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. 2025 Jan 2;15:325. doi: 10.1038/s41598-024-83460-x

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. 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-nc-nd/4.0/.

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Fig. 2 — Lipid dyshomeostasis in NPC2^−/− HEKs. a Representative epifluorescence images of untreated WT HEKs, NPC2^−/− HEKs and HP-β-cyclodextrin treated NPC2^−/− HEKs, labelled with the fluorescent probe filipin (blue) to mark free cholesterol, DRAQ5-647 (red) to stain the nucleus and an antibody to LAMP1 to mark the late endosomal/lysosomal membrane (green). Insets (bottom row) show late endosomes of the NPC2^−/− HEKs co-stained with LAMP1 and filipin (unesterified cholesterol) whereas no filipin is detectable in late endosomes of WT HEK. b Quantification of the filipin fluorescence intensity (unesterified cholesterol) detected in WT andNPC2^−/− HEKs. AU = arbitrary units c–g Mass spectrometry quantitation of the levels of: c Sphingosine (Sph d18:1); d Glucosylsphingosine (GlcSph d18:1); e Total Glucosylceramide (sum of GlcCer chain length species d16:0, d18:0, d20:0, d22:0, d24:0, d24:1); f Total Sphingomyelin (sum of SM chain length species d16:0, d18:0, d20:0, d22:0, d24:0, d24:1) and g Total Ceramide (sum of Cer chain length species d16:0, d18:0, d20:0, d22:0, d24:0, d24:1) in WT and NPC2^−/− HEKs. Mass- spec data are reported as pg/cell and expressed as mean +/- standard deviation (SD). ****p < 0.0001; **p < 0.01; ns = not significant. p values were calculated with the Student’s T test. Each dot of the graphs indicates a technical replicate (10) replicates used.