Fig. 4: Deficiency of CIB2 resulted in impaired phagolysosomal processing of OS.

a RPE whole mounts from 3- to 4-months-old mice euthanized at denoted times after light onset and immunostained with opsin Ret-P1 antibody (cyan) shows initial binding and ingestion of opsin-phagosomes is similar across genotypes (top panels), however, the phagolysosomal digestion is slower in mutant mice (bottom panels) as compared to the WT control. Scale bar, 10 µm. b Quantification of average number of opsin-phagosomes shown in a and Supplementary Fig. 4a. c TEM micrographs of 2-months-old mice euthanized 8 h after light onset show accumulation of undigested material, and fused remnants of phago-melanosomes in mutant mice. i–vi Insets show magnified images of normal phagosomes in WT mice and improperly processed phagosomes only in Cib2^KO/+ and Cib2^KO/KO mice. Scale bar, 1 μm. d Quantification of individual phagosomal (left) and mitochondrial (right) areas (as control) for images shown in c further confirms marked accumulation of phagosomal area in mutant mice. All phagosomes and 15 mitochondria/image in 3–5 images per mouse were counted (n = 3 per genotype). e TEM micrographs from 2-months-old mice show marked thickening and accumulation of debris under the RPE in mutant mice. Yellow arrows indicate the boundary between the basal surface of RPE and choroid. Scale bar, 1 μm. f Representative RPE whole mount images from 3-to-4-months-old mice euthanized 8 h after light onset and immunostained with opsin Ret-P1 antibody (cyan) show slower phagolysosomal clearance, quantified in g, in RPE-specific Cib2-mutant mice. Scale bar, 10 µm. At least three images per mouse (~30 cells/ image) were quantified and the average per animal is shown for denoted genotypes. Data presented as mean ± SEM. Each data point represents an average per individual mouse unless specified. One-way ANOVA and Bonferroni post hoc test (b, d) or unpaired two-tailed t-test (g), p < 0.05 (*), <0.01 (**), or <0.001 (***), NS not significant.