(A) Left: Complement C3 (yellow arrow, green) is localized particularly along Bruch’s membrane under RPE cells (white arrow) in WT mice. Right: Strong C3 immunolabeling (green) is also observed in CNV lesions of Vegfahyper mice, where it co-localizes with CD31+ neovessel protrusions (red; white arrows). 12-months-old mice. Scale bars, 50 μm. Location of ONL, choroid, and RPE are shown. (B) Left: C3 (green, yellow arrow) is detected in CNV lesions in eyes of Vegfahyper mice but not in CNV lesions in VegfahyperC3-/- mice (CNV lesion shown by a red arrow), confirming loss of C3 protein in C3-/- mice. Right: CNV lesions in Vegfahyper mice show immunolabeling for C5b-9 (red, white arrows), whereas no C5b-9 immunolabeling is detected in CNV lesions in VegfahyperC3-/- mice. 12-months-old mice. Scale bars, 50 μm (left), 20 μm (right). C5b-9 immunolabeling (red) from co-immunolabeling image shown in Figure 5—figure supplement 1. Locations of ONL, choroid, and RPE are shown. (C) VegfahyperC3-/- mice show significantly fewer CNV lesions compared to littermate Vegfahyper mice (VegfahyperC3+/+ mice), but this difference was only observed in male mice. Male Vegfahyper mice have more CNV lesions compared to females, whereas there was no marked difference in lesion numbers for male and female VegfahyperC3-/- mice. Inactivation of C3 did not significantly affect CNV lesion sizes. 6-weeks-old mice. Values represent total CNV lesion numbers/eye for each mouse and average lesion area/eye for each mouse. Absolute numbers of mice per group are indicated in parentheses. Graphs show mean ± SEM. P-values are shown (two-tailed) and were determined with a Mann-Whitney test. (D) Representative choroidal flat mount images of 6-weeks-old male mice show multifocal CD31+ CNV lesions (white arrows) in Vegfahyper mice and significantly fewer CNV lesions in VegfahyperC3-/- mice. Staining with phalloidin in green and immunolabeling for CD31 in red. Scale bars, 200 μm. (E) Lack of complement C3 does not prevent inflammasome activation in eyes of VegfahyperC3-/- mice. Two RPE/choroid tissue lysates were pooled from 15 to 17 months-old mice. Ratios of p10/β-actin band intensities normalized to WT values are shown. (F) C3 deficiency does not cause significant retinal morphological defects at 6 weeks of age. Representative sections through posterior eyes of 6-weeks-old male WT mice and C3-/- mice are shown. Scale bars, top 20 μm, bottom 5 μm. Outer nuclear layer (ONL) thickness was measured at seven distances along the meridian in eyes of 6-weeks-old mice. Values represent average ONL thickness in μm per group. SEMs are shown for each mean. Student’s t-tests were performed for values measured at equal distances from the optic nerve (ON). No significant difference in ONL thickness between WT mice and C3-/- mice was observed. PR: photoreceptors; CHOR: choroid.