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. 2017 Jul;58(7):3073–3085. doi: 10.1167/iovs.16-20083

Figure 4.

Figure 4

Cell-associated ApoE accumulation was dependent on MAC deposition. RPE cells were primed with or without S58 (1.2 mg/mL) for 30 minutes and then treated with 6% serum for 30 minutes (A), 6 hours (B, C), or 5 hours (D, E). (A) Induction of MAC accumulation on RPE cells from a 62-year-old donor with ApoE phenotype E3/E3 and CFHYY402 variant. After serum treatments in the presence or absence of anti-C5 antibody (10 μg/mL), cells were fixed in 4% PFA for 15 minutes and stained with mouse anti-human C5b-9 (aE11) antibody. Data are representative of two separate experiments in two donors with similar results. Red stain indicates MAC deposition. Blue stain corresponds to 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei. Scale bar: 100 μm. (B) Accumulation of ApoE was prevented when C5 was blocked by anti-C5 antibody. Total proteins (15 μg) obtained from RPE cells of a 61-year-old donor with ApoE phenotype E3/E4 and CFHYH402 variant were separated by SDS-PAGE 6 hours post complement challenge in the presence or absence of anti-C5 antibody (10 μg/mL). (C) The quantity of ApoE relative to GAPDH shown in (B) was determined by densitometry. *P = 0.001 vs. C1q-Dep and C5 Ab+C1q-Dep. **P = 0.002 vs. S58+C1q-Dep. Data are representative of three separate experiments in three donors with similar results. (D) Accumulation of ApoE was blocked by absence of C6. Total proteins (30 μg) obtained from RPE cells of a 51-year-old donor with ApoE phenotype E3/E3 and CFHHH402 variant were separated by SDS-PAGE after treatment with C6-Dep in the presence or absence of C6 protein at 7.3 or 65 μg/mL. (E) The quantity of ApoE relative to GAPDH shown in (D) was determined by densitometry. *P < 0.05 vs. C6q-Dep and S58+C6-Dep. Data are representative of two separate experiments in two donors with similar results.