Supplementary Materials

Supplementary Material for:

The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection

Mélanie Dieudé, Christina Bell, Julie Turgeon, Deborah Beillevaire, Luc Pomerleau, Bing Yang, Katia Hamelin, Shijie Qi, Nicolas Pallet, Chanel Béland, Wahiba Dhahri, Jean-François Cailhier, Matthieu Rousseau, Anne-Claire Duchez, Tania Lévesque, Arthur Lau, Christiane Rondeau, Diane Gingras, Danie Muruve, Alain Rivard, Héloise Cardinal, Claude Perreault, Michel Desjardins, Éric Boilard, Pierre Thibault,* Marie-Josée Hébert*

*Corresponding author. E-mail: marie-josee.hebert.chum{at}ssss.gouv.qc.ca (M.-J.H.); pierre.thibault{at}umontreal.ca (P.T.)

Published 16 December 2015, Sci. Transl. Med. 7, 318ra200 (2015)
DOI: 10.1126/scitranslmed.aac9816

This PDF file includes:

  • Fig. S1. Serum deprivation significantly increased caspase-3 activation and the percentage of cells with chromatin condensation in the absence of cell membrane permeabilization.
  • Fig. S2. Small-particle flow cytometry size calibration.
  • Fig. S3. Characterization of small particles secreted by apoptotic ECs sensitive to detergent treatment.
  • Fig. S4. Inhibition of autophagy with bafilomycin does not modulate exosome-like vesicle secretion by serum-starved ECs.
  • Fig. S5. Electron micrographs of apoptotic bodies released by serum-starved HUVECs and isolated by sequential ultracentrifugation.
  • Fig. S6. Electron micrographs of exosome-like vesicles released by serum-starved HUVECs and isolated by sequential ultracentrifugation.
  • Fig. S7. Gene ontology analysis of cellular components and biological processes for proteins unique to exosome-like vesicles and apoptotic bodies.
  • Fig. S8. Characterization of the presence of classical exosomal markers in the apoptotic exosome-like vesicles proteome.
  • Fig. S9. Redundant peptide count of perlecan in exosome-like vesicles and apoptotic bodies.
  • Fig. S10. Injecting equal amounts of proteins from preparations of apoptotic bodies and preparations of apoptotic exosome-like vesicles fail to unmask immunogenic activity in apoptotic bodies.
  • Fig. S11. Apoptotic exosome-like vesicles, unlike apoptotic bodies, favor B cell responses and autoimmunity.
  • Fig. S12. Intima/media ratio in murine allografts 3, 6, or 9 weeks after aortic transplantation.
  • Fig. S13. Characterization of anti-LG3 IgG subclasses.
  • Fig. S14. Healthy ECs secrete exosome-size vesicles lacking caspase-like proteasome activity.
  • Fig. S15. Apoptotic vascular smooth muscle cells and tubular epithelial cells also secrete active 20S proteasome in exosome-size EVs.
  • Fig. S16. Bortezomib treatment does not affect cell death levels.
  • Fig. S17. The proteasome is not detected in serum membrane vesicles isolated before surgery.

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Other Supplementary Material for this manuscript includes the following:

  • Table S1 (Microsoft Excel format). Quantitative proteomic identifications based on spectral counts in nanovesicles and apoptotic bodies from serum-starved apoptotic HUVECs.
  • Table S2 (Microsoft Excel format). Quantitative proteomic identifications based on spectral counts in nanovesicles from serum-starved apoptotic HUVECs reated with the proteasome inhibitor bortezomib or vehicle.
  • Table S3 (Microsoft Excel format). Ubiquitylated proteins identification by MS in nanovesicles from serum-starved HUVECs treated with the proteasome inhibitor bortezomib or vehicle.
  • Data S1 (.pdf format). Source data, unedited gels.
  • Data S2 (.pdf format). Source data, flow cytometry gating.

[Tables S1 to S3]