Fig 2: MERTK siRNA inhibited cell proliferation and induced morphological changes and phagocytic dysfunction in HsRPE cells. (A) The primary cultured HsRPE cells were observed with an inverted microscope at different magnifications (4X, 10X, and 20X). Scale bars: white bar 100 μm; red bars 10 μm. (B) The HsRPE cells were identified by immunofluorescence staining with RPE65 antibody. Scale bars: 50 μm. (C) HsRPE cells were treated with NC, MERTK siRNA1, or MERTK siRNA2 (20 nM) for 24 h, and the expression of MERTK was analyzed by RT-PCR. (D) HsRPE cells were treated with NC, MERTK siRNA1, or MERTK siRNA2 (20 nM) for 48 h, and MERTK expression was determined by Western blotting. (E) HsRPE cells were treated with NC or MERTK siRNA2 (MERTK siRNA) for 48 h, and cell proliferation was analyzed by CCK8. (F) HsRPE cells were treated the same as in (E), and cell morphology was observed with an inverted microscope. Scale bars: 100 μm. (G) HsRPE cells were treated the same as in (E), and wound-healing assays were performed to assess the wound-healing capabilities of HsRPE cells. Scale bars: 200 μm. (H) HsRPE cells were treated the same as in (E), and Transwell assays were performed to evaluate the migration activity of HsRPE cells (purple). Scale bars: 100 μm. (I) HsRPE cells were treated the same as in (E), and their phagocytic ability was examined using phagocytosis assays. The red dots represent the particles and the green dye represents the MERTK protein. Scale bars: 5 μm. **P < 0.01; *** P < 0.001.

Fig 3: Similarities and differences between non-pigmented rabbit RPE cells and CECs in situ. a Schematic diagram of the morphology and location of RPE cells and CECs in rabbits created with BioRender.com. b qRT-PCR analysis of OTX2, CRELBP, BEST1, MITF, RPE65, ATP1A1, and TJP1 between Rb-RPE and Rb-CEC. Quantification represented the levels of relative mRNA expressions normalized to GAPDH. c Representative immunofluorescent images of Rb-RPE and Rb-CEC in situ were collected, including ZO1 (green), ATP1A1 (red) and retinal pigment epithelial markers MITF (green), and RPE65 (red). Nuclei were stained with DAPI (blue) (scale bar: 50 µm). d Cell density, hexagonality, and coefficient of variation analysis were based on ZO1 immunostaining. e Scanning electron microscope showed a regular hexagonal shape in both Rb-RPE and Rb-CEC, which were well formed and with distinct cell boundaries (scale bar: 10 µm) (Upper). Transmission electron microscope showed that both types of cells were attached to their respective basement membrane, named BM and DM, by hemidesmosomal junctions (?) (scale bar: 500 nm) (Middle). Adjacent cells were joined with numerous well-developed tight junctions (?) (scale bar: 500 nm) (Lower). Data are mean ± SEM. All results were obtained from three independent experiments. Significance (*P < 0.05, **P < 0.01 and ns: nonsignificant) relative to Rb-CEC. RPE, retinal pigment epithelium; CEC, corneal endothelial cell; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BM, Bruch’s membrane; DM, Descemet’s membrane; Rb-CEC, rabbit CECs; Rb-RPE, rabbit RPE cells; SEM, standard error of the mean

Fig 4: Therapeutic effects of primary pigmented RPE cells for corneal recovery. a Corneal transparency was observed at days 1, 3, 7, and 14 postoperatively. b Transplanted pigmented RPE cells were stained with ZO1 (green) and RPE65 (green) at day 14 after surgery. Nuclei were stained with DAPI (blue) (scale bar: 50 µm). In vivo experiments were performed using three independent animals per group. RPE, retinal pigment epithelium

Fig 5: Confluent cultures of primary RPE cells and primary CECs taken from non-pigmented rabbit eyeball. a Cell morphology of Rb-CEC and Rb-RPE at passages 0–2 were assessed with an inverted phase-contrast microscope (scale bar: 100 µm). b Representative immunofluorescence staining images of corneal endothelial markers ZO1 (green), ATP1A1 (red), retinal pigment epithelial markers MITF (green), and RPE65 (red) in Rb-RPE and Rb-CEC. Nuclei were stained with DAPI (blue) (scale bar: 50 µm). c Cell permeabilities of Rb-RPE and Rb-CEC using HRP tracer at 5 min, 10 min, 15 min, and 30 min, respectively. d The Na + /K + -ATPase activities of Rb-RPE and Rb-CEC at passage 2. Data are mean ± SEM. All results were obtained from three independent experiments. Significance (**P < 0.01, ns: nonsignificant) relative to Rb-CEC. RPE, retinal pigment epithelium; CEC, corneal endothelial cell; Rb-CEC: rabbit CECs; Rb-RPE, rabbit RPE cells; SEM, standard error of the mean