FIG 1.

Depletion of SM-sequestered and accessible cholesterol suppressed IAV internalization by ∼70%. (A) Experimental scheme for quantifying the amounts of internalized IAV. Biotin was removed from the IAV remaining on the cell surface using a membrane-impermeable reducer, 2-mercaptoethanesulfonate (MESNA). The removal efficiency is >99.9%, which greatly improved the precision in the estimation of internalized IAV from the previously reported method (15) (see also Fig. S1 in the supplemental material). The cells were fixed, permeabilized, and then labeled with Cy3-SA to evaluate the amounts of internalized IAV. (B) Typical confocal images (among 20 images) showing that the binding of OlyA-mEGFP (probe for SM-sequestered cholesterol) to the PM outer leaflet is decreased after MβCD (top) and SMase (bottom) treatments (time courses) (nuclei stained with Hoechst 33342 are shown in blue). (C) SMase treatment rapidly reduced OlyA-mEGFP binding (SM-sequestered cholesterol) but not mEGFP-D4 binding (accessible cholesterol). The MβCD treatment reduced both accessible and SM-sequestered cholesterol, but the accessible cholesterol is removed faster than the SM-sequestered cholesterol. The MβCD treatment employed here reduced the total cellular cholesterol in a time course similar to those reported previously (13). Errors bars represent standard errors of the means (SEMs) throughout this report. (D) Typical confocal images of internalized IAV (see panel B) in cells treated with MβCD (among 25, 23, and 23 images from left to right) and SMase (among 20 images) for 0, 5, and 30 min. (E) IAV and Tf internalizations were decreased with prolonged MβCD and SMase treatments.