Fig. 4.

The cellular localization and expression of junctional proteins. A: the localization of ZO-1, N-cadherin, and β-catenin was determined by immunofluorescence staining. Mouse RPE cells were plated on fibronectin-coated (2 μg/ml) chamber slides in different glucose conditions and stained with specific antibodies as detailed in materials and methods. No staining was observed in the absence of primary antibody (not shown). Please note reduced junctional localization of ZO-1 as well as its increased nuclear localization in RPE cells in high glucose. B: the localization of ZO-1 in human RPE cells. Similar to mouse RPE cells, high glucose resulted in increased nuclear localization of ZO-1 in human RPE cells. C: Wholemount staining of RPE layer in RPE-choroid tissues prepared from wild-type (WT) (nondiabetic) and Akita/+ (diabetic) mice. Please note increased ZO-1 nuclear staining in RPE-choroid tissues from Akita/+ mice compared with wild-type mice. These experiments were repeated with eyes from five mice with similar results. Control is staining with no primary antibody. D: the quantitative assessment of A and B. **P < 0.01, ***P < 0.001. E: Western blot analysis of junctional proteins. Total cell lysates were prepared from RPE cells in different glucose conditions and analyzed for expression of ZO-1, N-cadherin, β-catenin, P120-catenin, and β-actin. Please note a significant decrease in expression of β-catenin in RPE cells cultured in high glucose. There were no significant changes in the levels of N-cadherin, ZO-1, and P-120 catenin under different glucose conditions. The β-actin was used for loading control (*P < 0.05, n = 3).