DGKε regulates the expression of complement regulatory proteins but does not promote C3 deposition on ECs. (A) HUVECs and HMECs were transfected with 20 nM of control siRNA (shaded histogram) or with 10 nM (dashed histogram) or 20 nM (open histogram) of DGKE siRNA, and expression of MCP (CD46), DAF (CD55), and CD59 was analyzed 48 hours later by flow cytometry. Representative histograms are shown, and the bar graphs represent the mean fold change in MFI (± SEM) vs control siRNA–transfected cells from at least 4 independent experiments. **P < .01, ***P < .001. ns, not significant. (B) HUVECs and HMECs were transfected with control or DGKE siRNAs. After 48 hours, cells were incubated with 1:4 normal human serum (NHS) for 30 minutes at 37°C, and the deposition of C3c at their surface was evaluated by flow cytometry. Cells incubated for 20 minutes with 100 μM heme before NHS exposure were used as a positive control for C3 deposition, as described elsewhere.28 HUVECs treated for 30 minutes with a blocking anti-MCP antibody or a control IgG1 isotype (50 μg/mL) before NHS exposure were used to determine whether MCP blockade affects C3 deposition at the surface of ECs. A representative histogram is shown, and the bar graphs represent the mean fold change in MFI (± SEM) from at least 3 independent experiments. **P < .01.
