Expansion of differentiated ECs by ROCK suppression is mainly the result of the promotions of EC proliferation and Flk1⁺ MPC adhesion. (A) Diagram of time points (6, 12, and 36 hours) for assays of proliferation, cell cycle, apoptosis, and adhesion in differentiating Flk1⁺ MPCs incubated with VEGF-A (50 ng/mL; Control) or VEGF-A (50 ng/mL) plus Y27632 (10μM; Y72632). (B) Immunofluorescence images showing PHH3⁺/CD144⁺ proliferative EC colonies and DAPI⁺ nuclei at 36 hours. Scale bars represent 100 μm. (C) Number of PHH3⁺/CD144⁺ ECs in cultured area (cm²) at 36 hours. Each group, n = 5. *P < .05 versus Control. (D) Representative FACS analysis showing cell cycle in CD144⁺ ECs from differentiating Flk1⁺ MPCs at 36 hours. (E) Percentage of CD144⁺ ECs under each phase of cell cycle at 36 hours. Each group, n = 3. *P < .01 versus Control. (F) Representative FACS analysis showing annexin V⁺/ PI⁻ apoptotic cells. (G) Percentage of annexin V⁺/PI⁻ apoptotic cells at 6 and 36 hours. Each group, n = 4. n.s represents statistically nonsignificant. (H) Percentage of adherent cells onto plate at 6 hours. Each group, n = 5. *P < .05 versus Control.