ROS and superoxide generation in HUVECs exposed to high glucose.

<p>NG, constant normal glucose (5 mM); HG, constant high glucose (22 mM); and N/HG, 5 mM alternating with 22 mM glucose. (A) Intracellular ROS was measured by visualizing the use of the fluorescent probe CM-H₂DCFDA. (B) Superoxide was detected via DHE and was analyzed using flow cytometry. (C) Expression of p22^<i>phox</i> protein levels. In the top, typical Western blots are shown. β-Actin served as loading control. (D) Transfection of p22^<i>phox</i> siRNA effectively eliminated p22^<i>phox</i> protein expression. (E) Transfection of p22^<i>phox</i> siRNA negated the increase in superoxide production in the fluctuating-glucose condition. (F) Transfection of p22^<i>phox</i> siRNA blunted the fluctuating glucose-induced SA-β-gal activity. (G) Transfection of p22^<i>phox</i> siRNA blunted DNA damage of APsite. The values of the three independent experiments are mean ± S.D. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001 vs. NG; ###<i>p</i><0.001 vs. HG.