Figure 6. B1 enters/traffics through cells using multiple endocytic pathways, and the ability of these pathways to mediate cellular uptake does not correlate with the ability to support payload delivery to sites where bioactivity can be realized.

(A) Effect of temperature on cellular internalization of B1. TZM-bl cells were incubated with 2 μM GFP-L-B1 or 2 μM +36GFP at 4, 16, 22 or 37°C for one hour, washed with DPBS containing 0.04% Trypan Blue to quench extracellular GFP and analyzed by flow cytometry. (B) Role of different endocytic pathways in cellular uptake of B1. TZM-bl or 293T cells were pretreated with the endocytic inhibitors amiloride (5 mM) ⁽⁵⁰⁾, chlorpromazine (55 μM) ⁽⁵²⁾, dynasore (50 μM) ⁽⁵¹⁾, mannan (100 μg/ml) ⁽⁵³⁾, nystatin (50 nM) ⁽⁵⁸⁾ or cytochalasin B (4 μM) for one hour prior to the addition of GFP-L-B1 (2 μM). Part (D) indicates the endocytic pathways inhibited by these small molecules. One hour later, these cells were washed with DPBS containing 0.04% Trypan Blue and analyzed by flow cytometry. (C) Fluorescence microscopic images of TZM-bl cells transfected with GFP-L-B1 in the presence of the indicated inhibitors. Data is representative of 4 independent experiments. (D) Role of different endocytic pathways in supporting B1-mediated functional delivery of a RNA payload. 293T cells were pretreated with the indicated inhibitors for 1 hour prior to pIRF mRNA transfection using GFP-L-B1 or Lipofectamine 2000. The activity of Fluc deriving from translation of the delivered RNA was measured 6 hours post transfection. Error bars represent the standard deviation of two independent experiments.