Figure 6. Syringaresinol induces FOXO3 nuclear accumulation, upregulation of FOXO3 target genes expression, and decreased production of ROS.

H9c2 cells were treated with syringaresionl for 24 h. (A) Cellular localization of FOXO3 were determined by immunofluorescence. Cells were fixed and stained with anti-FOXO3 (green) and DNA was stained by 4′,6-diamidino-2-phenylindole (DAPI) (blue). Bar: 50μm. (B) Equal cell equivalents of nucleus and cytosol were separated by gel electrophoresis and processed for Western blotting with the indicated antibodies. (C) Levels of phosphorylation of FOXO3 were examined by Western blotting. (D) The expression of the indicated FOXO3 target genes were measured by real time PCR. The levels were quantified and normalized by GAPDH. (E) H9c2 cells were pre-treated with increasing concentrations of syringaresinol (0, 2, 10, 25, 50 and 1000 μM) for 9 h and then exposed to 150 μM H₂O₂ for 6 h. Cellular ROS levels (percentage compared to Ctrl) were analyzed by carboxy-H₂DCFDA staining followed by flow cytometry. (F) H9c2 cells were treated with 25 μM Syringaresinol for 15 min or 9 h prior to incubation with or without 150 μM H₂O₂ for 6 h. γH2AX formation (the phosphorylated form of H2AX) was examined by Western blotting with anti-γH2AX antibody. For positive control, cells were treated with indicated concentration of etoposide. (G) H9c2 cells were transfected with control or FOXO3 siRNA. After 24 h cells were pre-treated with or without 25 μM syringaresinol for 9 h and then exposed to 150 μM H₂O₂ for 6 h. Cellular ROS levels (percentage compared to Ctrl) were analyzed by carboxy-H₂DCFDA staining followed by flow cytometry. (H) ROS levels in H2c9 exposed to normoxia, SYR, H/R or H/R+SYR were determined by DCFDA staining followed by flow cytometry. All results are representative or means ± SD of six independent experiments. #P < 0.001 versus control group *P < 0.001 versus H₂O₂ treatment group. Ctrl:control; H/R: hypoxia/reoxygenation; SYR: syringaresinol.