Fig. 7. A proportion of CLN3 protein localizes to RPE microvilli in both (i) control hiPSC-RPE cells with and without CLN3 overexpression and (ii) primary human and mouse RPE cells.

A Representative confocal microscopy image of the orthogonal view of control hiPSC-RPE monolayer post immunocytochemical analyses with antibodies against CLN3 and EZR (an RPE microvilli protein) showing apical presence of endogenous CLN3 and co-localization of endogenous CLN3 and EZR (scale bar = 10 µm) (n ≥ 3). B Representative confocal microscopy image post microvilli isolation with lectin-agarose beads and immunocytochemical analyses with CLN3 and EZR antibody showing co-localization of endogenous CLN3 and EZR in the control hiPSC-RPE microvilli-bound to lectin-agarose beads (scale bar = 10 µm) (n = 3). C Representative confocal microscopy images post immunostaining with a GFP antibody showing robust expression of EGFP in control CLN3 hiPSC-RPE cells transduced with pHIV-FLAG-IRES-EGFP lentiviral vector (scale bar = 10 µm) (n ≥ 3). Of note, the observed EGFP localization in the nucleus is due to the nuclear and kinetic entrapment of EGFP homomultimers, and has been previously reported⁵³. D Representative confocal microscopy images post immunocytochemical analyses with FLAG and CLN3 antibodies showing co-localization of FLAG-CLN3 in control hiPSC-RPE cells transduced with pHIV-FLAG-CLN3-IRES-EGFP lentiviral vector (scale bar = 10 µm) (n ≥ 3). Of note, nuclei were stained with DAPI and are excluded in the top panel showing the D image to better visualize the CLN3-EZR co-localization. In the bottom panel showing the orthogonal view, DAPI is included to illustrate the apical localization of both FLAG-CLN3 and EZR. E Confocal microscopy analyses of mouse retina sections after immunocytochemical analyses with CLN3 and EZR antibody showed co-localization of endogenous CLN3 with EZR (top panel). Notably, CLN3 antibody fails to detect CLN3 expression but EZR can be visualized in the RPE cells of CLN3^−/− mice (E, middle panel). Furthermore, no specific CLN3 staining was seen in WT mouse retina sections in the no primary controls that excluded incubation with primary antibody (E, bottom panel). Of note, because the host of CLN3 antibody is mouse, we utilized mouse on mouse (M.O.M ®) kit in these experiments (scale bar = 10 µm) (n ≥ 1). F Confocal microscopy images post immunocytochemical analyses showed co-localization of endogenous CLN3 and EZR in primary human RPE wholemounts and orthogonal view (scale bar = 10 µm) (n ≥ 1). G Representative western blot image showing presence of endogenous CLN3 protein (50 kDa) using CLN3 specific antibody in WT mouse, primary human and hiPSC-RPE samples (n ≥ 1). Also note a distinct pattern of CLN3 in mouse versus human RPE cells. Specifically, multiple bands for CLN3 were seen in native mouse RPE (~15–20 µg total protein) compared to human RPE (primary, hiPSC) possibly due to non-specific background due to the use of mouse CLN3 antibody.