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. 2021 Sep 9;12:5354. doi: 10.1038/s41467-021-25621-4

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© The Author(s) 2021

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. 4 — a, b Normalized rate of mitochondrial division (number of mitochondrial divisions normalized by time and volume) in HeLa cells (a) expressing wild-type or mutants 3HA-Rab7. Cells from three independent experiments. One-way ANOVA with Dunnett’s Multiple Comparison Test; b when VAPs expression levels were downregulated using siRNAs. Cells from three independent experiments. Two-sided unpaired t-test. The graphs show the mean ± SEM. c Live-cell imaging of HeLa cells expressing GFP-ORP1L and mApple-TOM20. The white and yellow arrows show an ORP1L positive lysosome-mitochondria contact and a mitochondrial fission event. Scale bar: 1 µm. d Percentage of mitochondrial division events that were marked by GFP-ORP1L (cells from two independent experiments) in HeLa cells. e Representative maximum projection images of mitochondrial morphology in HeLa cells treated with indicated siRNAs. Scale bars: 10 µm and 5 µm (inset). f–h Mitochondrial morphology was quantified for f mean area per mitochondrion, g mitochondrial number per region of interest (ROI), and h number of junctions per mitochondria. Cells from three independent experiments. One-way ANOVA with Dunnett’s Multiple Comparison Test. The graphs show the mean ± SEM. i, j Normalized rate of mitochondrial division of i HeLa cells treated with indicated siRNAs. Cells from three independent experiments. One-way ANOVA with Dunnett’s Multiple Comparison Test and of j ORP1L WT and KO HeLa cells. Cells from three independent experiments. Two-sided unpaired t-test. The graphs show the mean ± SEM. k ORP1L mutants used and the loss of function caused by the mutations or deletions. l Normalized rate of mitochondrial division in ORP1L KO HeLa cells expressing GFP or GFP-ORP1L constructs and mApple-TOM20. The graphs show the mean ± SEM, cells from three independent experiments. One-way ANOVA with Dunnett’s Multiple Comparison Test. m Normalized rate of mitochondrial division in ORP1L KO HeLa cells expressing mTq2-GAI-ΔANKORP1L, iRFP-GID1-Rab7, and mApple-TOM20 before and after GA₃-AM (10 µM) treatment. Cells were imaged from 5 to 20 min after GA₃-AM treatment. Cells from three independent experiments. Two-sided unpaired t-test. The graphs show the mean ± SEM.