FIG. 1.

PRDX1 binds to FOXO3 in an H₂O₂ dose-dependent manner and regulates FOXO3 nuclear localization. (A) Immunoprecipitation of precleared lysate with PRDX1 or IgG antibodies found PRDX1 bound FOXO3. 293T cells underwent serum starvation for 30 min and were then treated with the 0 or 100 μM H₂O₂ for an additional 30 min. (B and C) 293T cells were transfected with pcDNA3-FLAG-FOXO3A or EV and treated with increasing concentrations of H₂O₂ for the indicated times. Before lysis, cells were washed with 20 mM NEM in phosphate-buffered saline to block lysis-induced disulfide bond formation. FLAG-labeled proteins were immunoprecipitated and detected by immunoblot with FLAG and PRDX1 antibodies. (D and E) The percentage of 293T cells displaying nuclear FOXO3-EGFP localization was enhanced with reduction of PRDX1; 150 or more cells analyzed per sample, p < 0.0001 (t-test). Cells infected with shPRDX1A or control lentivirus for 48 h, followed by transfection with FOXO3-EGFP for 24 h. H₂O₂ was added during the last 30 min. (F) HA-PRDX reduced FLAG-FOXO3 activity when transiently cotransfected into MEF and analyzed utilizing a dual-luciferase reporter assay. Values (mean + SE) were normalized to vehicle treatment. *p < 0.05, t-test (N = 3). (G) qPCR gene transcription of SESN3 and P27 was increased in PRDX1-deficient 293T cells (white bars) treated with 250 μM H₂O₂ compared to pLKO.1 control cells (black bars) after 16 h. Values (mean + SE) were normalized to vehicle treatment. *p < 0.05, t-test (N = 3). MEF, murine embryonic fibroblast; NEM, N-ethylmaleimide; SE, standard error.