Intracytoplasmic delivery of anionic proteins Deniz Dalkara, Guy Zuber, Jean-Paul Behr  Molecular Therapy  Volume 9, Issue 6, Pages 964-969 (June 2004) DOI: 10.1016/j.ymthe.2004.03.007 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Time course of intracellular R-phycoerythrin delivery with DOGS. BHK cells were incubated with phycoerythrin/DOGS complexes at a charge ratio of 720. The red fluorescent protein was traced by confocal microscopy (scale bar, 50 μm). Molecular Therapy 2004 9, 964-969DOI: (10.1016/j.ymthe.2004.03.007) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 DOGS-mediated intracellular delivery of anti-β-actin IgG and BSA–FITC10 to CHO, BHK, and Jurkat cells. (A) 2 μg of IgG was delivered with DOGS as described previously. CHO cells show diffuse intracytoplasmic fluorescence. (B) 2 μg of BSA–FITC10 was delivered with DOGS. Cells display a more punctate pattern of intracellular fluorescence. (C) The fluorescent IgG was delivered to Jurkat cells as for CHO cells. There was no visible intracellular fluorescence. Scale bar, 50 μm. Molecular Therapy 2004 9, 964-969DOI: (10.1016/j.ymthe.2004.03.007) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Evidence for the involvement of cholesterol in protein delivery. BHK cells were incubated for (4 + 4) h with 2 μg R-phycoerythrin complexed to 5 μl of 2 mM DOGS as described for Fig. 1. Incubation was performed in the absence (top) or presence (bottom) of 20 mM β-cyclodextrin, a cholesterol-sequestering agent. Cells were observed with a confocal laser scanning microscope. The distance between consecutive focal planes was 1 μm. Starting from the glass bottom, the first five stacks were overlaid to obtain the final image. Scale bar, 50 μm. Molecular Therapy 2004 9, 964-969DOI: (10.1016/j.ymthe.2004.03.007) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 DOGS-mediated intracellular delivery of anti-β-actin and anti-α-tubulin IgG's. Cells show diffuse intracytoplasmic fluorescence with no evidence for cytoskeleton labeling (A–C) after delivery of (A) anti-β-actin and (B, C) anti-α-tubulin IgG's. (D) Microtubules can be visualized in taxol-permeable human HeLa cells (rodent cells are not [17]) when preincubated for 1 h with 20 μM taxol prior to delivery of the anti-tubulin IgG. Scale bar for all pictures, 50 μm. Molecular Therapy 2004 9, 964-969DOI: (10.1016/j.ymthe.2004.03.007) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions