Fig 2: OPN is expressed in cells of the posterior retina and human donor eyes.A Real-time PCR of the SPP1 gene (OPN) in ARPE19, primary RPE cells (hRPE, 93-year-old, female donor), RF/6A cells, and freshly isolated RPE, retina, and choroid tissues from a 74-year-old, female donor. B Western blot showing OPN protein expression in ARPE19, RF/6A cells, and freshly isolated retina, RPE, and choroid. C Immunofluorescence staining of retinal sections from an aged non-AMD donor shows OPN expression throughout the retinal layers, RPE, and choroid (Scale bar = 20 µm). No primary control is shown in the inset. (BrM Bruch’s membrane, GCL ganglion cell layer, INL inner nuclear layer, IPL inner plexiform layer, IS photoreceptor inner segments, NFL nerve fiber layer, ONL outer nuclear layer, OPL outer plexiform layer, OS photoreceptor outer segments, RPE retinal pigment epithelial cells).

Fig 3: Extracellular OPN levels secreted from human primary RPE cells in response to AMD-risk factors.(A) OPN levels were measured by ELISA in media collected from human primary RPE cells, from a 93-year-old donor, treated with AMD-risk factors. (*p < 0.05, n = 3, One-way ANOVA, multiple comparisons). (B) Cell viability of hRPE cells in response to treatment. (AA arachidonic acid, DHA docosahexaenoic acid, LDL low-density lipoprotein, OxLDL oxidized LDL, CSE cigarette-smoke extract, HQ hydroquinone, NaIO3 sodium iodate, VEGF vascular endothelial growth factor.

Fig 4: OPN immunoreactivity within basal deposits.A, B, C Immunofluorescence staining of human AMD donor eyes showing OPN puncta within diffuse basal deposits and RPE cells. D, E, F, G, H High magnification images of AMD donor eyes showing punctate OPN staining within small- and medium-sized drusen. Scale bar (C, H) = 20 μm, Scale bar (F) = 40 μm (OS photoreceptor outer segments, RPE retinal pigment epithelial cells). OPN green in (A–H); α-smooth muscle actin: red in (A–F).

Fig 5: Changes in the ECM composition of NHDF after a three-day RPM-exposure: Immunofluorescence images of 1g-control cells, RPM-AD and RPM-MCS of laminin (A–C) and fibronectin (D–F). Transcriptional and translational laminin analysis: Quantitative gene expression levels of LAMA3 (G), intracellular laminin levels (H) and flow cytometric analysis of laminin-labeled cells (I) displaying the percentage of laminin-positive cells as well as alteration of the median fluorescence intensity (MFI). Transcriptional and translational fibronectin analysis: Quantitative gene expression levels of FN1 (J), intracellular fibronectin levels (K) and flow cytometric analysis of fibronectin-labeled cells (L). Transcriptional and translational aggrecan analysis: Quantitative gene expression level of ACAN (M), intracellular aggrecan levels (N) and flow cytometric analysis of chondroitin sulfate-labeled cells (O). Transcriptional and translational osteopontin analysis: Quantitative gene expression levels of SPP1 (P), intracellular osteopontin levels (Q) and flow cytometric analysis of osteopontin-labeled cells (R). Full-length blots of cropped Western blot images are presented in Supplementary Fig. S1. *p < 0.05 1g vs. RPM; #p < 0.05 AD vs. MCS. Scale bars: 50 µm.