Journal: Frontiers in Virology

Article Title: Brief Research Report: Ebola Virus Differentially Infects Human Iris and Retinal Pigment Epithelial Cells

doi: 10.3389/fviro.2022.892394

Figure Lengend Snippet: FIGURE 1 | (A) Representative fluorescence photomicrographs of donor-matched human iris and retinal pigment epithelial cells immunolabelled to detect the presence of cytokeratin 8 (CK8) and absence of alpha-smooth muscle actin (SMA), plus human retinal pigment epithelial cells immunolabelled for retinal pigment epithelium-specific protein 65 (RPE65), and zonula occludens 1 (ZO1), with negative controls labelled with species-matched immunoglobulin (IG1 and IG2). Alexa Fluor 488 (green) with DAPI nuclear counterstain (blue). Original magnification: 400x. (B) Representative light photomicrographs of EBOV-, RESTV- and ZIKV- infected, plus uninfected donor-matched human iris and retinal pigment epithelial cell monolayers 72 hours following inoculation at a multiplicity of infection of 1. Original magnification: 10x.

Article Snippet: Additional retinal pigment epithelial cell monolayers were labelled with: mouse anti-human RPE65 (Novus Biologicals, Centennial, CO; catalogue number NB100-355; working concentration, 4 μg/mL), rabbit anti-human ZO1 (Thermo Fisher Scientific-Invitrogen, catalogue number 40-2200; working dilution, 1:100, equivalent to 2.5 μg/mL) and mouse immunoglobulin (BD Pharmingen-BD Biosciences, San Diego, CA; catalogue number 555746, working concentration, 4 μg/ mL).

Techniques: Infection

Journal: International Journal of Molecular Sciences

Article Title: Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing

doi: 10.3390/ijms131114053

Figure Lengend Snippet: Cell surface proteins expressed by RPE cells of horses. Proteins listed were identified by LC MS/MS with a probability score that is significant with p < 0.05 if the confidence score was >30 at a significance threshold for the Mascot result of p ≤ 0.01. N = native RPE cells; P4 = passage-4 RPE cells; C = cytoplasmatic; M = membrane associated; E = extracellular.

Article Snippet: For verification, we used the following primary antibodies: mouse anti-human beta-actin (Sigma-Aldrich, Deisenhofen, Germany; dilution: 1:7000), rabbit anti-human RPE65 (Santa Cruz, Heidelberg, Germany; dilution: 1:800), anti-equine CRALBP antiserum (self-made; dilution: 1:1000) [ ]), rat anti-human CD49c (Ascenion, München, Germany; reported cross-reactivity to horse, dilution: 1:500) and rabbit anti-human Fibronectin (Thermo Scientific, Schwerte, Germany; dilution: 1:1000).

Techniques: Sequencing, Activation Assay, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing

doi: 10.3390/ijms131114053

Figure Lengend Snippet: Gel electrophoresis of PCR products of native (N) and passage-4 (P4) equine RPE cell cDNA on a 2% agarose gel. Beta-actin is equally expressed by both cell states. Native RPE cells express CRALBP and RPE65, whereas no signal could be detected in passage-4 RPE. Fibronectin and CD49c are expressed distinctly by passage-4 RPE cells, while only a faint signal can be seen in native cells.

Article Snippet: For verification, we used the following primary antibodies: mouse anti-human beta-actin (Sigma-Aldrich, Deisenhofen, Germany; dilution: 1:7000), rabbit anti-human RPE65 (Santa Cruz, Heidelberg, Germany; dilution: 1:800), anti-equine CRALBP antiserum (self-made; dilution: 1:1000) [ ]), rat anti-human CD49c (Ascenion, München, Germany; reported cross-reactivity to horse, dilution: 1:500) and rabbit anti-human Fibronectin (Thermo Scientific, Schwerte, Germany; dilution: 1:1000).

Techniques: Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis

Journal: International Journal of Molecular Sciences

Article Title: Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing

doi: 10.3390/ijms131114053

Figure Lengend Snippet: Immunocytochemistry of equine RPE cells. Upper panel ( A – E ) shows native equine RPE cells. Lower panel ( F – J ) shows cultured equine RPE cells of passage-4. Native cells show positive immunoreactivity for CRALBP ( B ) and RPE65 ( C ). Native cells show no immunoreactivity for fibronectin ( D ) and CD49c ( E ). In passage-4 RPE cells, CRALBP ( G ) and RPE65 ( H ) do not show immunoreactivity, other than fibronectin ( I ) and CD49c ( J ), which is found positive in passage-4 cells. Nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI). A and F show Nomarski imaging of the respective RPE cells, native ( A ) and passage-4 ( F ).

Article Snippet: For verification, we used the following primary antibodies: mouse anti-human beta-actin (Sigma-Aldrich, Deisenhofen, Germany; dilution: 1:7000), rabbit anti-human RPE65 (Santa Cruz, Heidelberg, Germany; dilution: 1:800), anti-equine CRALBP antiserum (self-made; dilution: 1:1000) [ ]), rat anti-human CD49c (Ascenion, München, Germany; reported cross-reactivity to horse, dilution: 1:500) and rabbit anti-human Fibronectin (Thermo Scientific, Schwerte, Germany; dilution: 1:1000).

Techniques: Immunocytochemistry, Cell Culture, Staining, Imaging