Transduction of MPG-EGFP is dependent on an amiloride-sensitive endocytic pathway. A. Flow cytometric analysis of the MPG-EGFP uptake in the presence of various endocytosis inhibitors. The following concentrations were used: 10 μM cytochalasin D, 4 mM amiloride, and 5 mM MβCD. An inhibitor or DMSO (vehicle, 0.2%) was added to HeLa cells 30 min prior to the addition of MPG-EGFP, except for amiloride, which was added 10 min or 30 min prior to the addition of MPG-EGFP. MPG-EGFP at 40 μg/ml was treated to cells for 30 min in the presence of an inhibitor (see the diagram in B.). All cells were washed with the acid buffer before the analysis. Note the decreased MPG-EGFP signal in the amiloride-treated cells. MβCD treatment increased the intensity of EGFP signal significantly. These experiments were repeated at least three times with similar results and one representative set of data is shown. B. Confocal live images of HeLa cells treated with an endocytosis inhibitor as indicated. Photomicrographs were taken at 40X and zoomed in 2X. C. A barogram showing the averaged transduction efficiencies in the presence of various inhibitors. Errors bars represent the standard deviations. C, no protein added; EGFP, 40 μg/ml EGFP protein; MPG-EGFP, 40 μg/ml MPG-EGFP; -, no drug added; D, 0.2% DMSO (vehicle); A10, 4 mM amiloride for 10 min; A30, 4 mM amiloride for 30 min; C10, 10 μM cytochalasin D; M5, 5 mM MβCD.