Human arpe 19 cells

Human ARPE19 cells (ATCC, Wesel, Germany) were grown in a culture medium that consisted of DMEM-F12 solution (Sigma-Aldrich, St Louis, MO, USA), supplemented with 2% antibiotic penicillin/streptomycin (Sigma-Aldrich, St Louis, MO, USA)) and 10% FBS, and kept in a humidified atmosphere of 5% CO₂, at 37 °C. Doubling growth time was approximately 60 h. Either 100 µL or 2 mL of cell culture (approximately 10 × 10⁴ cells/mL) were taken and placed in 96-well plates or T75 flasks, respectively.
After allowing the cells to settle (approximately 24 h in the case of the 96-well plates and 72 h in that of the T75 flasks) the samples were subjected to the treatment regimens indicated in Table 1.
Blue light LEDs (Electro DH SL, Barcelona, Spain) were used to deliver light (465–475 nm, 400 lux, 18 W/m²) to the cultures and the temperature was monitored to maintain it at 37 °C.
The aim of giving cells a one-hour pretreatment in the dark was to let them settle in the new culture medium.

Primary mouse RPE cells were cultured from 3‐month‐old C56BL/6J mice as described previously (Chen et al., 2008; Luo et al., 2011). Briefly, after removing the anterior segment of the eye, the vitreous and retina, the RPE/choroid/sclera eyecups, were incubated with 0.5% (w/v) trypsin–EDTA (ICN Flow, Irvin, UK) at 37°C for 30 min. RPE single‐cell suspension was collected and seeded into culture plates with complete DMEM (DMEM supplemented with 10% FCS, 100 μg mL⁻¹ primocin). Cells were subcultured when they reached confluence. The phenotype of RPE cells was confirmed by RPE65 immunostaining at passage. Only cells with >95% purity were used in the study. Passage 3–5 cells were used for experiments.
The B6‐RPE07 mouse RPE cell was cultured in DMEM with 10% FCS as previous described (Chen et al., 2008). Cells were passaged at a ratio of 1:4 once a week. Human ARPE19 cells (originally purchased from ATCC, CRL‐2302, Middlesex, UK) were cultured in DMEM/F12 (Life Technologies Ltd, Paisley, UK) supplemented with 15% FCS and subcultured at a ratio of 1:3 once every week.

Human ARPE-19 cells (ATCC, Manassas VA) were grown in HEPES-buffered DMEM and Ham’s F12 (1:1) supplemented with 10% fetal bovine serum (FBS; HyClone) and 1% anti-anti (Life Technologies) at 37°C/5% CO₂. Cells (between passages 3 and 18) were plated at a density of 10,000 cells per well and grown in DMEM/F12 + 10% FBS for 24 hours, then starved in DMEM/F12 + 0.1% FBS for 16–18 hours before treatment to remove any response to TGFβ found in FBS. Salinomycin (Sigma, S4526) dissolved in DMSO was added to media with 0.1% FBS and then cells were assayed after 48 hours, unless otherwise indicated. SB-431542 (Sigma) and (5Z)-7-oxozeaenol (Tocris) were dissolved in DMSO and used at concentrations of 10uM and 1uM, respectively. Human primary RPE cells (Sciencell) were grown in EpiCM media supplemented with 2% FBS; starved cells were treated in EpiCM with no FBS added. Cells were plated and treated the same as ARPE-19 cells.