2.1. Retinal Pigment Epithelial (RPE) Cell Culture

Primary human fetal RPE cells (ScienCell, Carlsbad, CA, USA) from three donors with unknown clinical or genetic background were used for our experiments at passage number three (P3). Cells were seeded onto Corning 6-well transwell inserts (10 µm thick polyester inserts with 0.4 µm pore size, 4*10⁶/cm² pore density, Corning, Wiesbaden, Germany) in epithelial cell medium (EpiCM, ScienCell, Carlsbad, CA, USA). After seven days, the cell culture medium was replaced with Miller medium with 1% FBS [45,46] for an additional four weeks in the absence or presence of 125 µM externally added zinc (as zinc sulphate; Thermo Fisher Scientific, Waltham, MA, USA), resulting in ~10 nM bio-available or free zinc [38,44]. Cellular differentiation was monitored through the development of cobblestone morphology and increase in pigmentation using light microscopy and the increase in transepithelial electrical resistance (TEER) was measured by using the EVOM2 Epithelial Voltohmmeter and STX2 electrodes (World Precision Instruments, Sarasota, FL, USA). The obtained results were analyzed using two-way ANOVA and Tukey’s test and a p-value < 0.05 was considered significant.

At the end of the experiments, cells were washed with PBS (Thermo Fisher Scientific, Waltham, MA, USA) 3 times for 2 min. Then differentiation media were replaced with serum-free Minimum Essential Medium Eagle Alpha Modifications (Merck, Darmstadt, Germany). After 24 h, apical and basal media (approximately 600 and 900 µl, respectively) were collected, snap frozen in liquid nitrogen and stored at −80 °C until proteomics analysis was conducted. The transwell membranes containing the cells were excised and divided into five equal-sized wedges. These were either immediately snap frozen and kept at −80 °C or fixed for 15 min in 4% PFE (Merck, Darmstadt, Germany) diluted in PBS (Thermo Fisher Scientific, Waltham, MA, USA) or kept in Karnovsky fixative (Agar, Essex, UK) comprising 3% (v/v) glutaraldehyde, 1% (v/v) paraformaldehyde in 0.08 M sodium cacodylate buffered to pH 7.4 with 0.1M HCl.

For immunofluorescence analysis, the cells on the transwell membrane were permeabilized in 0.5% Triton-X (Merck, Darmstadt, Germany) in PBS for 10 min at 4 °C and then washed in 0.1% Tween20 in PBS (PBST) (Merck, Darmstadt, Germany) and blocked with PBST with 5% goat sera (Merck, Darmstadt, Germany) for one hour at room temperature. Next, samples were incubated with primary antibodies PMEL17 (Agilent, Santa Clara, CA, USA, dilution 1:25), ZO-1 (BD Biosciences, San Jose, CA, USA, 1:200), BEST-1 (Merck, Darmstadt, Germany, 1:50), and RPE65 (Merck Millipore, Darmstadt, Germany, 1:50) diluted in PBST containing 1% goat sera. Following washing with PBST, the samples were incubated with secondary antibodies in 1:200 in PBST with 1% goat sera for one hour in the dark at room temperature. After, the secondary antibody incubation samples were washed with PBST for 5 min, followed by 5 min washing with PBS. Cell nuclei were then labelled for 15 min with DAPI (Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:1,000 in PBS. Finally, the samples were washed for 5 min in PBS, before mounting onto Menzel-Glaser slides (Thermo Fisher Scientific, Waltham, MA, USA) in Vectashield (Vector Laboratories, Burlingame, CA, USA). The flat mounts were sealed by applying nail polish around coverslips. For negative control, the primary antibody labelling was replaced by incubation with PBST only. Results were visualized using a Leica SP8 confocal microscope (Leica, Wetzlar, Germany) with a 40x/1.25 oil immersion objective. Images were obtained and analyzed with Leica Application Suite X Image software (Leica, Wetzlar, Germany).

For transmission electron microscopy, the 1% glutaraldehyde (Agar, Essex, UK) and 2.5% paraformaldehyde-fixed samples were post-fixed in 1% (w/v) osmium tetroxide (Agar, Essex, UK) in 0.1 M PBS for 50 min, dehydrated and embedded in Araldite (Agar, Essex, UK). Semi-thin sections were generated using Leica ultra-microtome (Leica, Wetzlar, Germany), stained with 1% (w/v) uranyl acetate and Reynolds’ lead citrate and viewed with a JEOL JEM-1010 electron microscope (JEOL USA, Peabody, MA, USA) and a Gatan Orius CCD camera (Gatan, Pleasanton, CA, USA).