Fig. 2.
LNO2 activates Kelch-like ECH-associating protein 1 (Keap1)/nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway in VSMCs. A: quiescent RASMCs were stimulated with LNO2 (2.5 μM), LA (2.5 μM), or ethanol (0.1%) as control solvent for 3 h, and then nuclear translocation of Nrf2 was analyzed as described in METHODS. B: 3× antioxidant response element (ARE)-luc and 3× mutARE-luc reporter vectors were transiently transfected into RASMCs. Cells were then treated with LNO2 (2.5 μM), LA (2.5 μM), or ethanol (0.1%) as control solvent for 24 h, and luciferase activity was measured. Values are means ± SD (n = 6). *P < 0.05 vs. control (−) in the same experimental groups. C: RASMCs were infected with green fluorescent protein adenovirus [Ad-GFP; 10 plaque-forming units (pfu)/cell] and Ad-Keap1 (10 pfu/cell) and then transiently transfected with 3× ARE-Luc. After the transfection (24 h), the cells were treated with or without LNO2 (2.5 μmol/l) for 24 h, and luciferase activity was measured. Values are means ± SD (n = 6). *P < 0.05 vs. Ad-GFP (−). D: RASMCs infected with Ad-GFP (10 pfu/cell) or Ad-Keap1 (10 pfu/cell) were stimulated with LNO2 (2.5 μM), LA (2.5 μM), or ethanol (0.1%) for 1 h, and Nrf2 nuclear protein levels were determined by Western blot. The result is representative of three separate experiments.