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. 2022 Apr 6;13:1875. doi: 10.1038/s41467-022-29481-4

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© The Author(s) 2022

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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Fig. 3 — a Schematic outline of lysosomal repair assay. b Time-lapse images of LysoTracker-labeled/EqtSM-expressing HeLa cells pulse-treated with GPN (200 μM, 2 min). c Time-course plotting LysoTracker- and EqtSM-positive puncta in cells treated as in (b). Data are means ± SD. n = 18 cells over four independent experiments. d Time-lapse images of LysoTracker-labeled WT and SMS-KO HeLa cells pulse-treated with GPN. e Time-course plotting LysoTracker-positive puncta in cells treated as in (b). Data are means ± SD. n = 29 cells for WT, 24 cells for SMS-KO over four independent experiments. f Time-lapse images of LysoTracker-labeled SMS-KO cells transduced with doxycycline-inducible SMS1 (pInd-SMS1) and pulse-treated with GPN following 48 h preincubation without (–doxy) or with 1 µg/ml doxycycline (+doxy). g Time-course plotting LysoTracker-positive puncta in cells treated as in (d). Data are means ± SD. n = 20 cells for +doxy, 17 cells for –doxy over three independent experiments. h Time-lapse images of SMS-KO cells expressing EqtSM treated with 1 mM LLOMe for the indicated time following 16 h preincubation with 160 μM PC or PC/SM (70/30 mol%). i Time-course plotting LysoTracker-positive puncta in SMS-KO cells pulse-treated with GPN following preincubation with PC or PC/SM as in (h). Data are means ± SD. n = 50 cells per condition over three independent experiments. j Survival rate of WT and SMS-KO cells after 5 h exposure to LLOMe at the indicated concentration. Data are means ± SD. n = 4 independent experiments. k Survival rate of WT cells pre-treated with siRNAs targeting GFP or ALIX/TSG101 for 72 h and exposed for 5 h to LLOMe at indicated concentrations. Data are means ± SD. n = 3 independent experiments. l Survival rate of SMS-KO cells pre-treated with siRNAs targeting GFP or ALIX/TSG101 for 72 h and exposed for 5 h to LLOMe at indicated concentrations. Data are means ± SD. n = 3 independent experiments. P values were calculated by unpaired (e, g, i) or paired two-tailed t test (j, k, l). Scale bar, 10 µm.