Fig. 1.

NOTCH activation increases hematopoiesis from D4 HE. a NOTCH1 receptor expression is first detected on D4 CD144⁺ cells. DLL4 expression is first detected on D5 CD144⁺ endothelial cells. b Schematic diagram of experiments. Cells were differentiated for 4 days on collagen IV, D4 CD144⁺CD43⁻CD73⁻ HE cells were purified using CD31 microbeads and plated in 3 different NOTCH conditions. c Western blot of D4 HE cultured for 24 h (D4 + 1) in presence of DAPT or DLL1-Fc shows a decrease in the activated cleaved form of NOTCH1 in DAPT-treated cells, and an increase in the activated cleaved form of NOTCH1 in cells plated on DLL1-Fc. d qPCR analysis shows decreased HES1 mRNA expression in D4 HE cultured for 12 h (D4 + 0.5) with DAPT, while HES1 mRNA expression is increased in cells plated on DLL1-Fc. Results are mean ± s.e.m. for three independent experiments; two-way ANOVA Bonferroni post-hoc test, ***p < 0.01. e Flow cytometry on each day from D4 + 1 to D4 + 4 shows decreased CD43⁺ HPs in the cultures treated with DAPT, and increased HPs in the cultures plated on DLL1-Fc. Representative contour plots from three independent experiments are shown. f Total numbers of CD43⁺ HPs and CD144⁺CD43⁻ endothelial cells in cultures plated on DLL1-Fc. Results are mean ± s.e.m. for three independent experiments; two-way ANOVA Bonferroni post-hoc test, ***p < 0.001