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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. 7 — a Time-course plotting LysoTracker-positive puncta in HeLa cells pre-treated with siRNA targeting GFP (72 h) and GW4869 (10 µM) or DMSO (0.5%, 30 min) and pulse-treated with GPN. Data are means ± SD. n = 52 cells for DMSO and 39 cells for GW4869 over three independent experiments. b Time-course plotting LysoTracker-positive puncta in HeLa cells pre-treated with siRNAs targeting ALIX/TSG101 and GW4869 or DMSO and pulse-treated with GPN. Data are means ± SD. n = 46 cells for DMSO and 44 cells for GW4869 over three independent experiments. c Survival rate of HeLa cells pre-treated with siRNA targeting GFP after 5 h of exposure to LLOMe at indicated concentrations in the presence of GW4869 or DMSO. Data are means ± SD. n = 3 independent experiments. d Survival rate of HeLa cells pre-treated with siRNA targeting ALIX/TSG101 after 5 h exposure to LLOMe at indicated concentrations in the presence of GW4869 or DMSO. Data are means ± SD. n = 3 independent experiments. e Time-course plotting LysoTracker-positive puncta in HeLa cells pre-treated with siRNAs targeting ALIX/TSG101 and GFP or nSMase-1 and pulse-treated with GPN. Data are means ± SD. n = 28 cells for siGFP and 28 cells for sinSMase-1 over three independent experiments. f Time-course plotting LysoTracker-positive puncta in HeLa cells pre-treated with siRNAs targeting ALIX/TSG101 and GFP or nSMase-2 and pulse-treated with GPN. Data are means ± SD. n = 28 cells for siGFP and 40 cells for sinSMase-2 over three independent experiments. g Time-course plotting LysoTracker-positive puncta in WT or nSMase-2 KO HeLa cells pulse-treated with GPN. Data are means ± SD. n = 58 cells for WT and 66 cells for nSMase-2 KO over three independent experiments. h Survival rate of wild-type (WT) or nSMase-2 KO HeLa cells after 5 h exposure to LLOMe at the indicated concentration. Data are means ± SD. n = 4 independent experiments. P values were calculated by paired (c, d, h) or unpaired two-tailed t test (a, b, e, f, g). i Model illustrating how Ca²⁺-activated SM scrambling and turnover may promote restoration of damaged lysosomes.