C3 deposits on heme-treated endothelial cells are increased with sera from aHUS patients compared with normal sera. Endothelial cells were treated as in Figure 2A with 100 μM heme and 1:4 dilutions of various sera representing aHUS relevant conditions. (A) Resting or heme-exposed GEnCs were incubated with NHS (n = 30) or sera from aHUS patients with FH (n = 3) or C3 (n = 5) mutations, and the level of C3 deposition was measured by fluorescence-activated cell sorter. (B) HUVECs were exposed to 100 µM heme and 1:4 dilutions of NHS and sera from two aHUS patients with C3 mutations (P1 and P2) and labeled with anti-CD31 (green) and anti-C3 (red) for immunofluorescence, as in Figure 2C. Scale bar = 50 µm. (C-E) Heme-exposed HUVECs were treated with normal sera (gray) or different model conditions for aHUS (red). (C) FB-depleted serum complemented with recombinant FB either wild-type or with aHUS-related mutation D254G, (D) FI-depleted serum, (E) FH-depleted serum, and (F) normal serum complemented with blocking anti-FH antibodies against the N-terminus (Ox24, AbD Serotec; blue) or C-terminus (C18, Santa Cruz; red) or nonblocking antibody (Ox23, dashed line). The isotype control is in black. A representative histogram from 1 of at least 3 independent experiments is presented. Statistical significance, **P < .01, ***P < .001.