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. Author manuscript; available in PMC: 2017 May 1.
Published in final edited form as: DNA Repair (Amst). 2016 Mar 29;41:32–41. doi: 10.1016/j.dnarep.2016.03.009

Figure 5.

Figure 5

Nicorandil and/or daidzin do not inhibit APE1 redox signaling activity in a cell-based reporter transactivation or redox EMSA assays. Pa02c cells were cotransfected for 24 hours (37°C, 5% CO2) with AP-1 Luciferase Firefly vector and control pRL Renilla-TK vector in a 20:1 ratio, respectively, using 3.0 μL/mL of Lipofectamine®2000 (Invitrogen). Following media exchange and 24 hour recovery, cells were treated in doses for 24 hours of either E3330, Nicorandil (Sigma-Aldrich), daidzin (Indofine Chemical), or nicorandil with 10 μM daidzin. Firefly luciferase and Renilla activities were measured using the Promega Dual-Luciferase®system. For each dose, AP-1 luciferase RLU was normalized with Renilla-TK RLU, and then further normalized to media response. Data shown is percent normalized to media with mean standard error and includes vehicle (DMSO) response. Experiments were done in duplicate and repeated twice. In a dose-dependent manner, the APE1 redox inhibitor E3330 (A) reduced AP-1 activity as expected (50% response = 22.9 μM), while nicorandil (B), daidzin (C), and nicorandil with 10 μM daidzin/dose (D) did not elicit any APE1 redox inhibition. In a redox EMSA assay (E), E3330 (positive control) blocked APE1 redox signaling function while nicorandil had no effect.