Fig. 1: Mst1 phosphorylates FoxO1, thereby enhancing nuclear translocation of FoxO1.

a Cardiomyocytes treated with Ad-Mst1 or Ad-LacZ in the presence or absence of H₂O₂ (10 μM) were stained with a FoxO1 antibody (green), a troponin T antibody (red), and 4′,6-diamidino-2-phenylindole (DAPI; blue). b Coimmunoprecipitation assays with myocyte lysates. After immunoprecipitation with control IgG or an Mst1 antibody, immunoblots for endogenous FoxO1 were performed. c Left: interaction between Mst1 and FoxO1 was examined by pull-down assays with indicated recombinant proteins. Middle: Coomassie brilliant blue (CBB) staining of the gel after SDS–PAGE. Right: diagrams of mouse FoxO1 recombinant proteins (full-length and partial) used as preys of pull-down assays or substrates of in vitro kinase assays. DBD DNA-binding ___domain, NLS nuclear localization signal, NES nuclear export signal, TA transactivation ___domain. d Left: in vitro kinase assays were carried out by incubating recombinant Mst1 with recombinant mouse FoxO1 proteins (full-length and partial) as substrates. Right: CBB staining of the gel after SDS–PAGE. e Upper: alignment of sequences in the forkhead (DNA-binding) ___domain of mouse/human FoxO1 and FoxO3, Drosophila dFOXO, and C. elegans DAF-16. Lower: cardiomyocytes treated with adenoviruses harboring GFP-tagged wild-type FoxO1 or FoxO1-Ser209/Ser215/Ser218/Thr228/Ser232/Ser243A (PR) mutant in the presence or absence of Mst1 were stained with a GFP-tag antibody (green), a troponin T antibody (red), and DAPI (blue). Left: representative immunostaining pictures. Yellow arrows indicate nuclei-localized FoxO1 and blue arrows indicate cytosol-localized FoxO1 in cardiomyocytes. Right: the results of quantitative analyses (n = 6). f Myocardial sections of the ischemic border zone from indicated mice with or without I/R were stained with anti-FoxO1 antibody (green), anti-troponin T antibody (red), and DAPI (blue). FoxO1-positive nuclei were counted in the LV tissues. Upper: representative pictures of immunostaining in the LV tissues. Lower: the results of quantitative analyses (n = 3). All experiments were repeated at least three times, with n representing biologically independent replicates. P values were determined by two-sided unpaired Student’s t test in (e) or one-way ANOVA followed by Tukey’s multiple comparison test in (f). Data are mean ± SEM. Source data are provided as a Source Data file.