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Page 49 Plössl et al. J Transl Genet Genom 2022;6:46-62 https://dx.doi.org/10.20517/jtgg.2021.39

Microscopy images of BEST1 and ZO-1 stainings were taken on an Olympus FV3000 confocal microscope
and processed with the FV31S-SWPMS software set. The 40× magnification images of SI treated cells were
visualized using a Fluorescence Microscope Axioskop 2 (Carl Zeiss GmbH, Jena, Germany). Image editing
was done in ImageJ 1.52n.

Photoreceptor outer segment phagocytosis assay
[23]
In short, POS were isolated from porcine retinae by sucrose density gradient centrifugation as described
and their concentration was determined applying the Roti®-Quant Bradford reagent (Carl Roth, Karlsruhe,
2
Germany). A POS concentration of 4 μg/cm for analysis of POS phagocytosis in RPE cells served as point of
reference [12,27] , which is in accordance with the typical number of 20 POS per hiPSC-RPE cell [4,23,24] . POS were
diluted in Opti-MEM® (Thermo Fisher Scientific) with 1% Pen/Strep and fed to iPSC-RPE cultured in a 12-
well format for 2 h. Subsequently, excess POS were removed with PBS and the cells were covered with fresh
maintenance medium. Samples for subsequent Western blot analyses were taken after the 2 h feeding time
point (0 h of degradation, confirming successful internalization of POS) and after 4 and 6 h in total
(revealing POS degradation 2 and 4 h after the feeding was stopped). Samples for Western blots were
harvested in 1× PBS + complete™ protease inhibitor (Sigma Aldrich, St. Louis, MO, USA), sonicated at 30%
intensity for 10 s, mixed with Laemmli buffer, and heated to 95 °C for 5 min.

Western blotting
Proteins were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis in 12.5% gels and
then transferred to polyvinylidene difluoride membranes (Immobilon, Millipore, Schwalbach, Germany) by
semidry blotting (24 V, 40 min). Membranes were blocked in TBST (Tris-buffered saline: 20 mM Tris,
150 mM NaCl, pH 7.4 with 0.1% Tween-20) containing 5% non-fat dry milk for 1 h at RT. Subsequently,
membranes were incubated with primary antibodies in TBST containing 5% non-fat dry milk at 4 °C ON.
Antibodies used for Western blot were rabbit polyclonal anti-hBEST1-334 (diluted 1:2500), mouse
monoclonal anti-RPE65 (diluted 1:5000, Abcam, Cambridge, UK), and anti-ACTB (dilution 1:10,000,
Sigma-Aldrich). The Rhodopsin primary antibody was kindly provided by Prof. Robert Molday, University
of British Columbia, Vancouver, Canada and was diluted 1:10,000. Primary antibodies were detected with
anti-rabbit or anti-mouse IgG horseradish peroxidase-linked antibodies (1:10,000, Calbiochem, Merck
Chemicals GmbH, Schwalbach, Germany). All antibodies were diluted in TBS-T containing 5% non-fat dry
milk. Clarity™ Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA) and an Odyssey FC
imager (LI-COR, Lincoln, NE, USA) were utilized for visualization of protein bands. Densitometric
evaluation was carried out with ImageStudio Software LI-COR.

Quantitative reverse transcriptase polymerase chain reaction
RNA isolation was done applying the PureLink® RNA Mini Kit (Thermo Fisher Scientific) according to the
manufacturer’s protocol with only minor changes. In brief, for homogenization, samples were transferred
onto QIAshredder homogenizers (Qiagen, Hilden, Germany) and centrifuged for 3 min at 12,000 g. Lysed
samples were subjected to Steps 1-5 of the manufacturer’s protocol. In Step 6, 350 µL Wash Buffer I was
added to the spin cartridges and centrifuged at 12,000 g for 30 s, followed by a 15 min DNAse digestion with
80 µL DNAse diluted 1:8 in Buffer RDD, before columns were washed with 350 µL of Wash Buffer I. RNA
was eluted in 30 µL of RNAse-free water and concentrations were measured on the NanoDrop® ND1000
Spectrophotometer. Then, 500 ng of RNA were transcribed into cDNA with RevertAid M-MuLV Reverse
Transcriptase (Thermo Fisher Scientific) and poly(dT) primers according to the manufacturer’s
instructions. Reaction mixtures for quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)
amplifications contained Takyon™ ROX Probe 2X MasterMix dTTP (Eurogentec, Seraing, Belgium),
200 nM primers, 0.25 µL dual-labeled probe (Roche ProbeLibrary, Roche Applied Science, Mannheim,