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Buckwheat trypsin inhibitor enters Hep G2 cells by clathrin-dependent endocytosis |
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| Authors: | Xiaodong Cui Zhuanhua WangYuying Li Chen Li |
| Affiliation: | Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, PR China |
| Abstract: | Recombinant buckwheat trypsin inhibitor (rBTI) was studied to evaluate if it could enter cancer cells and to determine the mechanism. Fluorescein isothiocyanate-labelled buckwheat trypsin inhibitor (FITC-BTI) entered Hep G2 cells in a concentration-dependent manner. FITC-BTI colocalised with labelled transferrin (Tf) in the punctate structure, implying that rBTI enters Hep G2 cells by clathrin-dependent endocytosis. Incubation of Hep G2 cells with different chemical inhibitors abolished diffuse, but not punctate fluorescence, thus indicating that membrane potential plays a critical role in this process. Impairment of clathrin-mediated endocytosis by RNAi with clathrin heavy chain greatly reduced or completely abolished both diffuse and punctate fluorescence, further supporting a theory of a single route of endocytosis. Consistent with our working hypothesis, Hep G2 cells which were arrested in the M phase did not show any vesicular or diffuse FITC-BTI. We conclude from these results that both endocytosis and membrane potential are required for rBTI entry into Hep G2 cells. |
| Keywords: | rBTI recombinant buckwheat trypsin inhibitor Tf transferrin DAPI 2-(4-amidinophenyl)-1H-indole-6-carboxamidine MDC monodansylcadaverine FITC fluorescein isothiocyanate DMSO dimethyl sulfoxide Noc nocodazole HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid uPA urokinase-type plasminogen activator PAI-1 plasminogen activator inhibitor type-l |
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