AMPK-Dependent Angiogenesis by Nitrated Lipids Figure 6. CaMKK is the upstream AMPKK that mediates OA-NO2-induced AMPK activation. A) HUVECs were treated with STO-609 (1 mM) or KN-93 (3 mM) for 1 h followed by incubation with OA-NO2 for 16 h. AMPK and eNOS phosphorylation and protein expression were assayed as described in Materials and Methods. The blot is representative of three blots obtained from three independent experiments. Lower panels: summary data (*p,0.05 vs. control; #p,0.05 vs. OA-NO2 group; n = 3). B) HUVECs were incubated with CaMKKb-specific siRNA or control siRNA for 48 h and then treated with OA-NO2 for 16 h. After treatment, cell lysates were analyzed for AMPK and eNOS phosphorylation and protein levels. Lower panels: summary data (*p,0.05 vs. control; #p,0.05 vs. OA-NO2 group; n = 3). C) HUVECs were pre-treated with YC-1 (30 mM) for 30 min followed by incubation with OA-NO2 for 16 h. An immunoblot of AMPK precipitated with an anti-CaMKK antibody is shown. The blot is representative of three blots obtained from three independent experiments. Lower panels: summary data (*p,0.05 vs. control; #p,0.05 vs. OA-NO2 group; n = 3). doi:10.1371/journal.pone.0031056.g006 Figure 7. AMPK mediates OA-NO2-induced eNOS phosphorylation and NO production in BAECs. BAECs were treated with (A) different concentrations of OA-NO2 for 16 h or (B) 2.5 mmol/L OA-NO2 for the indicated times. Lysates were analyzed by western blot for the indicated proteins. The blot is representative of three blots obtained from three separate experiments. C) Western blot of phosphorylated AMPK and eNOS in OA-NO2- stimulated BAECs infected with Ad-DN-AMPK (MOI = 50). Non-infected cells or cells infected with Ad-GFP served as controls. For A–C, the corresponding densitometric analyses are shown. *p,0.05 vs. control; #p,0.05 vs. GFP with OA-NO2-treated group. D) NO release by OA-NO2-stimulated BAECs infected with Ad-DN-AMPK (MOI = 50) or Ad-GFP (control). *p,0.05 vs. non-transfected, no OA-NO2 group; #p,0.05 vs. OA-NO2-treated, Ad-GFP group. E) AMPK activity corresponding to C and D above. *p,0.05 vs. no OA-NO2 treatment, Ad-GFP group; #p,0.05 vs. OA-NO2-treated, Ad-GFP group. F) The proposed signaling pathway involved in AMPK/eNOS activation in response to OA-NO2 treatment in endothelial cells. doi:10.1371/journal.pone.0031056.g007 PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e31056