αB Crystallin Is Apically Secreted within Exosomes by Polarized Human Retinal Pigment Epithelium and Provides Neuroprotection to Adjacent Cells
Figure 5
αB Crystallin secretion in polarized RPE cells under conditions of oxidative stress.
(A) Polarized RPE cells were challenged with severe (500 µM H2O2-24 h) or mild (150 µM H2O2 - 24h) oxidative stress and αB crystallin secretion was determined. Values marked on the top of the blot are densitometric ratios, normalized to apical secretion of unstressed RPE taken as unity. (B) Protein expression of the exosomal marker CD63 in the medium of RPE treated with 150 µM and 500 µM H2O2. (C) Immunogold labeling of CD63 with 15 nm gold particles in the exosomes isolated from apical (Panel a) and basolateral (Panel b) medium. (D) Immunogold labeling of αB crystallin (15 nm gold particles) in the exosomes isolated from apical (panel a) and from basolateral medium (panel b, c). (E). Gold particles were counted from individual exosomes positive for αB crystallin and data are expressed as average number of gold particles per exosome (n = 61–100). No significant difference in gold particles/exosome was observed for exosomes isolated from apical media from unstressed and stressed cells. However, a significant (p<0.001) increase in αB crystallin localization in exosomes was observed in exosomes isolated from basal medium of RPE cells subjected to severe oxidative stress. Scale bar = 100 nm for C and D.