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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. 1 — SEIP-1 regulates LD size. a Schematic representation of seip-1. Exons and untranslated regions are indicated by black and white boxes, respectively. The region deleted in the tm4221 allele is indicated. Scale bar = 100 bp. b Label-free quantification of neutral lipid content of individual animals by Stimulated Raman Scattering (SRS). Number of animals analyzed: wild type (WT) = 22, daf-22(−) = 22, seip-1(−) = 24. c Visualization of LDs using mRuby::DGAT-2 (hjSi112) in a WT or seip-1 (tm4221) mutant larval L4 stage animal. mRuby is in red and autofluorescence from lysosome-related organelles (LROs) is pseudocolored cyan. A projection of 7.5 μm z stack centering at the second intestinal segment is shown. d The expression levels of SEIP-1::GFP and endogenous SEIP-1 were determined by SDS-PAGE and immunoblotting with anti-SEIP-1 antibodies. A non-specific band at ~95 kDa served as a loading control. The expression level of endogenous SEIP-1 in WT animals served as a reference for normalization of signals in other samples. Mean ± SD is presented from three independent experiments. e Frequency distribution of LD diameter. The curve was fitted twice using Fit Spline/LOWESS (20 points in smoothing window, 4000 segments) method based on a histogram with a bin size of 0.3 μm (see Supplementary Fig. 1d, applicable to all subsequent frequency distribution curves). Inset: a scatter plot summarizing LD diameter. Total number of LDs measured: WT = 1680, seip-1 (tm4221) = 3934, hjSi189;seip-1 (tm4221) = 2224, and hjSi3;seip-1 (tm4221) = 1209. f Visualization of intestinal peroxisomes in a WT or seip-1 (tm4221) larval L4 stage animal. Signals from tagRFP::DAF-22 peroxisomal thiolase were pseudocolored magenta. A projection of 6.5 μm z stack centering at the second intestinal segment is shown. g The total number of intestinal peroxisomes in a normalized volume of 80 × 80 × 6.5 μm (xyz). n = 10 animals. For all fluorescence images, scale bar = 5 μm. For all scatter plots, median with interquartile range is displayed. For all statistics, *p < 0.05, **p < 0.01, ***p < 0.001; ****p < 0.0001 (unpaired t-test), which applies to all subsequent statistical analysis