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. 2019 Oct 17;2:380. doi: 10.1038/s42003-019-0615-z

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© The Author(s) 2019

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 — High cytosolic calcium levels cause accumulation of cholesterol in late endosomes/lysosomes in the absence of spatacsin. a Quantification of the number of LAMP1-positive tubules in Spg11^+/+ and Spg11^−/− fibroblasts expressing LAMP1-mCherry, analyzed by live imaging. The graphs show the mean ± SEM. N > 60 cells analyzed in four independent experiments. Two-way ANOVA: *p = 0.034, ***p < 0.0001. b Western blots of TFEB in cytosolic and nuclear fractions of Spg11^+/+ and Spg11^−/− fibroblasts transfected with control siRNA or a specific siRNA that downregulates TFEB. c Quantification of the number of LAMP1-positive tubules in Spg11^+/+ and Spg11^−/− fibroblasts transfected with control siRNA or siRNA that downregulates TFEB. The graph shows the mean ± SEM. N > 60 cells analyzed in four independent experiments. Two-way ANOVA: ***p < 0.0001. d Downregulation of STIM1 decreases the amount of cholesterol colocalized with LAMP1 in Spg11^−/− fibroblasts. The graph shows the mean ± SEM. N > 95 cells analyzed in three independent experiments. Two-way ANOVA: ***p < 0.001. e Lowering intracellular calcium levels with EGTA-AM (1 h) decreases the amount of cholesterol colocalized with LAMP1 in Spg11^−/− fibroblasts. The graph shows the mean ± SEM. N > 45 cells analyzed in three independent experiments. Two-way ANOVA: ***p < 0.001. f Downregulation of TFEB decreases the amount of cholesterol colocalized with LAMP1 in Spg11^−/− fibroblasts. The graph shows the mean ± SEM. N > 45 cells analyzed in three independent experiments. Two-way ANOVA: *p < 0.05. g Quantification of plasma membrane cholesterol with the probe GFP-D4, performed on live Spg11^+/+ and Spg11^−/− fibroblasts, showing that downregulation of STIM1 restores normal levels of cholesterol in the plasma membrane in Spg11^−/− fibroblasts. The graph shows the mean ± SEM. N > 180 cells analyzed in three independent experiments. Two-way ANOVA: ***p < 0.0001