Figure 5.

Effect of BEST1 mutations on lysosomal homeostasis in BD_IN and ARB hiPSC-RPEs. (A) Schematic protocol used to assess lysosomal pH (pH_Lys) after daily photoreceptor outer segment (POS) feeding. (B) Box plot showing pH_Lys in hiPSC-RPEs from +/+ #1 and +/+ #2 (control), +/Q238R, and +/I295del #2 (BD_IN) and the two ARB patients after two weeks of POS feeding (~20 POS/cell for 2 h/day). Ratios of fluorescence excited at 385 nm and 330 nm were determined through fluorescence measurements on a plate reader using the ratiometric indicator dye LysoSensor^TM Yellow/Blue DND-160. Absolute values of pH_Lys were calculated relative to a standard curve for each cell line. Mean values are given as mean ± SD from five technical replicates per individual sample (n = 2). Statistical analysis was performed applying Kruskal-Wallis test, following post-hoc Dunn’s multiple comparisons test including Benjamini-Hochberg procedure: * = p < 0.05. (C and D) Analysis of proteolytic enzyme cathepsin D (CTSD) maturation. Representative Western blot images of whole cell lysates from BD_IN and ARB hiPSC-RPEs (C) after one week of daily POS feeding for 2 h or 24 h and (D) before (−) and after (+) treatment with 100 µM CQ for 12 h. Antibody against CTSD was used to detect two mature (matCTSD; ~34 kDa and ~14k Da) and a short-lived form (proCTSD; ~55 kDa) of CTSD. Please note that proCTSD (edited) in (C) represents an overexposed image to demonstrate absence of the 55 kDa band in the ARB samples. A summary of hiPSC clones used in the experiments is given in Supplementary Table S4.