Fig. 4.

Altered localization of Occludin protein expression in MVECs co-cultured with OPTN(E50K) RGCs and astrocytes. (A-I) Representative images of immunostaining of MVEC monocultures (A-C), triple co-cultures of MVECs with isogenic control RGCs and astrocytes (D-F), or triple co-cultures of MVECs with OPTN(E50K) RGCs and astrocytes (G-I) for Occludin, Claudin-5, and ZO-1. (J) A comparison of tight junction protein continuity was quantified by measuring the area fraction index, in which a significant increase was observed in continuous junctions in Occludin expression in triple co-cultures of MVECs with isogenic control RGCs and astrocytes compared to MVEC monocultures or triple co-cultures of MVECs with OPTN(E50K) RGCs and astrocytes. No significant differences were observed in the localization of other tight junction proteins such as Claudin-5 and ZO-1. ***p = 0.0010 for monocullture vs. isogenic control, ***p = 0.0003 for isogenic control vs. OPTN (E50K) RGCs and astrocytes. (K-L) No significant alterations were observed in the overall expression of tight junctions analyzed by western blot. Data represents mean values ± SEM from at least three independent differentiation experiments. Statistical analyses included two-way ANOVA followed by Tukey’s multiple comparison test in J and L. Scale bar represents 100 μm