Inhibitory effects of KOR agonists U50,488H and TRK820 on EC induction from Flk1⁺ cells. Flk1⁺ cells after 3 days of culture (Flk-d3). (A) Flow cytometry. X-axis: CD31; y-axis: side scatter (SSC). Percentages of CD31⁺ ECs among total Flk1⁺ cell–derived cells are indicated. (B) Quantitative evaluation of the effect of VEGF on CD31⁺ EC induction from Flk1⁺ cells by FACS. Percentages of CD31⁺ cell population among total Flk1⁺ cell–derived cells. Control (n = 7) and VEGF (50 ng/mL; n = 7) treatments are shown (**P < .01 vs control). (C) Double fluorescent staining for CD31 and αSMA at Flk-d3. Left, CD31 (pan-ECs, green) and DAPI (blue); middle, αSMA (MCs, red) and DAPI (blue); and right, merged. Flk1⁺ cells were stimulated with VEGF (50 ng/mL), DAMGO (10μM), SNC80 (10μM), U50,488H (10μM), or TRK820 (10μM) as indicated. Scale bars, 200 μm. (D) Flow cytometry. X-axis: CD31; y-axis: SSC. Percentages of CD31⁺ ECs among total Flk1⁺ cell–derived cells are indicated. (E) Percentages of CD31⁺ cell population among total Flk1⁺ cell–derived cells. Treatments with VEGF alone (50 ng/mL; n = 7), and VEGF plus DAMGO (1, 3, 10μM; n = 4), SNC80 (1, 3, 10μM), U50,488H (1, 3, 10μM; n = 4), or TRK820 (1, 3, 10μM; n = 4) are shown (**P < .01, *P < .05 vs VEGF). (F) CD31⁺ cell number that appeared from 1.5 × 10⁵ of Flk1⁺ cells. (**P < .01, *P < .05 vs VEGF).