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. 2022 Mar 9;96(5):e01813-21. doi: 10.1128/jvi.01813-21

FIG 4.

FIG 4

The CME of IAV depended on SM-sequestered cholesterol more than the CME of Tf. (A) The amounts of internalized IAV and Tf via CME are not simply related to the amounts of accessible cholesterol, as measured by mGFP-D4 binding. For the raw data, see Fig. 1C and E and Fig. S6 in the supplemental material. (B) The amounts of internalized IAV and Tf via CME monotonically decreased with decreasing amounts of SM-sequestered cholesterol, as measured by OlyA-mGFP binding. IAV internalization via CME depended on the amounts of SM-sequestered cholesterol in a manner independent of the cholesterol depletion methods (MβCD and SMase treatments) (the black cubic function is to facilitate visualization). Meanwhile, the CME of Tf depended not only on the amounts of SM-sequestered cholesterol but also on the methods of cholesterol depletion, suggesting that it depends on the accessible and essential cholesterol pools (green and blue straight lines). (C) Fractions of IAV and Tf internalized via CME with dependences on the three cholesterol pools. MβCD and SMase treatments were performed for 60 min. Chol, cholesterol; S, sensitive; I, insensitive. The fraction of CIE was taken from the sixth bar from the left in Fig. 3D (Tf internalization via CME is assumed to be 100%). Direct experimental and calculated results are indicated by the solid and dotted lines enclosing parts of the bars, respectively. The difference between the fraction sensitive to the MβCD treatment (cyan) and that sensitive to the SMase treatment (light green) gives an estimate for the fraction sensitive to only the accessible cholesterol pool (dark green bars). The fractions sensitive to essential cholesterol and cholesterol-insensitive CME (dark blue bars) were calculated by subtracting the fractions of SM-sequestered cholesterol (light green) and accessible cholesterol (dark green) from the fraction of CME (orange open bar). (D) Increased membrane tension by using diluted medium enhanced IAV internalization but not Tf internalization. The requirement of SM-sequestered cholesterol for IAV internalization remained the same. Horizontal bars, crosses, boxes, and whiskers indicate the median values, mean values, interquartile ranges (25 to 75%), and 10 to 90% ranges, respectively.