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. 2022 Dec 30;132(2):187–204. doi: 10.1161/CIRCRESAHA.122.321398

Figure 1.

Figure 1.

Generation of NOTCH1 knockout (N1KO) human induced pluripotent stem cells (iPSCs) and endothelial differentiation. A, Schematic overview of deleting a 2.4-skb segment including exon 1 and exon 2 in the NOTCH1 gene using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 genome editing. B, Quantitative PCR (qPCR) analysis shows mRNA levels in N1KO iPSCs (t test, n=3). C, Western blot indicates the absence of NOTCH1 in the homozygous N1KO iPSCs at the protein level. GAPDH is used as an internal control. D, Endothelial differentiation efficiency measured by the percentage of CD31+ cells at D10 of differentiation (1-way ANOVA, n=6). E, Expression of endothelial markers CD31 and CD144 in wild-type (WT) and N1KO iPSC-ECs. F, qPCR analysis shows reduced expression of arterial endothelial genes (DLL4 and EFNB2) and enhanced expression of venous endothelial genes (NR2F2 and EPHB4) in homozygous N1KO iPSC-ECs. G, qPCR data indicate downregulation of NOTCH1, NOTCH4, HEY1, and HEY2 in N1KO compared with WT iPSC-ECs (t test, n=3). All bar graphs show mean±SEM. *P<0.05, **P<0.01, ***P<0.001. Scale bars = 50 μm. EC indicates endothelial cell.