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. 2020 Jun 16;11(3):e00589-20. doi: 10.1128/mBio.00589-20

FIG 4.

FIG 4

ESCRT-III subunits decrease LMP1 packaging into extracellular vesicles. HEK293 cells expressing inducible GFP as a control and GFP-tagged dominant negative constructs against the ESCRT-III subunits (CHMP2A, CHMP3, CHMP4A, CHMP4B, and CHMP6) were transfected with SNAP-LMP1. (A and B) Whole-cell (A) and EV (B) lysates were separated by SDS-PAGE and analyzed by immunoblot analysis for LMP1 and common EV markers (Alix, HSC70, TSG101, Flotillin-2, and CD81). (C) Semiquantitative Western blot analysis of results from more than three independent experiments showing LMP1 relative EV secretion for HEK293 cells expressing ESCRT-III dominant negative constructs. (D) Internalization and degradation of fluorescently labeled EGF in cells expressing dominant negative constructs against ESCRT-III dominant negative constructs. (E to L) HEK293 cells stably expressing shRNAs against ESCRT-III subunits (CHMP1A, CHMP2A, CHMP2B, CHMP3, CHMP4A, CHMP5, and CHMP6) were generated and transfected with SNAP-LMP1. (E, F, I, and J) Immunoblot analysis of cell (E and I) and vesicle (F and J) lysates from cells expressing shRNA. (G and K) Relative mRNA levels of CHMPs (CHMP1A, -2A, -2B, -3, -4A, -5, and -6) in cells stably expressing shRNAs. (H and L) Semiquantitative Western blot analysis of results from more than 3 independent experiments. Data are presented relative to the values for wild-type LMP1 packaged into EVs (*, adjusted P < 0.005; **, adjusted P < 0.0001; ***, adjusted P < 0.05).