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Plössl et al. J Transl Genet Genom 2022;6:46-62 https://dx.doi.org/10.20517/jtgg.2021.39 Page 50

Germany) and 25 ng of cDNA. The amplification program consisted of 2 min hold at 58 °C, 30 min hold at
60 °C, and 5 min hold at 95 °C, followed by 45 cycles of 20 s, 94 °C melt, and 1 min anneal/extension at
60 °C. All amplifications were done in technical triplicates, and the obtained data were analyzed by the ΔΔcT
[28]
method . HPRT1 was used as a housekeeping gene for normalization. If the standard deviation within
technical triplicates was greater than 0.3, the value deviating most from the mean value was excluded from
the analysis.

Measurement of transepithelial electrical resistance
After six weeks of cultivation on Transwell inserts, transepithelial electrical resistance (TEER) of iPSC-RPE
was measured with an epithelial Volt/Ohm Meter (World Precision Instruments, Sarasota, FL, USA)
according to the manufacturer’s instructions. In preparation for the measurements, the electrodes were
sterilized in 70% ethanol for 15 min and then immersed in a 150 mM NaCI solution for 15 min to allow
equilibration. Blank measurements were taken from Matrigel® coated Transwell filters which did not
contain any cells. After deducting the mean of the blank values from the resistance measurements, cell
specific resistance values were multiplied with the surface area of the Transwell filter to obtain values in
2
Ω*cm .

Enzyme-linked immunosorbent assay
Secreted VEGFA protein in apical and basal supernatants of iPSC-RPE cells cultured on Transwell inserts
for six weeks was analyzed applying the Human vascular endothelial growth factor (VEGF) Quantikine
Enzyme-linked immunosorbent assay Kit (R&D System, Minneapolis, MI, USA) according to the
manufacturer’s instructions. Supernatants were diluted 1:10 in PBS prior to analysis.

Oxidative stress induction
SI-mediated oxidative stress treatment
SI was dissolved in iPSC-RPE maintenance medium without KOSR (KnockOut™ Serum Replacement,
Thermo Fisher Scientific) and diluted to the desired concentration. After evaluating cytotoxicity of SI on
iPSC-RPE, a concentration of 0.5 mM SI was chosen for the 24 h stress experiments. For an extended
duration stress model, cells were exposed to concentrations of 0.125 and 0.25 mM SI over a period of 72 h
and the media was replaced daily. In both experimental settings, cells cultivated in iPSC-RPE maintenance
medium without KOSR served as controls.

Cytotoxicity assay (LDH Release Assay)
To determine if SI treatment was cytotoxic to iPSC-RPE, cells were seeded on 96-well plates, matured for
four weeks, and then subjected to oxidative stress treatment. SI-induced cytotoxicity was measured applying
the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA) according to the
manufacturer’s instructions. Absorbance measurements were performed with a TECAN SPARK®
Multimode Microplate Reader (TECAN, Männedorf, Switzerland) at 490 nm with a reference wavelength of
620 nm.

Statistical evaluation
[29]
Statistical analysis was performed with R (version 3.6.0) . Gaussian distribution was determined by a
Shapiro-Wilk normality test. These data were analyzed with the Student’s t-test (2 experimental groups) or
the ANOVA test with Tukey’s multiple comparison test (> 2 experimental groups). Data not following a
Gaussian distribution were analyzed with the Mann-Whitney U-test (2 experimental groups) or Kruskal-
Wallis test with post hoc Dunn’s multiple comparison test following the Benjamini-Hochberg method
[30]
implemented in the Fisheries Stock Analysis package (> 2 experimental groups).