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. 2020 Sep 16;12(9):1032. doi: 10.3390/v12091032

Figure 4.

Figure 4

Multiple transient recruitments of VPS4 into HIV Gag WT and Gag (YP-) VLPs under ALIX depletion. HeLa cells were treated with two rounds of siRNA against ALIX. (A–C) HeLa ALIX–h37–eGFP cell line treated with siRNA against ALIX and then transfected with 1500 ng of Gag–mCherry WT. An individual multinucleated cell was chosen (A) and a representative HIV Gag–mCherry VLP assembly in this cell is shown in (B) with intensity plots (as gray dots and fitted with gray line) and cropped TIRF images of the Gag (top, gray) and ALIX (bottom, Blue). In 40 VLPs analyzed, there was no recruitment of ALIX, as shown in (C). HeLa cells were treated with siRNA against ALIX and then transfected with 1200 ng of ΔCMV–VPS4–h37–mCherry and 300 ng of HIV Gag–eGFP (D–F) and Gag (YP-)–eGFP (G–I). (D,G) Multinucleated cells chosen for experiments with (E&H) showing a representative HIV Gag–mCherry (E) or HIV Gag (YP-)–mCherry (H) VLP assembly with intensity plots and cropped TIRF images of the Gag (top, gray) and VPS4 (bottom, Red). (F,I) Histograms of the number and timing of the first (dark red) and later recruitment (light red) of VPS4 in (F) HIV Gag–mCherry VLPs and (I) HIV Gag (YP−)–mCherry VLPs.