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. 2025 Jul 29;44(18):5086–5111. doi: 10.1038/s44318-025-00517-x

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© The Author(s) 2025

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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(A) 3D-SIM live-cell imaging of stable HeLa LAMP1-BFP cells loaded with lysotracker and expressing ATXN3-GFP. Cells were treated with 1 mM LLOMe for 12 min, washed, and followed during recovery in medium containing lysotracker for 3 h. Scale bar, 5 µm. (B) Quantification of 2D-SIM images taken in experiments shown in (A). n = 2 biologically independent experiments with >20 cells quantified per experiment. Lines indicate the median. (C) HeLa cells expressing ATXN3-mCherry and the PI(4,5)P2 sensor GFP-PH (PLCD1) were mock or LLOMe-treated as indicated and immuno-stained for LAMP1. Note the colocalization of ATXN3 and GFP-PH on a subpopulation of lysosomes. Scale bar, 10 µm. (D) Quantification of (C), n = 3 biological replicates with >20 cells per condition per experiment. One-way ANOVA with Tukey’s multiple comparison test. The line indicates the mean. (E) DQ-BSA assay for proteolytic activity in lysosomes. HeLa parental and ATXN3 knockout cells were LLOMe-treated for 1 h. DQ-BSA was added 13 h after washout, and DQ-BSA fluorescence in lysosomes was imaged at a total of 20 h after LLOMe washout. Scale bar, 10 µm. (F) Quantification of (E), n = 3 biological replicates with >40 cells per condition per experiment. Mixed effects analysis with Tukey’s multiple comparison test. Error bars represent the mean with SD. Source data are available online for this figure.