One possible explanation for the differing results is the differences between these cell culture systems. wild-type (WT)- or R345W-Fibulin-3. Barrier function was assessed by evaluating zonula occludens-1 (ZO-1) distribution and trans-epithelial electrical resistance (TER). Polarized secretion of vascular endothelial growth factor (VEGF), was measured by Enzyme-linked immunosorbent assay (ELISA). Differentiation status was assessed by qPCR of genes known to be preferentially expressed in terminally differentiated RPE cells, and conversion to an epithelialCmesenchymal Mogroside IVe transition (EMT) phenotype was assessed by a migration assay. Results Compared to RPE cells expressing WT-Fibulin-3, ZO-1 distribution was disrupted and TER values were significantly lower in RPE cells expressing R345W-Fibulin-3. In cells expressing mutant Fibulin-3, VEGF secretion was attenuated basally but not in the apical direction, whereas Fibulin-3 secretion was reduced in both the apical and basal directions. Retinal pigment epithelial signature genes were downregulated and multiple genes associated with EMT were upregulated in the mutant group. Migration assays revealed a faster recovery rate in ARPE-19 cells overexpressing R345W-Fibulin-3 compared to WT. Conclusions The results suggest that expression of R345W-Fibulin-3 promotes EMT in RPE cells. Luciferase (GLuc) and GLuc tagged wild type or R345W Fibulin-3 were described previously (Hulleman et al., 2013). ViraPowerTM Lentiviral Expression systems (Thermo Fisher Scientific, Waltham, MA, United States) were used to produce Lentiviruses in 293T cells by calcium phosphate transfection. Cell Culture Human fetal RPE (hfRPE) cells were generously provided by Dr. Sheldon S. Miller, Dr. Kapil Bharti, and Dr. Arvydas Maminishkis (National Eye Institute, Bethesda, MD, United States) and cultured following the protocol published previously (Maminishkis et al., 2006). In brief, hfRPE cells were maintained in MEM medium ( modification) with N1 supplement, glutamine, non-essential amino acid, penicillinCstreptomycin, taurine, hydrocortisone, triiodothyronine, and 5% fetal bovine serum (heat inactivated) at 37C with 5% CO2. Human fetal RPE cells were seeded on human ECM (#354237, Corning Life Sciences, Tewksbury, MA, United States) coated 12 mm polyester (PET) Transwell? inserts with 0.4 m pores in 12-well plate (#3460, Corning Life Sciences, Tewksbury, MA, United States) with 150K cells per well. Medium was changed twice a week. At the beginning of seven weeks after seeding, hfRPE cells were infected with Lentiviral GLuc-tagged WT-Fibulin-3, GLuc-tagged R345W-Fibulin-3, or GLuc tag only at MOI 10 with 6 g/ml hexadimethrine bromide (#H9268, Mogroside IVe MilliporeSigma, Burlington, MA, United States) for 4 h each day for 5 days, resulting in a copy quantity of 55 9 (imply SEM) in WT group versus 57 3 (imply SEM) in mutant group. ARPE-19 Tet-On cells with Lentiviral GLuc, GLuc-tagged WT- or R345W-Fibulin-3 were explained previously (Hulleman et al., 2013). Put genes were expressed only in the presence of Doxycycline (1 g/ml, Dox, #D9891, MilliporeSigma, Burlington, MA, United States). ARPE-19 Tet-On cells were managed at 37C with 5% CO2 in DMEM (Dulbeccos Modified Eagles Medium)/Hams F-12 50/50 Blend (#10-092-CV, Corning Existence Akt3 Sciences, Tewksbury, MA, United States) supplemented with 10% fetal bovine serum (FBS, #100106, BenchMarkTM GeminiBio, Western Sacramento, CA, United States) and penicillinCstreptomycin. Immunocytochemistry Cells in Transwell? inserts were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min at space temperature. Cells were washed twice with PBS, then Mogroside IVe treated with 0.1 M glycine for 15 min and permeabilized with 0.1% Triton X-100 for three times, 2 min each. Cells were clogged with 10% normal donkey serum for 2 h at space temperature then incubated with rabbit polyclonal anti- zonula occludens-1 (ZO-1) (1:100, #61-7300, Thermo Fisher Scientific, Waltham, MA, United States).
