Figure 11.

Repetitive sublytic complement challenge caused ApoE deposition in ECM. RPE cells from a 51-year-old donor with ApoE phenotype E3/E3 and CFH_HH402 variant were primed with either normal sheep IgG (0.4 mg/mL) or S58 (0.4 mg/mL) for 30 minutes and then treated with 6% C1q-Dep or HiC1q-Dep for 48 hours. Repetitive sublytic challenge was produced as described in the Methods section. RPE cells were removed after the third complement challenge by incubating cells with 0.02 N ammonium hydroxide (NH₄OH). (A) Similar cell morphology was observed across treatment groups prior to adding NH₄OH and complete cell removal after adding NH₄OH. (B) Equal lysate volumes were separated by SDS-PAGE and probed with ApoE. (C) The quantity of ApoE shown in (B) was determined by densitometry and normalized to protein concentration. *P < 0.05 vs. S58+HiC1q-Dep and IgG+C1q-Dep. Data are representative of four separate experiments in two donors with similar results. (D) Following three repetitive complement challenges, as described in the Methods section, RPE cells from a 62-year-old donor with ApoE phenotype E3/E3 and CFH_YY402 variant were fixed in 4% PFA for 12 minutes and costained with rabbit anti-ZO-1 (green) and phalloidin (red). Blue stain corresponds to DAPI-stained nuclei. Twelve-micrometer Z-stacks were captured. Confocal horizontal (X–Y) sections are shown above and vertical (X–Z) sections are shown below. Data are representative of two separate experiments in two donors that showed similar results. Scale bar: 20 μm. (E) Following three repetitive challenges as described in the Methods section, cell viability was determined by WST-1 assay. (F) RPE cells from two donors were repetitively primed with S58 (0.4 mg/mL) and treated with 6% C1q-Dep and removed as described in (A, C). Equal lysate volumes were separated by SDS-PAGE and probed with fibronectin.