Extended Data Fig. 8: HGE3 KO and HGE TKO human pluripotent stem cell knockout lines display reduced cardiomyocyte differentiation and mesoderm-specific ZIC3 expression defects.
From: Human-gained heart enhancers are associated with species-specific cardiac attributes
All individual PCR data for WT and ZIC3 HGE knockout (KO) and HGE triple knockout (TKO) lines are shown with statistical significance indicated (ZIC3, n= 9 for all lines; HOPX, n = 9 for WT/HGE1-3 KO lines, n = 12 for WT/HGE TKO lines; APLNR, n = 6 for WT/HGE1 and HGE2 KO lines, n = 9 for WT/HGE3 KO lines, n = 12 for WT/HGE TKO lines; NODAL, n = 6 for WT/HGE1 and HGE2 KO lines, n = 12 for WT/HGE3 KO and HGE TKO lines; LEFTY2, n = 6 for WT/HGE1 and HGE2 KO lines, n = 9 for WT/HGE3 KO lines, n = 12 for WT/HGE TKO lines. TBP gene expression was used for gene expression normalization. b, qPCR analysis of ZIC3 HGE3 KO and HGE TKO human PSC lines reveals no significant changes in ZIC3 expression at the PSC stage compared to WT cells (n = 9). c, qPCR analysis reveals significant changes in the expression of ZIC3 but not neighboring genes, RBMX and FGF13, in HGE TKO human PSC lines when compared to WT human PSC lines at the mesoderm stage (n = 6). d, Representative examples of flow cytometry analyses for TNNT2 staining (x-axis) show that ZIC3 HGE3 and TKO lines (but not HGE1 nor HGE2) display reduced cardiac differentiation. Data is represented as the mean ± SEM with individual data points. Data was analyzed using a two-tailed Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. HGE, human-gained enhancer; expr., expression; PSC, pluripotent stem cells; Mes, mesoderm.
