Fig 1: Iron-loaded iPS-RPE accumulate lysosomes with impaired enzyme activity. (A) Western blot analysis of human iPS-RPE loaded with 50 μM FeSO4 for 6 and 9 weeks. AzureRed total protein stain served as a loading control. Each lane represents one experimental replicate. (B) Representative confocal images of human iPS-RPE cells loaded with LysoTracker (red) to detect lysosomes and DQ-BSA (green) to detect lysosomal proteolysis. Nuclei are labeled with DAPI (blue). Scale bars: 10 μm. (C) Quantification of LysoTracker-positive puncta per cell. (D) Quantification of DQ-BSA-positive puncta per cell. (E) Measurement of lysosomal pH using LysoSensor Yellow/Blue, with higher values of the 340/380 ratio correlating to increased pH. (F-J) Enzyme activity assays for cathepsin D (F), cathepsin B (G), glucosylceramidase (GCase) (H), acid sphingomyelinase (aSMase) (I) and lysosomal acid lipase (LAL) (J). Cells were incubated with 0, 50 or 100 μM FeSO4 for 6 weeks in B-J. Values in graphs represent the mean±s.e.m. RFU, relative fluorescence units. ns, not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (two-tailed unpaired Student's t-test).