Figure 8.

Effect of FoxO1 on β-cell antioxidative function. A, Effect of FoxO1 on β-cell antioxidative gene expression. Islets from FoxO1-tg and WT littermates (male, 10 wk old, n = 6/group) were subjected to real-time qRT-PCR analysis for determining Sod1, Cat, Gpx1, and FoxO1 mRNA levels. B, Effect of FoxO1 on antioxidative gene expression in INS-1 cells. INS-1 cells were transduced with Adv-Empty and Adv-FoxO1 vectors (100 pfu/cell). C, Immunoblot of INS-1 cells pretransduced with Adv-Empty and Adv-FoxO1 vectors. D, Effect of FoxO1 on antioxidative protein levels in INS-1 cells. After a 24-hour incubation, cells were subjected to real-time qRT-PCR and immunoblot assays. E, The mouse Sod1 promoter. F, ChIP analysis of FoxO1-Sod1 promoter association. G, The mouse Cat promoter. H, ChIP analysis of FoxO1-Cat promoter interaction. Pancreatic islets were subjected to ChIP assay using anti-FoxO1 or anti-β-galactosidase IgG for determining FoxO1-bound DNA. As off-target control, immunoprecipitated DNA was analyzed by real-time PCR, using primers that are complementary to the mouse Sod1 or Cat coding region. I, Sod1 promoter activity. J, Cat promoter activity. The mouse Sod1 (−880/+1 nt) or Cat (−755/+1 nt) promoter was cloned into the pGL3-basic luciferase reporter system for determining Sod1 or Cat promoter activity in INS-1 cells that were pretransduced with Adv-FoxO1 or Adv-Empty vector (100 pfu/cell). K, Effect of FoxO1 on apoptotic gene expression in INS-1 cells. INS-1 cells pretransduced with Adv-FoxO1 and Adv-Empty vectors were subjected to H₂O₂ treatment, followed by real-time qRT-PCR analysis of prosurvival and proapoptotic gene expression. Data were from 3–5 independent experiments. *, P < .05 and **, P < .001 vs control.