Dual up-regulation of Flk1 and NRP1 by PKA activation. (A-D) Two-dimensional culture of Flk1⁺ cells. (A) RT-PCR showing mRNA expression of VEGF₁₆₄, VEGF₁₂₀, Flk1, NRP1, ephrinB2 (arterial marker), and COUP-TF II (venous marker) after 1 (Flk-d1) and 3 (Flk-d3) days of culture of Flk1⁺ cells with DM alone in the presence or absence of Dox (1 μg/mL). (B) Quantitative RT-PCR showing mRNA expression of Flk1, NRP1, VEGF₁₆₄, and VEGF₁₂₀ at Flk-d1 and Flk-d3 in the presence or absence of Dox. mRNA expression at Flk-d1 with Dox was set as 1.0. (C) Time course of Flk1⁺ cell appearance evaluated by FACS. Flk1⁺ cell appearance was examined on differentiation day 4.5 before and after sorting, and at Flk-d1, Flk-d2, and Flk-d3 cultured with DM alone in the presence or absence of Dox (1 μg/mL). (Top panels) Dox treatment. (Bottom panels) Dox-free. Percentages of Flk1⁺ cells are indicated. (D) Double fluorescent staining for CD31 and NRP1 at Flk-d3. (Left 6 panels) Dox treatment. (Right 6 panels) Dox-free. Flk1⁺ cells stimulated with vehicle (top panels), VEGF (50 ng/mL; middle panels), or VEGF and 8bromo-cAMP (0.5 mM; bottom panels). Scale bars represent 100 μm. (E) Vascular formation from Flk1⁺ cell aggregates in three-dimensional culture in Dox-free condition at Flk-d5. In-gel double fluorescent staining for Flk1 and NRP1. (Left panel) Flk1 (red). (Right panel) NRP1 (green). Scale bars represent 100 μm.