DGKE knockdown in EC impairs their angiogenic responses in vitro by inhibiting migration and increasing apoptosis. (A) Control siRNA– or DGKE siRNA–transfected HUVECs (10 nM and 20 nM, respectively) were seeded on top of a Matrigel matrix and cultured for an additional 6 hours to allow the formation of tubelike structures. A representative photomicrograph of each condition is shown (×10). The bar graph shows quantitative analysis of the mean fold change in the number of junctions between tubes per field (± SEM) vs control siRNA–transfected cells from 4 independent experiments. (B) HUVECs were transfected with control or with 2 concentrations of DGKE siRNAs, and after 48 hours, a linear “scratch” was created in cell monolayers. Migration of cells into this wound was measured after 6 additional hours. Representative photomicrographs (×20) of wounds at 0 hours and after 6 hours are shown; the white lines highlight the linear wound for each group of cells. The bar graph shows the mean fold change in percentage of wound closure vs control siRNA–transfected cells (± SEM), from 3 independent experiments. (C) HUVECs and HMECs transfected with control or DGKE siRNAs were stained with Annexin V and 7-AAD 48 hours after transfection to evaluate apoptosis by flow cytometry. Representative dot plots are shown on the left, and the bar graph represents the mean percentage (± SEM) of apoptotic cells (Annexin V⁺ and 7-AAD⁻) from 4 independent experiments. *P < .05, **P < .01.