Fig. 2

Effects of Notch1 activation on differentiation of ESC-derived ECs. a Timeline for differentiation of iICN1 ESCs into endothelial cells. b FACS analysis of Flk1-positive cells isolated by magnetic-activated cell sorting. Double staining of cells with PE-conjugated anti-Flk1 and Alexa Fluor® 647 anti-CD31 antibodies. Numbers denote percent of cells per quadrant. c Effects of γ-secretase inhibitor DAPT on endothelial differentiation of Flk1-positive iICN1-ECs. Flk1-positive cells treated 10 μM DAPT for indicated times, and expression levels of endothelial markers (Flk1, CD31, and VE-cadherin), ICN1, and GAPDH determined by western blotting analysis. d Representative photographs of iICN1-ECs after treatment with or without Dox for 3 days. Scale bars: 100 μm. e Effect of ICN1 induction on expression of endothelial markers in Flk1-positive cells. Flk1-positive cells treated with Dox for indicated periods. Representative western blotting data from three independent experiments. f Effects of ICN1 induction on endothelial differentiation of iICN1-ECs. Flk1-positive cells treated with or without Dox for 3 days, and mRNA levels of ICN1, CD31, VE-cadherin, and Flk1 determined by qRT-PCR. mRNA level of each gene normalized to GAPDH, and data represent mean ± SD (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001. bFGF basic fibroblast growth factor, BMP4 bone morphogenetic protein-4, Dox doxycycline, GAPDH glyceraldehyde 3-phosphate dehydrogenase, iICN1 inducible intracellular domain of Notch1, mESC mouse embryonic stem cell, PE phycoerythrin, VEGF vascular endothelial growth factor, DAPT  N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester