Fig. 3: Mutation of the human LPAR4 promoter region to which human SOX17 binds and a gain or loss of function study for SOX17.

a Schematic figure for SOX17 binding site mutations in the LPAR4 promoter region. A point mutation (C to A) in AACAAG(T), the consensus sequence that binds SOX17, was performed. Based on the ChIP assay results, mutation (mut.) sites 1 and 3 were used as negative controls, and site 5 was excluded because these sites do not bind SOX17, whereas mut. sites 2 and 4 are sites known to bind SOX17. A luciferase assay was performed with these point mutation constructs. b Activation of the human LPAR4 gene promoter in hiPSC-derived cells at cardiac differentiation Day 4 using the luciferase assay. ns; not significant, ***p < 0.001. All experiments were conducted at least in triplicate. c Real-time PCR analysis of mRNA expression levels of SOX17, LPAR4, and the early cardiac markers MESP1 and Nkx2.5 after cardiac differentiation using SOX17 knockdown (using SOX17 siRNA) hiPSCs. Statistical analyses were performed using one-way ANOVA (Newman‒Keuls). **p < 0.05, ***p < 0.001. d Real-time PCR analysis of mRNA expression levels of SOX17, LPAR4, Nkx2.5, and the mature cardiac marker αMHC after cardiac differentiation using SOX17-overexpressing hiPSCs (SOX17 OE). One-way ANOVA (Newman‒Keuls) was performed to assess significant differences (*p < 0.01, ***p < 0.001). All experiments were conducted at least in triplicate.