NOTCH4in28 is required for arterial specification. (A) Representative dot plots show impaired expression of arterial markers in D5 HE in generated from 2 NOTCH4^{Δin28−/Δin28−} hPSC clones (C2^–/– and C3^–/–). (B-C) Graphs show the percentages VEC⁺ cells, and HE with arterial phenotype in D5 differentiation cultures, respectively (means ± SDs, n = 4 experiments); ∗∗∗∗P < .0001, 1-way ANOVA, Dunnett multiple comparisons test (B); and ∗P < .05 and ∗∗∗∗P < .0001, 2-way ANOVA, Tukey multiple comparisons test (C). (D) quantitative reverse transcription PCR (qRT-PCR) analysis compares the expression of indicated genes in D5 HE generated from WT and NOTCH4^{Δin28−/Δin28−} hPSC clones. (E) Representative dot plots show impaired expression of arterial markers in D5 HE from DOX-treated cultures of differentiated iSOX17/NOTCH4^{Δin28−/Δin28−} hPSC clones (iSOX17C18^–/– and iSOX17C48^–/–). (F-G) Graphs show the percentages of VEC⁺ cells and HE with arterial phenotype in D5 differentiation cultures treated with DOX; ∗∗∗P < .001, 2-way ANOVA, Tukey multiple comparisons test. (H) Representative dot plots and (I) histograms show flow cytometric analysis of NOTCH4 expression in D5-gated HE cells from iSOX17 hPSC with intact and deleted NOTCH4in28. (J) Mean fluorescence intensity of NOTCH4 expression in D5 HE. (K) Percentages of NOTCH4⁺ and NOTCH4^– cells within DLL4^+/− populations. (L) qRT-PCR analysis of NOTCH pathway-associated genes (HEY1, NOTCH4, NOTCH1, DLL4, and CXCR4) in D5 HE from DOX-treated culture (means ± SDs, n = 9 experiments); ∗∗∗∗P < .0001, t test. dMFI, delta mean fluorescence intensity; ns, not significant.