Figure 1.

Cholesterol and TR codistribute in control and CD-treated CHO cells, and cholesterol depletion increases TR on the cell surface. Cholesterol distribution detected with filipin staining (A) and TR distribution detected with Cy3-transferrin (B) in the same field of cells. Filipin stains both the plasma membrane and the transferrin-containing, pericentriolar endocytic recycling compartment of CHO cells (arrows in A and B). Cholesterol distribution detected with filipin staining (C) and TR distribution detected with Cy3-transferrin (D) in the same field of cholesterol-depleted CHO cells. The endocytic recycling compartment (arrows in C and D) is diminished in intensity when labeled by both filipin and Cy3-transferrin, and the amount of Cy3-transferrin on the cell surface is increased. Cells were incubated with Cy3-transferrin for 1.5 hr at 37°C. Cholesterol was depleted by adding 10 mM of CD for the last 30 min of incubation. The cells were washed, fixed, and stained with filipin. The focal planes were chosen by using the filipin staining. (E) Cells were incubated in serum-free medium (Control) or in serum-free medium with 10 mM of CD for 30 min at 37°C and incubated in serum-free medium (Control and Cholesterol depleted) or serum-free medium with 10 mM of CD preloaded with cholesterol (Cholesterol replete) for 10 min. Cells were washed, incubated with iodinated transferrin in serum-free medium for 8 min at 37°C, placed on ice, and washed extensively with medium (4°C), and surface-bound transferrin was removed by incubation in a pH 2.0 buffer (24). The data are the averages of three determinations ±SD from a representative experiment.