Figure 6.

High glucose treatment induced nuclei aggregation through foxo1a-klf2a signal in zebrafish embryos. A, Real-time PCR analysis of klf2a expression in control (Ctrl) and AS1842856-treated embryos. Each dot represents data from an independent experiment (n=3). t test. B, Real-time PCR analysis of KLF2 expression in human umbilical vein endothelial cells (ECs) treated with dimethyl sulfoxide (DMSO) and AS1842856. Each dot represents data from an independent experiment (n=3). t test. C, A potential Foxo1 (forkhead box protein O1)-binding sequence presented in JASPAR database. D, Three Foxo1a different candidate binding sites (BSs) upstream of the transcription start site of klf2a in zebrafish. E, Results of the chromatin immunoprecipitation–PCR assay demonstrated that the predicted sequence ATGTAAACATT is a Foxo1a BS of klf2a in zebrafish. Schematic diagram showing the Foxo1a BS in the klf2a promoter region. Input sonicated genomic DNA samples without immunoprecipitation as a positive control. IgG, sonicated, and IgG-immunoprecipitated genomic DNA samples as a negative control. F and G, Luciferase reporter activity in foxo1a overexpressed or knocked down embryos, respectively. Each dot represents data from an independent experiment (n=3). t test. H, Confocal imaging analysis of Ctrl embryos, high glucose–treated embryos, and Tg(hsp70l:mApple-klf2a-DN) and high glucose–treated embryos at 72 hours post-fertilization (hpf). I, Statistical analysis of the EC nuclei nearest neighbor distance (NND) in the Ctrl (n=22), high glucose–treated (n=32), and Tg(hsp70l:mApple-klf2a-DN) and high glucose–treated embryos (n=24) at 72 hpf. One-way ANOVA. Scale bars, 100 µm. DN indicates dominant negative; and PCR, polymerase chain reaction.