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. 2019 Jul 1;10:2902. doi: 10.1038/s41467-019-10835-4

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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. 2 — SEIP-1 localizes to peri-LD cages. a Visualization of SEIP-1::GFP (hjSi189, lane 3 in Fig. 1d) in a larval L4 stage animal. Autofluorescence is pseudocolored blue. The LD marker mRuby::DGAT-2 (hjSi112) was used and the mRuby signal is pseudocolored magenta. A projection of 4.5 μm z stack centering at the second intestinal segment is shown. The boxed area was magnified 5× and shown on the right. Scale bar = 5 μm. b Visualization of human seipin::GFP (hjSi223) in a seip-1 (tm4221) mutant larval L4 stage animal. The human seipin isoform 2 was expressed from a single-copy transgene. A projection of 3 μm z stack centering at the second intestinal segment is shown. The LD marker and autofluorescence signals are displayed as in (a). The boxed area was magnified 5× and shown on the right. Scale bar = 10 μm. c Frequency distribution of LD diameter in larval L4 stage animals of indicated genotypes. The curve was fitted as in Fig. 1e. Inset: a scatter plot summarizing LD diameter. Total number of LDs measured: WT = 4600; seip-1(tm4221) = 6069; seip-1 (tm4221);hjSi223 = 4258. d Label-free quantification of lipid concentration of individual LDs by SRS. LDs with a diameter of 0.8–1.7 μm in the second intestinal segment were measured. Total number of LDs analyzed: SEIP-1::GFP (−) = 34; SEIP-1::GFP (+) = 22. e Visualization of SEIP-1::GFP in LDs purified from hjSi3 animals. LDs were stained with LipidTox Deep Red (pseudocolored magenta). f As in (a), but with an animal overexpressing SEIP-1::GFP (hjSi3, lane 4 in Fig. 1d). g Percentage of recovered fluorescence of ACS-22::GFP (resident ER protein, hjSi29) and SEIP-1::GFP (hjSi3) in wild-type animals. The connecting curves show the means and the filled areas show the SEM range. Number of photobleaching events: ACS-22::GFP = 17; SEIP-1::GFP full cage = 15; SEIP-1::GFP partial cage = 20. h As in (f), but in daf-22(ok693) mutant background. i The percentage of LDs associated with SEIP-1::GFP in the second intestinal segment in wild-type and daf-22(ok693) mutant animals. Number of animals analyzed: wild type (WT) = 11, daf-22(−) = 14. For all scatter plots, median with interquartile range is displayed