1. Gliomagenezės proceso reguliatoriaus semaforino Sema3C veikimo molekulinių mechanizmų tyrimai
- Author
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Stakaitis, Rytis and Kazlauskas, Arūnas
- Subjects
animal structures ,semaphorin ,Sema3C ,U87 ,glioma ,angiogenesis - Abstract
The function of Sema3 proteins is partially regulated by protease family of furins, which recognize RxRR amino acid sequence in Sema, PSI and basic domains of semaphorins. In order to establish the importance of furin cleavage to Sema3C function in angiogenesis, two recombinant Sema3C variants were synthesized and purified: one of the proteins (termed SCL) was constructed to mimic the Sema3C basic domain, which is cleaved at the hypothetical furin recognition site 742RNRR745, whereas the other recombinant protein represented the basic domain of Sema3C in a non-cleaved state (termed SWT). Purified recombinant SCL and SWT proteins were used in in vitro angiogenesis system to determine their impact on endothelial HUVEC cell ability to form microcapillary structures. Results of this experiment revealed significant difference between SCL and SWT in their effect on microcapillary network structure, thus confirming the hypothesis that furin cleavage at 742RNRR745 Sema3C basic domain is crucial to its function. In order to fully understand mechanisms of Sema3C function, a glioblastoma U87 cell line was modified to have a combined gene expression system “Flp-In” and “T-REx”. Newly generated cell line, termed U87frt/tet, will be of great use for future studies as an efficient tool for quick generation of multiple stable glioblastoma cell lines each encoding different types of Sema3C variants
- Published
- 2018