Figure 4.

R-59-022 inhibits macropinocytosis in Vero cells. (A) Vero cells were pre-treated with R-59-022 (5 µM), EIPA (30 µM), or vehicle for 30 min followed by addition of high molecular weight fluorescein dextran and incubation at 37 °C for another 30 min. Cells were stained with Cell Tracker Blue CMAC fluorescent dye, which was added concomitantly with the fluorescent dextran. Cells were fixed and imaged with a LSM 800 confocal microscope (Zeiss). Images are displayed as maximum intensity z-projections, bar = 10 µm; (B) Number of dextran-positive puncta per cell volume (µm³) was determined using the Imaris software (Bitplane). (C) GFP-Utrophin transfected Vero cells were pre-treated with R-59-022 (5 µM) or vehicle for at least 30 min. The cells were then placed in an environmental chamber (37 °C, 5% CO₂) and mCherry EBOV VLPs added. Immediately after VLP addition, cells were imaged with a LSM 880 confocal microscopy (Zeiss) using AiryScan FAST on a single z plane. Images were analyzed using tracking algorithms on Imaris software (Bitplane). Each track represents the movement of VLPs over time. Bar = 5 µm; (D) Track length and displacement were measured (per VLP) and the number of VLPs with a displacement greater than 2 µM were counted per time lapse image. Data are representative of 3 independent experiments. * p < 0.05, p < 0.01, *** p < 0.001.