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. 2018 Jul 3;8:10004. doi: 10.1038/s41598-018-28296-y

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Spermine clears prion aggregates by enhancing autolysosomal flux. SMB.s15 cells were either left untreated or treated with 10 μM CQ or treated with 5 μM Spermine alone or 5 μM Spermine and 10 μM of CQ for 24 hrs. (a) Representative images showing increased number of LC3 positive vesicles on spermine treatment (N = 3). (b) Significantly increased LC3 positive vesicles on spermine + CQ treatment compared to control + CQ treatment. (c) Representative images of increased number of Lyso-ID positive lysosomes on spermine treatment (N = 3). (d) Significantly increased lysosomes on spermine treatment. (e) Representative immunoblot to show increased conversion of LC3I to LC3II on spermine and CQ treatment (lanes 3, 4) compared to untreated cells (Lanes 1, 2) (N = 3). (f) Quantification of LC3II/ LC3I ratio as a marker of autophagic flux. SMB.s15 cells were treated with 5 μM spermine for 0, 24 and 72 hrs and 10 μM of CQ was given 16 hrs before the end point of spermine treatment to visualise the autolysosomes. (g) Representative immunofluorescence images of SMB.s15 cells stained for PrP^Sc and LC3 antibodies (N = 3). (h) Representative TEM image after 72 hrs of spermine treatment showing vesicles with reduced aggregates, area inside the circle is enlarged to show the vesicles (N = 2). (i) Vesicles with significantly reduced PrP^Sc aggregates on 72 hrs of spermine treatment. Scale bar for confocal images = 10 μm. Respective full length blots are shown in Supplementary Fig. 3. Abbreviations used: Chloroquine (CQ), Transmission Electron Microscopy (TEM).