| Popis: |
Background and aims: Claudin-19 is the most abundant claudin protein in human retinal pigment epithelia. There have been only few observations of ciliary localization of claudin-19, and its role in developmentally important primary cilia has not yet been established. Our previous work has identified that human embryonic stem cell derived retinal pigment epithelial cells possess claudin-19 immunopositive primary cilia during cell maturation and after certain stimuli, such as epithelial-mesenchymal transition. The aim of this study was to establish the role and more precise location of claudin-19 in primary cilia and also, whether epithelial-mesenchymal transition affects ciliary localization of claudin-19. Methods: The role of claudin-19 in primary cilia was studied by transiently knocking down its expression with RNA interference. The change in protein levels was examined with Western blot. It was studied whether inducing cells to epithelial mesenchymal transition with TGF-β2 treatment would increase claudin-19 localization in primary cilia and also it was used to find the precise localization site within primary cilia. The cells were immunostained against claudin-19 and ciliary marker protein and imaged using confocal and expansion microscopy. Results: It was not verified whether the claudin-19 knockdown would affect primary cilia maintenance, as the claudin-19 knockdown was not successful. The exact localization of claudin-19 in primary cilia was not identified, but it was shown that epithelial-mesenchymal transition induces claudin-19 localization in primary cilia. Conclusion: There were several different methods optimized during the study and protocols for Western blot, epithelial-mesenchymal transition induction, and expansion microscopy were established. This study showed that claudin-19 is occasionally located in primary cilia. It has been noticed from previous studies that claudin-19 associated diseases and primary cilia associated diseases have some similarities. Based on this evidence it is possible that claudin-19 has a meaning and role in primary cilia, even though it was not verified in this study. |
