Fig. 2
The protective effect of an A2AR agonist is mediated by TRAX. a-c PC12-pSuper and PC12-shTRAX cells were treated with an A2AR agonist (CGS21680, CGS; 10 μM) or vehicle for 1 h to activate the A2AR and then treated with H2O2 (100 μM) for 4 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX, anti-TRAX and anti-α-Tubulin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times (a). The extent of DNA damage was analyzed via the neutral comet assay. The mean tail moment was quantified with COMETscore.v1.5 software, scale bar, 50 μm (b). Apoptosis was assessed with the Annexin V apoptosis detection kit. Cells were co-stained with Annexin V-FITC and PI for 10 min followed by flow cytometric analysis. These experiments were repeated three times (c). d, e Primary hippocampal neurons (DIV14) from TRAX-WT and TRAX-null mice were treated with CGS (10 μM) or vehicle for 1 h and then treated with H2O2 (100 μM) for 2 h. DNA damage was assessed by determining the number of DNA foci per cell by immunofluorescence staining using the anti-γH2AX antibody (green) in neurons identified by a neuronal marker (TUJ1, red). The percentage of cells with >5 γH2AX foci per cell in at least 100 cells were determined in each condition. Scale bar, 10 μm. f Human MSN neurons were infected with lentivirus expressing TRAX shRNA or control shRNA for 3 days. Human MSN neurons were treated with an A2AR agonist (CGS21680, CGS; 10 μM) or vehicle for 1 h to activate the A2AR and then treated with H2O2 (100 μM) for 4 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX, anti-TRAX and anti-β-Actin antibodies as indicated. The amount of target protein was quantified and normalized to that of β-Actin, the loading control. These experiments were repeated three times. Data are presented as the mean ± SEM from at least three independent experiments. *P < 0.05, **P < 0.01 compared to control
