Overexpression of optineurin-GFP (OPTN_WT-GFP) but not myocilin-GFP (MYOC-GFP) impedes transferrin (Tf) uptake.

<p>(<b>A</b>) Fluorescent images of GFP (Aa and Ab)- and OPTN_WT-GFP (Ac and Ad)-expressing RPE cells after incubation with Texas red-Tf (TR-Tf) for 15 min. The cells were fixed and the fluorescent images were captured under a Leica confocal microscope. Transfected cells (green) are seen in Aa and Ac and the internalized TR-Tf (red) is seen in Ab and Ad. Scale bar, 20 µm. (<b>B</b>) Quantification of TR-Tf uptake in GFP (green filled circles)-, and OPTN_WT-GFP (pink squares)-expressing RPE cells. Fluorescence intensity of TR-Tf in the cells was measured at 0-, 2-, 5-, 8-, 10-, and 15-min time points. Images in seven randomly selected 20× fields, each containing at least 8 transfected cells, were analyzed. TR-Tf internalization for samples at each time point is expressed as percentage relative to that of the 15-min uptake in GFP-expressing mock control cells. Data presented are mean ± standard deviation (SD) from a representative of three independent experiments. *, P<0.0001 (n = 8) compared to GFP controls. (<b>C</b>) Quantification of TR-Tf uptake in GFP (green filled circles)-, or MYOC-GFP (blue diamonds)-expressing TM cells. (<b>D</b>) Fluorescent images of GFP (Da and Db)- and OPTN_WT-GFP (Dc and Dd)-expressing RGC5 cells (green) after incubation with TR-Tf (red, Db and Dd) for 15 min. Scale bar, 20 µm. (<b>E</b>) Quantification of TR-Tf uptake in GFP (green filled circles)-, and OPTN_WT-GFP (pink squares)-expressing RGC5 cells. Fluorescence intensity of TR-Tf in the cells was measured at the 0-, 8-, and 15-min time points. The uptake is expressed as percentage (mean ± SD) relative to that of the 15-min uptake in GFP-expressing control cells. Experiments were repeated at least three times with similar results. *, P<0.001 compared to GFP controls.