FIG. 5.

Cholesterol-dependent Ad2-stimulated macropinocytosis and delivery of fluid phase endosomal contents to the cytosol. (A) Control cells, mβCD-treated cells, or mβCD-treated cells with cholesterol replaced (mβCD → 0.1mM Ch) were incubated with Ad2 in the cold, washed, internalized at 37°C in the presence of dextran-FITC for 15 min, and analyzed by flow cytometry. (B) The internalized dextran was compared to the dextran internalization in PMA-treated cells and was normalized to uninfected, unstimulated cells. Dextran release to the cytosol of cells pulsed with dextran-FITC for 15 min and chased in the absence of dextran for 30 min was analyzed by fluorescence microscopy, and the number of cells with dextran-positive nuclei was determined. (C) Examples of control cells containing endosomal dextran (punctate signals [a, c, and g]) and cytosolic dextran (e and i) were imaged by CLSM, and total projections are shown, including the corresponding Nomarski images (b, d, f, h, and k). Bar = 10 μm. The error bars indicate the corresponding standard errors of the mean.