Fig 2: Expression of TSPAN4 in human PVR membranes clinical sample, normal donated retina and vitreous bodies. A Immunofluorescence analysis of TSPAN4 (green) in normal donated retina section and normal RPE in RBCC. RPE65 is the marker of the RPE cells. The Müller cells are labeled with GFAP (red) and microglia cells labeled with CD11b (red). Scale bars, 25 μm. B Immunofluorescence analysis of TSPAN4 (green) in PVR membranes. Scale bars, 25 μm. C The expression of TSPAN4 in vitreous bodies from donated human eyes (normal control group), idiopathic macular epiretinal membrane (IERM) and PVR patients, and subretinal fluid from PVR patients were detected by western blots. The percentage of TSPAN4+ samples, as detected by western blot, in each group was calculated. n = 5 in each group. D Observation of PVR membrane by TEM. Yellow arrows indicated the vesicles out of the cell. The extracellular vesicles released by cells are shown in the amplified picture. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; M, melanin

Fig 3: RPE65 protein expression analysis in different passages of dog primary RPE cells. RPE cells were stained with anti-RPE65 MoAb and analyzed by flow cytometry. a representative histogram (left) of the percentage of RPE65 expressing cells in dog RPE cell cultures in passage 1; the bar graph (right) shows the mean of three independent experiments; b percentage of RPE65 protein expressing cells in passages 1, 2, and 3; c gene expression comparison among different passages of cultured RPE cells (passages 1, 2, 3, and 4). RPE65 expression in passage 1 was regarded as maximum expression. Representative results from three separate experiments

Fig 4: Expression of RPE65 protein in cultured dog RPE cells. Cells (in passage 1) were fixed and stained with RPE65 antibody to label RPE65 protein (green) and DAPI to label nuclei (blue). a DAPI was used to visualize cell nuclei in RPE cells; b RPE65 expression in canine epidermal keratinocytes (MSECK), as control group; c RPE65 expression in dog primary RPE cells

Fig 5: Effects of CTGF on EMT and the ECM synthesis. (A) Immunohistochemistry of surgically excised human PVR specimens stained for CTGF, fibronectin and collagen III. Original magnification, ×400 and ×800 (enlargement of boxed area). (B) Immunofluorescence co-staining of PVR membranes for CTGF (red) and RPE65 (green). Original magnification, ×400 (C). Relative mRNA expression levels of biomarkers for EMT and the ECM synthesis in ARPE19 cells treated with CTGF (15, 30 and 60 ng/ml), including specific markers of epithelial cells (ZO-1 and E-cadherin), mesenchymal cells (fibronectin, N-cadherin and a-SMA) and the ECM (collagen type III). (D) Representative western blot analysis of the expression levels of EMT markers in ARPE19 cells treated with CTGF (15, 30 and 60 ng/ml). (E) Quantification of relative protein expression of ZO-1, E-cadherin and N-cadherin. (F) Representative western blot analysis of the expression levels of EMT and ECM synthesis markers in ARPE19 cells treated with CTGF (15, 30 and 60 ng/ml). (G) Relative protein expression levels of fibronectin, a-SMA and collagen type III; n=3. *P<0.05, **P<0.01, ***P<0.001 vs. control group; ns, not significant; CTGF, connective tissue growth factor; EMT, epithelial-mesenchymal transition; ECM, extracellular matrix; PVR, proliferative vitreoretinopathy; RPE65, retinal pigment epithelium-specific 65 kDa protein; a-SMA, a-smooth muscle actin.