Fig 5.

siRNA-mediated depletion of Tsg101 or Alix decreases tetherin recruitment in HeLa cells. (A) Western blotting of Tsg101 and Alix in cells transfected with indicated siRNA and lysed at 64 h posttransfection. Alpha-tubulin is shown as a loading control. (B and C) HeLa cells were transfected sequentially with the indicated siRNA and a molecular clone encoding Gag-YFP without Vpu. At 64 h post-transfection with siRNA (16 h post-transfection with Gag-YFP expression plasmids), cells were fixed by 4% PFA for 30 min and immunostained for endogenous tetherin. (B) Representative images of the dorsal surface of cells are shown. Scale bar = 5.0 μm. (C) Degree of colocalization was calculated in the same manner as for Fig. 1. The result is shown for one of two independent experiments and displayed as mean R values ± SEM. *, P < 0.01. (D) HeLa cells were transfected with indicated siRNA, followed by transfection with a Vpu-deficient molecular clone expressing Gag-mEos3.2. Cells were then fixed, stained with Alexa Fluor 647 antitetherin antibody, and imaged by TIRF. Superresolution images were reconstructed as described in Materials and Methods. Representative images are shown. Magnified images for boxed areas in the left panels are shown on the right. Scale bars are 2 μm for the left panels and 500 nm for the right panels. (E) Images of five cells per condition were acquired in each of two independent experiments (total, 10 cells) and used for measurement of Gag-tetherin cross-correlation. Cross-correlation values are shown as means ± SEM. The dashed line indicates random distribution (cross correlation = 1). (F) Integrated cross-correlation was calculated as described in Materials and Methods and is shown as mean integrated cross-correlation values ± SEM. *, P < 0.05.