The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls.
By D. Vercauteren, R. Vandenbroucke, A. Jones, J. Rejman, J. Demeester, S. D. De Smedt, N. Sanders, K. Braeckmans. Molecular Therapy, 2010
669 citations recorded by the source corpus.
Nonviral gene complexes can enter mammalian cells through different endocytic pathways. For efficient optimization of the gene carrier it is important to profile its cellular uptake, because this largely determines its intracellular processing and subsequent transfection efficiency. Most of the current information on uptake of these gene-delivery vehicles is based on data following the use of chemical inhibitors of endocytic pathways. Here, we have performed a detailed characterization of four commonly used endocytosis inhibitors [chlorpromazine, genistein, methyl-beta-cyclodextrin (MbetaCD), and potassium depletion] on cell viability and endocytosis in five well-described cell lines. We found that chlorpromazine and to a lesser extent MbetaCD significantly decreased cell viability of some cell lines even after short incubation periods and at concentrations that are routinely used to inhibit endocytosis. Through analyzing the uptake and subcellular distribution of two fluorescent endocytic probes transferrin and lactosylceramide (LacCer) that are reported to enter cells via clathrin-dependent (CDE) and clathrin-independent (CIE) mechanisms, respectively, we showed poor specificity of these agents for inhibiting distinct endocytic pathways. Finally, we demonstrate that any inhibitory effects are highly cell line dependent. Overall, the data question the significance of performing endocytosis studies with these agents in the absence of very stringent controls.
Subfields: Cell Biology, Molecular Biology
View Paper: https://www.semanticscholar.org/paper/5c135c9557fb9ade3ead56edcbcafda4083ee661
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Citations in the corpus
- Cell‐penetrating peptides: 20 years later, where do we stand? (2013, background): “line used and even sometimes decrease in cell viability [146,147].”
- Understanding ultrasound induced sonoporation: definitions and underlying mechanisms. (2014, background): “(CDE) or receptor-mediated endocytosis and clathrin-indepent endocytosis (CIE), comprising lipid raft endocytosis like caveolin-mediated endocytosis [121].”
- Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity (2012, methodology): “inhibitor and quantitative methods used in delineating nanomaterial endocytic pathways, and issues associated with interpretation of these experimental methods, the reader is directed to the following literature [8-11].”