Zonula occludens 1

Antibodies against p-CaMKII (1:500, ab32678), p-RyR2(S2808) (1:500, ab59225), RyR2 (1:200. ab117840), α-SMA (1:1000, ab5694), and Desmoplakin (1:200, ab106342) were purchased from Abcam. Flag-M2 (1:1000, F1804) and α-Actinin (1:1000, A7737) were obtained from Sigma-Aldrich. Antibodies against Gapdh (1:1000, LFPA0018) and HA (1:1000, LFMA0048) were from AbFrontier. Collagen1 (1:1000, SC-8784) and HSP90 (1:1000, SC-7947) were obtained from Santa Cruz Biotechnology. PRMT1 (1:500, 07-404) and Oxi-CaMKII (1:500, 07-1387) were from Millipore. N-cadherin antibody (1:500, 610921) was from BD biosciences. ANP (1:500, PA5-29559) and Zonula Occludens-1 (1:200, 40-2200) were from Thermo Fisher Scientific. Antibodies against ASYM (1:200, 13522), Connexin43 (1:1000, 3512) and CaMKII (1:1000, 3362) were from Cell Signaling Technology. Anti-mouse (1:5000, 115-035-033), anti-rabbit (1:5000, 111-035-003) IgG peroxidase-conjugated secondary antibodies were purchased from Jackson Immunoresearch Laboratories and anti-goat (1:5000, 81-1620) IgG peroxidase-conjugated secondary antibodies was ordered from Thermo Fisher Scientifics.

Day 60 ESC-RPE (n = 3) and iPSC-RPE (n = 3) cultures grown on Transwell inserts (Corning) were fixed with 4% paraformaldehyde (pH 7.4) for 20 min at room temperature (RT), permeabilized with 0.2% Triton X-100 for 5 min and blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich) in phosphate-buffered saline (PBS) for 1 h. After removing blocking buffer, cells were incubated with primary antibodies diluted in 1% BSA in PBS overnight at 4 °C. After three washes to remove the primary antibodies, cells were incubated with respective secondary antibodies (Alexa Fluor 488, 1:1000, Thermo Fisher Scientific) against its host species and Hoechst 33,342 solution (1:1000, Thermo Fisher Scientific) for 30 min at RT. After three washes to remove the secondary antibodies, cells were mounted using Fluorsave (Millipore) and imaged using LSM700 confocal microscopy (Zeiss). The primary antibodies used were Zonula Occludens-1 (ZO-1, 1:100, Thermo Fisher Scientific, 617300), RPE65 (1:125, Abcam, ab13826), BEST1 (1:100, Abcam, ab2182) and Na⁺/K⁺ ATPase (1:100, Thermo Fisher Scientific, MA3-915).

The differentiation phenotype was assessed with immunofluorescence staining (n = 4) for K3 (1∶100, Millipore), P63 (1∶50, Millipore) and ABCG2 (1∶50, Millipore). Proliferative activity was measured via colony-forming efficiency (n = 10), as previously described [31] (link). Briefly, the epithelium of each specimen was trypsinized and planted at a density of 500 cells per 60 mm in dishes that had been preseeded with 3T3 fibroblast feeder layers. Colony-forming efficiency was calculated on day 6. Cell junction-related proteins zonula occludens-1 (1∶50, Invitrogen), desmocollin 2 (1∶100, Abcam), and integrin β4 (1∶50, Abcam) were detected using immunofluorescence staining (n = 4). Then, the contents of these proteins were quantitated using ELISA assays according to the manufacturer’s recommendations (n = 10, Cusabio Biotech). The ultrastructures of cell junctions in each group were observed using TEM (n = 4).