Analysis of the effects of Dll4 overexpression on cell-cycle distribution. (A) HUVECs transduced with Dll4 or vector only (60%-90% of cells expressing EGFP) were synchronized by 24-hour incubation in starvation medium supplemented with 2.5% serum and then stimulated with VEGF-A (50 ng/mL) for 24 or 48 hours. Cell-cycle distribution was evaluated by flow cytometric analysis of relative DNA content, after cell fixation in cold 70% ethanol and incorporation of propidium iodide (PI). The results, analyzed by MODFIT LT software, reflect the percentage of cells found in the G₀/G₁, S, and G₂/M phases of the cell cycle at time 0 (end of synchronization), 24 and 48 hours after culture with VEGF-A. Representative experiment of 4 performed. (B) Exponentially growing HUVECs transduced with vector only or Dll4 (60%-90% of cells expressing EGFP) were cultured in serum-reduced (2.5%) medium containing VEGF-A (50 ng/mL) for 24 to 72 hours. Cells were pulsed with BrdU (10 μM) over 1 hour prior to harvest. After fixation in 70% ethanol and Triton-X 100 permeabilization, the cells were stained with FITC-labeled mouse monoclonal anti-BrdU antibodies and subsequently allowed to incorporate PI. Cell-cycle distribution was evaluated by flow cytometry. The results reflect the percentage of cells in the S phase of cell cycle measured at time 0 (1 hour after culture in serum-reduced medium), 24, 48, and 72 hours after incubation in VEGF-A-supplemented serum-reduced culture medium.