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. 2010 Apr 19;189(2):325–338. doi: 10.1083/jcb.200904114

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© 2010 Yamamizu et al.

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Figure 3. — Arterial EC induction by dual activation of β-catenin and Notch signaling. (A) Experimental system for dual activation of Notch and β-catenin signaling. ES cell lines carrying CA-β-catenin regulated by Tet-Off system, and a fusion protein of N1ICD and estrogen receptor (ER), NERT^ΔOP, were established. CA-β-catenin was induced by depletion of Dox, and Notch activation was induced by nuclear translocation of NERT^ΔOP with addition of 4-hydrotamoxifen (OHT). 1 µg/ml Dox was added during the first 4.5 d of culture of ES cell differentiation to Flk1⁺ cells. After Flk1⁺ cells were sorted by MACS and plated on type IV collagen-coated dishes, cells were treated with or without Dox and/or OHT (150 ng/ml). (B and C) Activation of β-catenin together with VEGF. (B) Double-fluorescent staining for CD31 and ephrinB2 at Flk-d3. Left panels, Dox treatment. Right panels, Dox free (expression of CA-β-catenin). (C) Flow cytometry for CD31 and CXCR4 expression at Flk-d3. Percentages of CXCR4⁺/CD31⁺ arterial ECs and CXCR4⁻/CD31⁺ venous ECs in total ECs (CD31⁺ cells) are indicated. (D–F) Dual activation of Notch and β-catenin signaling. (D) Double-fluorescent staining for CD31 and ephrinB2 at Flk-d3. Top panels, CD31 (pan-ECs, red) and DAPI (blue). Bottom panels, EphB4-Fc (ephrinB2⁺ arterial ECs, green) and DAPI (blue). Flk1⁺ cells were treated with VEGF alone (50 ng/ml), together with Dox⁺ (control), Dox⁺/OHT⁺ (Notch activated), or Dox⁻/OHT⁺ (dual activated) condition. VEGF and 8bromo-cAMP (0.5 mM) treatment in Dox⁺ condition is shown as positive control. Bars: 100 µm. (E) Flow cytometry for CD31 and CXCR4 expression. Percentages of CXCR4⁺/CD31⁺ arterial ECs and CXCR4⁻/CD31⁺ venous ECs in total ECs (CD31⁺ cells) are indicated. (F and G) Expression profile of CXCR4 in CD31⁺ ECs by flow cytometry. Percentages of CXCR4⁺ arterial ECs in total ECs are indicated. (F) VEGF treatment alone (blue line), VEGF and 8bromo-cAMP (red line), and VEGF together with dual activation of β-catenin and Notch activation (Dox⁻, OHT⁺; green line) are shown. (G) VEGF treatment alone (blue line), VEGF and 8bromo-cAMP (red line), VEGF, 8bromo-cAMP, and LY294002 (7.5 µM; left panel), DAPT (2.5 µM; right panel, orange line), VEGF, 8bromo-cAMP, and LY294002 (left panel), or DAPT (right panel) together with dual activation of β-catenin and Notch activation (Dox⁻, OHT⁺; green line) are shown.