Buckwheat trypsin inhibitor enters Hep G2 cells by clathrin-dependent endocytosis |
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| Authors: | Xiaodong Cui Zhuanhua WangYuying Li Chen Li |
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| Affiliation: | Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, PR China |
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| 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. |
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| 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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