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Protein delivery into live cells by incubation with an endosomolytic agent


We report that a tetramethylrhodamine-labeled dimer of the cell-penetrating peptide TAT, dfTAT, penetrates live cells by escaping from endosomes with high efficiency. By mediating endosomal leakage, dfTAT also delivers proteins into cultured cells after a simple co-incubation procedure. We achieved cytosolic delivery in several cell lines and primary cells and observed that only a relatively small amount of material remained trapped inside endosomes. Delivery did not require a binding interaction between dfTAT and a protein, multiple molecules could be delivered simultaneously, and delivery could be repeated. dfTAT-mediated delivery did not noticeably affect cell viability, cell proliferation or gene expression. dfTAT-based intracellular delivery should be useful for cell-based assays, cellular imaging applications and the ex vivo manipulation of cells.

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Figure 1: Cytosolic delivery of dfTAT in live cells is efficient.
Figure 2: dfTAT penetrates the cytosol by escaping from the endocytic pathway.
Figure 3: dfTAT-mediated delivery does not substantially affect cell proliferation and transcription.
Figure 4: Delivery of intact and functional proteins using co-incubation with dfTAT.
Figure 5: dfTAT-mediated delivery improves the delivery and transcriptional output of a transcription factor.

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This article was supported by awards R01GM087227 and R01GM087981 from the US National Institute of General Medical Sciences, by the Norman Ackerman Advanced Research Program and by the Robert A. Welch foundation (grant A-1769). We are grateful to M. Lasagna for technical assistance with FRET assay as well as to R. Chapkin for access to the luminometer and flow cytometer in his laboratory. We thank L. Dangott for help with proteomic analysis; C. Cepko (Harvard Medical School) for pCALNL-GFP; K. Rajewsky (Max Delbrück Center for Molecular Medicine) for pTriEx-HTNC; G. Sauvageau (Montreal University) for pTAT-HA-HOXB4; P. Zandstra (University of Toronto) for the HOXB4 luciferase reporter, engineered 3T3 cells and β-gal vectors; and I.R. Correa (New England Biolabs) for pSNAP-H2B and Snap-Surface 488. We also thank R. Burghardt (Texas A&M University) for providing COLO 316 cells, J. Massagué (Memorial Sloan-Kettering Cancer Center) for HaCaT cells, J. Sacchettinni (Texas A&M University) for HDF and Neuro-2a cells, E. Shoubridge (Montreal Neurological Institute and Hospital) for MCH58 cells and G. Wu (Texas A&M University) for primary intestinal porcine epithelial cells.

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A.E.-O., K.N., L.D. and J.-P.P. designed experiments. A.E.-O., K.N. and L.D. generated and processed data. A.E.-O., K.N., L.D., T.-Y.W. and G.A.J. contributed reagents. A.E.-O., K.N. and J.-P.P. wrote, edited and approved the final manuscript.

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Correspondence to Jean-Philippe Pellois.

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Erazo-Oliveras, A., Najjar, K., Dayani, L. et al. Protein delivery into live cells by incubation with an endosomolytic agent. Nat Methods 11, 861–867 (2014).

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