Sensitivity of VEGF signaling is enhanced by PKA. Serum-free culture of Flk1⁺ cells on two-dimensional condition, at Flk-d3. (A-B) Flow cytometry for CD31 expression in the presence (Dox⁺; 1 μg/mL) or absence (Dox⁻) of Dox. x-axis: CD31; y-axis: SSC. Flk1⁺ cells were incubated with various concentrations of VEGF₁₆₅ or VEGF₁₂₁ in serum-free medium, SFO3. Percentages of CD31⁺ ECs in total Flk1⁺ cell–derived cells are indicated. (B) Quantitative evaluation of effects of PKA activation on EC differentiation. Percentages of CD31⁺ EC population in total Flk1⁺ cell–derived cells are evaluated (n = 3; *P < .05, **P < .01 vs corresponding values; NS indicates not significant). (C) Quantitative evaluation of the number of induced ECs. Total cell number that appeared from 12.5 × 10⁴ of plated Flk1⁺ cells at Flk-d3 is shown.