Figure 6.

Purified gal-3 does not promote binding or engulfment of outer segment particles by primary mouse RPE cells. Unpassaged polarized mouse RPE cells were fed with Texas Red-labeled POS in medium with no additional protein (A, B, top rows), added protein S (A, B, center rows), or added gal-3 (A, B, bottom rows). All samples received integrin ligand MFG-E8 to promote POS binding. Bound or engulfed POS material is shown in red. Bound particles only were counterstained post-fixation with rhodopsin antibody (green). Nuclei were counterstained in blue. Apically bound particles (green and red) are shown in apical maximal projections in A. Engulfed particles of the same fields as in A are shown in B in maximal projections of the center of the RPE as indicated by the RPE nuclei. Scale bar = 25 µm. (C, D) Bar graphs shows quantification of bound POS C and internalized POS D, respectively, relative to samples with only MFG-E8 added, which was set as 1. The bona fide MerTK ligand Protein S was added at 2 µg/mL (gray bars) to serve as positive control for MerTK-dependent POS internalization. Gal-3 was added at 2.5, 5, or 10 µg/mL (black bars, concentrations as indicated). Bars show mean ± s. e. m., n = 4 independent assays; 1-way ANOVA with Tukey's post hoc test; *p < 0.05. Differences between samples fed with MFG-E8 only or with MFG-E8 plus gal-3 at any concentration tested were not significant for either bound or internalized POS.