Fig. 4: Co-expressed FoxO1 and Mst1 reduce apoptosis of CMs by upregulating antioxidant genes but downregulating pro-apoptotic genes.

a Neonatal rat cardiomyocytes (CMs) were transduced with 10 MOI of adenovirus harboring either LacZ, FoxO1-WT, FoxO1 + Mst1-WT, or FoxO1 + DN-Mst1 in indicated combinations. mRNA expression of FoxO1 target genes was determined by qRT-PCR microarray. Upper: representative scatter plots of qRT-PCR microarray analysis of CMs transduced with indicated adenoviruses. FoxO1-associated genes showing increased expression compared to control are shown as red dots. Genes that are downregulated after transduction of indicated adenoviruses compared to control are shown as green dots. Lower: quantitative analysis of PCR microarray data. Results are presented as fold change compared to control and were calculated using the ΔΔCt method. 18S rRNA was used as a control. n = 3 per group. *P < 0.05 vs. Control group. b The effect of Mst1 on the transcriptional activity of FoxO1. Cultured CMs were transfected with reporter genes containing the FASLG promoter, PMAIP1 promoter, catalase promoter, or PRDX2 promoter, together with either pcDNA3.1-Mst1 or pcDNA3.1-DN-Mst1 at indicated concentrations (n = 3 for FASLG promoter and n = 5 for PMAIP1, catalase, and PRDX2 promoters). * vs. DN-Mst1 plasmid_0 ng of each group. c Mutations were introduced into the luciferase reporter construct of the catalase promoter (Supplementary Fig. 7b) that contains the region −521/+30 of the rat catalase gene. The DAF-16 binding element (DBE: ATAAATA) was mutated to AGCCCTA, CCAAT boxes (CCCAT) were mutated to CTGAT, and the C/EBP-β binding element (CEBE: CTCTTGCCTCACG) was mutated to CTCTCAACTCACG. Cardiomyocytes were transfected with the wild type or mutant catalase promoter reporter construct as illustrated in the left panel, with or without 360 ng of pcDNA3.1-Mst1 plasmid (n = 3–7/group). * wild type catalase promoter_Mst1 (−), † wild-type catalase promoter_Mst1 (+). 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 (a) or one-way ANOVA followed by Tukey’s multiple comparison test in (b, c). Data are mean ± SEM. Source data are provided as a Source Data file.