRNA was performed based on protocols provided by Santa Cruz Biotechnology (Santa Cruz, CA). Western blot was performed with indicated antibodies. All blots shown are representative of three independent experiments. *P,0.05 vs control (n = 3). NS: not significant vs control (at 0 mM of MGO). Casp-3: Caspase-3; c-Casp-3: cleaved Caspase-3. doi:10.1371/journal.pone.0046720.g002

Article Snippet: Transfection of control or Glo1 siRNA was performed based on protocols provided by Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Knockdown, Over Expression, Incubation, MTT Assay, Western Blot, Staining, Transfection, Control, Infection, Plasmid Preparation

Journal: PloS one

Article Title: Angiogenesis impairment in diabetes: role of methylglyoxal-induced receptor for advanced glycation endproducts, autophagy and vascular endothelial growth factor receptor 2.

doi: 10.1371/journal.pone.0046720

Figure Lengend Snippet: Figure 3. Inhibition of autophagy, but not proteasome or caspase, abolishes the reduction of VEGFR2 protein levels and angiogenesis by MGO. A–E: Suppression of autophagy, but not proteasome and caspase, prevented VEGFR2 reduction induced by MGO. (A–C and E) One hour prior to MGO challenge (25 mM for 16 h), BAEC were pre-incubated respectively with chloroquine (CQ: 100 mM), pepstatin A (Pep A: 10 mM), bafilomycin A1 (Baf A1: 5 nM), MG132 (0.5 mM), epoxomicin (Epo: 0.5 mM), lactacystin (Lact: 1 mM), and z-VAD-fmk (z-VAD: 20 mM); (D) Before MGO treatment (as above), BAEC were transfected either with control siRNA or siRNA targeting Beclin-1, based on instructions from Santa Cruz Biotechnology (Santa Cruz, CA); all cell lysates were subjected to Western blot with indicated antibodies. All blots shown are representative of three independent experiments. *P,0.05 vs control (n = 3). F: Administration of autophagy inhibitor rescued MGO-impaired tube formation. One hour before MGO stimulation (25 mM), endothelial cells were incubated respectively with CQ (100 mM), Pep A (10 mM), Baf A1 (5 nM) and subjected to tube formation assay. G: Knockdown of Beclin 1 by siRNA prevented MGO-reduced tube formation. All images presented are representative of three independent experiments. *P,0.05 vs control (n = 3). NS: not significant vs control. doi:10.1371/journal.pone.0046720.g003

Article Snippet: Transfection of control or Glo1 siRNA was performed based on protocols provided by Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Inhibition, Incubation, Transfection, Control, Western Blot, Tube Formation Assay, Knockdown