Fig 2: UBE2O ubiquitinates PTRF. A UBE2O-Myc-cs expression plasmid is a UBE2O mutation expression plasmid. UBE2O expressed by the UBE2O-Myc-cs expression plasmid could not bind to ubiquitin effectively. Flag-PTRF, His-HA-ubiquitin and UBE2O-Myc or UBE2O-Myc-cs expression plasmids were co-transfected into HEK293T cells. After cells were collected, His-HA-ubiquitin in cell lysates was immunoprecipitated with Ni–NTA superflow. The immunoprecipitates and the total cell lysates were immunoblotted for the indicated antibodies. B Flag-PTRF, UBE2O-Myc and His-HA-ubiquitin-KO or His-HA-ubiquitin wild type vectors were co-transfected into HEK293T cells. Cells were harvested after 40 h transfection and were analyzed by His immunoprecipitation ubiquitination assays and western blot

Fig 3: SDPR stabilizes PTRF secretion via exosomes and does not impede the effect of UBE2O. A Flag-PTRF and UBE2O-Myc were overexpressed in HeLa cells. Then, Flag-PTRF in the cell lysates was immunoprecipitated with Flag M2 beads. Flag-PTRF associated endogenous SDPR in HeLa cells was immunoblotted by western blot. B Flag-SDPR and UBE2O-Myc were co-transfected into HeLa cells. Flag-SDPR in the cell lysates was immunoprecipitated with Flag M2 beads. The immunoprecipitates and the total cell lysates were immunoblotted for the indicated antibodies. C, D UBE2O-Myc was transiently transfected into Flag-PTRF stable HeLa cell line or Flag-SDPR stable HeLa cell line. Serum-free culture medium which had culture HeLa cell lines for 40 h was collected to isolate exosomes. Then, exosomes were identified by transmission electron microscopy (C). Cell lysates and exosomes were analyzed by western blot as indicated (D)

Fig 4: The CR2 domain in UBE2O interacts with SDPR. A Flag-PTRF or Flag-SDPR expression plasmid was infected into HeLa cells. After cell lysates were prepared, Flag-PTRF or Flag-SDPR in cell lysates was immunoprecipitated with Flag M2 beads. PTRF-associated endogenous UBE2O or SDPR-associated endogenous UBE2O was detected by IB. B HeLa cells were transiently transfected with Flag-UBE2O vector. Flag-UBE2O-associated endogenous SDPR was detected by IP and IB. C Wild-type SDPR and SDPR deletion mutants D1 and D2 were shown as schematic representations. D UBE2O-Myc and Flag-SDPR or Flag-SDPR deletion constructs (D1–D2) vectors were transfected into HEK293T cells. After 40 h transfection, cells were harvested for IP and IB analyses. E Flag-SDPR and five UBE2O deletion constructs (D1–D5) were expressed in HEK293T cells. After cell lysates were precipitated with Flag M2 beads, immunoprecipitates and the total cell lysates were immunoblotted with the indicated antibody as shown

Fig 5: UBE2O controls the secretion of exosome-related PTRF and inhibits exosome secretion. A UBE2O was transiently transfected into PTRF or SDPR stable HeLa cell line with PEI. Transmission electron microscopy images of exosomes derived from overexpression HeLa cells were shown as indicated. Scale bar: 100 nm. B Exosomes from four groups of overexpressed cells were diluted in the same fold (1:1000). Then, the size distribution of exosomes was identified with nanoparticle tracking analysis (NTA). C, D HeLa cells were stably transfected with Flag-PTRF and transiently transfected with UBE2O-Myc. After 40 h of cell culture, exosomes in the cell supernatants were extracted. Exosome components and overexpressed cell components were analyzed by western blot. E, F, G, H Histograms showed densitometry analyses of exosomal PTRF (E), cell PTRF (F), CD63 (G) and TSG101 (H) expression relative to cell GAPDH expression. I Total protein of exosomes from four groups of overexpressed cells was measured by BCA assay. J The number of particles which with the size in 30–150 nm was determined by NTA. All statistical data are the means \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document}± SEM from more than 3 independent experiments. *P \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$<$$\end{document}< 0.05; **P \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$<$$\end{document}< 0.01; ***P \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$<$$\end{document}< 0.001