Fig 2: Knockdown of upstream ESCRT-dependent proteins reduces LMP1 vesicle secretion and packaging. HEK293 cells expressing inducible scramble, Alix, Hrs, Syntenin-1, or TSG101 shRNA constructs were transfected with GFP-LMP1. (A and B) Whole-cell (equal mass loaded) (A) and EV (equal volume loaded) (B) lysates were separated by SDS-PAGE and analyzed by immunoblot analysis for LMP1 and common EV markers (Alix, Hrs, calnexin, HSC70, TSG101, Syntenin-1, Flotillin-2, and CD63). (C) Semiquantitative Western blot analysis of results from more than 3 independent experiments. The data are presented relative to the values for wild-type LMP1 packaged into EVs. (D and E) EVs harvested from HEK293 cells expressing shRNAs transfected with GFP-LMP1 were analyzed by nanoparticle tracking analysis for size (D) and quantity (E). (F) HEK293 cells expressing the shRNAs and the NF-?B luciferase reporter were transfected with LMP1 and evaluated for activation of NF-?B signaling (*, adjusted P < 0.005; **, adjusted P < 0.0001; ***, adjusted P < 0.05; ns, not significant).

Fig 3: Syntenin-1 and Hrs knockdowns exhibit altered LMP1 endolysosomal trafficking. (A and C) Cells expressing shRNAs were either transfected with GFP-LMP1 and then stained with Lysotracker at 24 h posttransfection or cotransfected with GFP-LMP1 and Rab7. Live-cell confocal images were acquired at 24 h posttransfection on a Zeiss microscope. (B and D) Colocalization was quantified using Pearson’s correlation coefficient (n = 8 cells). Representative maximum-projection images are shown (*, adjusted P < 0.005; **, adjusted P < 0.0001; ***, adjusted P < 0.05). Bars, 10 µm.

Fig 4: LMP1 expression increases mRNA and protein levels of EV biogenesis components. mRNA was collected from normal HEK293 cells or HEK293 cells stably expressing an inducible LMP1 construct and subjected to RT-qPCR. (A and B) Relative mRNA levels of the different EV biogenesis components (CD9, CD63, CD81, Hrs, Alix, Syntenin-1, TSG101, CHMP1A, CHMP2A, CHMP2B, CHMP3, CHMP4B, CHMP4C, CHMP5, and CHMP6) were determined by normalization to GAPDH levels (for HEK293 cells) and normal HEK293 cells (for HEK293 cells stably expressing LMP1). (C) Relative mRNA levels of different EV biogenesis genes (Hrs, Alix, Syntenin-1, and TSG101) collected from EVs of HEK293 cells or HEK293 cells expressing LMP1. (D and E) Relative mRNA levels of EV biogenesis components collected from the nasopharyngeal carcinoma cell line HK1 or HK1 expressing inducible LMP1. (F) Relative mRNA levels of different EV biogenesis genes collected from EVs of HK1 cells or HK1 cells expressing LMP1. (G to J) Immunoblot analysis of cell and vesicle lysates from wild-type HEK293 or HK1 cells and HEK293 or HK1 cells expressing inducible LMP1 showing expression levels of the different EV biogenesis components from 3 independent experiments (*, P < 0.05; **, P < 0.001).