Extended Data Fig. 8: Removal of VEGF at day 7 of differentiation increases expression of aortic pre-hemogenic endothelial genes.
(a) Flow cytometry analysis of differentiating PB5.1 cells showing the increase in CD34+CXCR4+CD73lo/+ arterial cells in response to VEGF at 150 ng ml-1 (V150) from d3–7. (b) Continuing VEGF maintains CXCR4 expression. (c) VEGF removal leads to CXCR4 downregulation. (d) Negative control samples unstained for CD34 or for CXCR4 and CD73. (e) Real time PCR analysis of differentiated samples of PB5.1 analyzed from d5–11 for the indicated arterial endothelium, retinoid signaling and hematopoietic genes. Samples with continued VEGF signaling are compared to samples where the VEGF was removed after d7. Error bars, mean ± s.e.m., n = 3 independent experiments. V150 vs V150 d3-7 at d11: AGTR2, P = 0.0125; IL33, P = 0.0307; RUNX1, P = 0.0009; HLF, P = 0.0100; mixed-effects analysis (two-way ANOVA) with Sidak’s multiple comparisons test.
