Figure 1.

Lipofuscin-like AF formation in hfRPE cells. hfRPE cells pulsed with different concentrations of POS were monitored at the indicated time points after POS incubation. (A) AF levels were quantified by flow cytometry as in the Material and Methods and are represented as mean ± SEM of at least three independent experiments, performed in duplicate. Statistical differences relative to cells with no POS were assessed using one-way ANOVA followed by Kruskal Wallis test (***P < 0.001, *P < 0.05), and differences between cells treated with 50 and 200 µg/mL POS at each time point were evaluated using the Mann–Whitney t test (##P < 0.01; #P < 0.05). (B) AFGs were visualized by confocal immunofluorescence microscopy. Internalized rhodopsin-positive POS were detected using an anti-rhodopsin antibody (RetP1). Phalloidin staining was used to monitor cell limits and a Z-projection is represented. Arrows point to RetP1-positive POS aggregates that are attached to the apical cell membrane. Scale bar: 20 µm. (C) The number of intracellular AFGs were quantified using ImageJ and are represented as number of AFGs per field of view ± SEM of three independent experiments, where at least 10 random images were analyzed per assay. (D) Intracellular RetP1-positive POS were quantified using ImageJ and are represented as number of RetP1-positive POS per field of view ± SEM of 3 independent experiments, where at least 10 random fields were analyzed per assay. Statistical comparison was performed using one-way ANOVA followed by Dunnet's or Bonferroni's multiple comparison test (***P < 0.001, *P < 0.05). (E) POS degradation was assessed using an anti-rhodopsin antibody (1D4) by Western blot analysis. Images are representative of two independent experiments.