Fig 2: LAMP2A associates more closely to ESCRT-independent endosomal machinery.(A and B) Immunoprecipitation experiments using antibodies raised against LAMP2, LAMP2A, LAMP2B, CD63, Alix, and TSG101 show that LAMP2A, but not LAMP2B, coprecipitates with CD63 and Alix. TSG101 does not coprecipitate with either the LAMP2A or LAMP2B isoform. (C) Top: Schematic workflow of the experiment. EEs and LEs were further immunoprecipitated with antibodies raised against the LAMP2A or LAMP2B isoforms. WB shows (bottom) that VPS4B and TSG101 are enriched in LAMP2B pulled-down endosomes, while CD63; Alix; RAB31; mCherry-ExoSignal; and KFERQ motif–containing proteins EXOc2, STCH, and histone 3 are enriched in LAMP2A-enriched endosomes. (D) Confocal images show high colocalization coefficient of KFERQ motif–containing proteins EXOc2, STCH, STK25 (Serine/Threonine Kinase 25), and Calcoco1 with the EE marker EEA1 rather than with the LE marker Rab7. The results represent the mean ±SD of at least n = 3 independent experiments (n.s., nonsignificant; ***P < 0.001; ****P < 0.0001).

Fig 3: N16 (50 µM) prevents co-localization of Gag and Tsg101 on the plasma membrane. a–e Fluorescence microscopy images of cells that co-express Tsg101-Myc alone or GagWT-GFP and Tsg101-Myc. Cells grown on coverslips were co-transfected with DNA encoding Tsg101-Myc a or Tsg101-Myc and GagWT-GFP b, d, e or GagP7L-GFP c and treated with F15 d or N16 e. After 24 h the coverslips were processed for microscopy with Tsg101-Myc detected with anti-Myc antibody and Texas Red secondary antibody as described in Methods section. Scale bars = 10 microns. a Tsg101-Myc in cells treated with DMSO carrier (top) or N16 (bottom). b–e Top panels, show signal from Gag-GFP (green); middle panels, images showing Tsg101-Myc signal; bottom panels, merged images showing signals from Gag-GFP and Tsg101-Myc. Boxes frame a region of the plasma membrane. f Pearson’s coefficient of correlation values determined for the images shown. Error bars indicate the highest values obtained for similar samples. n = 6

Fig 4: TSG101 is required for PRRSV virion formation. (A) MARC-145 cells transfected with siTSG101 or siNC were infected with PRRSV at an MOI of 1 for 10 h. The infected cells and supernatants were harvested separately for detection of the intracellular and extracellular progeny virus titers by assessing TCID50. (B) HEK-293T cells grown in 12-well plates were transfected with siTSG101 or siNC for 24 h and then transfected with 2.5 µg of PRRSV infectious clone (HN07-GFP) for another 36 h. The transfected cells and supernatants were harvested separately for detection of the intracellular and extracellular virus titers by detecting TCID50. (C) TEM analysis of PRRSV virion formation. The MARC-145 cells transfected with siTSG101 or siNC were infected with PRRSV at an MOI of 2 at 37°C for 10 h. The infected cells were fixed and investigated using 80-kV TEM according to standard procedures. Representative electron micrographs of the infected cells are shown. The image on the right panels showed an enlargement of the selected area within the rectangle. Low magnification: scale bars, 2 µm; high magnification: scale bars, 500 nm. M, mitochondrion; V, vesicle; ER, endoplasmic reticulum. Red arrows indicated the typical PRRSV virions. The number of virions per 50 µm2 areas was counted. Data represent means ± the SEM from three independent experiments. Statistical analysis was carried out using the Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant (P > 0.05).

Fig 5: Characterization of plasma exosomes in healthy donor and ß-thalassemia major patients. (a) Particle size distributions of exosome as measured by DLS. (b) Exosome counts were determined by flow cytometry and FSC-SSC data were acquired in log mode (n = 10). (c) Western blot for exosomal marker expression (CD63, TSG101) in ß-TM or HD exosomes (d) Flow cytometric analysis for characterization of the cellular origin of exosomes (RBCs-CD235a; platelets-CD40L; monocytes-CD11b; Hematopoietic progenitor cells-CD34+ and exosomal marker- CD63).