Figure 1. Characterization of human retinal pigment epithelial cells, pCAGGS-FLAG-VP24 expression plasmid, and transfection procedures. A: Fluorescent photomicrographs of retinal pigment epithelial cells (isolate 4) immunophenotyped for five markers (cellular retinaldehyde-binding protein [CRALBP], cytokeratin 8 [CK8], retinal pigment epithelium–specific 65 kDa protein [RPE65], zonula occludens 1 [ZO], α–smooth muscle actin [α-SMA]) or species- and isotype-matched control antibodies (polyclonal rabbit IgG [pR IgG], monoclonal rabbit IgG [mR IgG], monoclonal mouse IgG1k [mM IgG]), with 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstain. Original magnification = 200×; scale bar = 100 μm. B: Schematic representation of the pCAGGS-FLAG-VP24 expression plasmid generated using PlasMapper version 3.0. C: Western blot of Zaire ebolavirus viral protein 24 (VP24) in protein extracted from ARPE-19 cells transfected with pCAGGS-FLAG-VP24 (arrowhead = expected molecular weight, 29 kDa). Ladder (L) molecular weight standards are in kDa. D: Fluorescent photomicrograph showing a retinal pigment epithelial cell transfected with green fluorescent protein (GFP) expression vector plus or minus rhodamine-labeled (Rho) polyinosinic-polycytidylic acid (poly I:C). Original magnification = 400×; scale bar = 50 μm. E: Interferon-β (IFN-β) concentration measured in culture supernatant after cellular transfection with VP24 or no-insert control (NI) expression plasmid for 48 h, followed by poly I:C for an additional 4 or 24 h (hours) by enzyme-linked immunosorbent assay. An additional control replacing poly I:C with water was included for the 4-h condition. Circles represent mean protein concentration for individual cell isolates, crossbars indicate mean, and error bars indicate standard deviation (n = 4 isolates/condition). ND, ≥2 of 4 isolates below detection limit (7.8 pg/ml); ns, not significant.