Fig. 6: UCE PAX6_Ta regulates cardiomyocyte differentiation in hESCs.
a, KEGG pathway enrichment analysis of all genes differentially regulated in UCE PAX6_Ta KO cells. The y axis represents the significantly enriched KEGG pathways, and the x axis represents their corresponding gene ratios, that is, the ratio between the number of unique genes in a specific pathway and the number of unique genes mapped to all of these pathways. Left: significantly activated pathways. Right: significantly suppressed pathways. The dot size denotes the number of genes mapped in a pathway, and the colour indicates the significance level of the enrichment. b, Silencer activities were measured by the repressive ability on the SC promoter driving the expression of GFP. A previously published silencer served as the positive control (validated silencer). The empty vector (SC promoter) was used as the control to normalize the respective repressive activities (n = 3 independent biological samples; values are shown as mean ± s.d. for each bar; validated silencer ***P = 0.0002 and PAX6_Ta ****P < 0.0001, calculated using two-tailed unpaired t-test). c, Knockout PAX6_Ta in NKX2-5eGFP/w hESCs. PCR results show the removal of PAX6_Ta in NKX2-5eGFP/w hESC PAX6_Ta KO bulk cells (PAX6_Ta_KO) compared to the NKX2-5eGFP/w hESCs (hESC_NKX). The blue and red arrowheads indicate the intact genomic regions and the NCRE deletions, respectively. d, The removal of PAX6_Ta caused a defect in cardiomyocyte differentiation, as measured by NKX2-5/GFP expression. Top: hESC_NKX after differentiation. Bottom: PAX6_Ta_KO after differentiation. Scale bar, 275 μm. e, Quantification of cardiomyocyte differentiation as shown in d using FACS. The cardiomyocyte differentiation efficiency measured by GFP in hESC_NKX was set to 100% (n = 3 independent biological samples; values are shown as mean ± s.d.; PAX6_Ta_KO ***P = 0.0006, calculated using two-tailed unpaired t-test). f, The transcription of gene RCN1 was quantified by qPCR and compared between hESC_NKX and PAX6_Ta_KO cells, before cardiomyocyte differentiation (hESC) and after cardiomyocyte differentiation (hESC-CM) (n = 3 independent biological samples; values are shown as mean ± s.d.; hESC **P = 0.001789 and hESC-CM **P = 0.005731, calculated using two-tailed unpaired t-test).
