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. 2021 Feb 23;12:1252. doi: 10.1038/s41467-021-21525-5

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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. 5 — a Confocal image of a Dox-inducible mitochondria-LDs MCSs reporter cell expressing mitoBFP (blue) and ACSL3-Halo (magenta). Yellow arrows denoted the reconstituted GFP (green) at MCSs. SplitGFP representing the reconstituted GFP fluorescence at mitochondria-LD junctions. b Cells as in a were treated with either scrambled or VPS13D siRNAs under OA/EBSS. Left: whole cell image; Right: one inset from a boxed region in whole cell image. c Quantification of mean GFP fluorescence intensity of mitochondria-LDs MCSs reporter cells upon treatment with either scrambled or VPS13D siRNAs by flow cytometry. A.U., arbitrary unit. d Confocal image of HEK293 cells expressing ACSL3-Halo (magenta) and mitoBFP (blue) upon treatments with scrambled (top panel) or VPS13D siRNAs (bottom panel). Left: whole cell image; Right: two insets from boxed regions in whole cell image with co-localized pixels in white representing MCSs. e Quantification of number of mitochondria-LDs MCSs per mitochondrial length; 18 scrambled siRNA treated cells and 14 VPS13D siRNA treated cells were calculated. f Percentage of LDs at peri-nuclear region; 36 scrambled siRNA treated cells and 29 VPS13D siRNA treated cells were calculated. The peri-nuclear region was defined as 10 μm distal to rims of nucleus. g, h Quantification of LDs size (g) and density (h); 25 scrambled siRNA treated cells and 21 VPS13D siRNA treated cells were calculated. i Duration of mitochondria-LDs interactions. 35 ROIs with well-resolved mitochondria and LDs from scrambled siRNA treated cells and 35 ROIs from VPS13D siRNA treated cells were calculated. j Representative time-lapse images of cells as in d with white pixels representing possible mitochondria-LDs junctions. At least three independent imaging sessions were performed with similar results in c, e–i. The statistic analysis was performed with two-tailed unpaired student’s t-test, and data are means ± SD in c, e–i. Time in sec. Scale bar, 10 μm in whole cell image and 2 μm in insets in a, b, d and 2 μm in j.