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. 2018 Mar 22;9:1195. doi: 10.1038/s41467-018-03531-2

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© The Author(s) 2018

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 — PHA-4 responses to nucleolar stress. a Nile Red staining of fixed worms. Synchronized smg-1(cc546);pha-4(zu225) eggs were hatched in M9 buffer to L1 worms at 24 ℃. Subsequently, L1 worms were transferred to NGM plates, raised at 20 ℃, and then collected when they reached 1 day of adulthood for analysis of lipid contents. Representative animals; the anterior is indicated on the left and the posterior is indicated on the right. Scale bar represents 20 μm. b Distribution of the lipid droplet size (% lipid droplets) as measured by Nile Red staining of fixed worms from a. Data are presented as the means ± SD of 10 animals for each worm strain. Significant difference between WT and a specific worm strain, ANOVA, ***P < 0.001. NS, no significant difference. c, d Percentage of triacylglycerol (% TAG) in total lipids (TAG + phospholipids, PL) analyzed by TLC/GC. Data are presented as the means ± SD of four biological repeats. Significant difference between WT and a specific worm strain, Student’s t-test, ***P < 0.001. NS, no significant difference. e Relative mRNA expression of pha-4 in WT and mutant worms. Data are presented as the means ± SD of three biological repeats. Significant difference between WT and a specific worm strain, ANOVA, ***P < 0.001, *P < 0.05. f Confocal microscopy of PHA-4::GFP {wgIs37[pha-4::GFP]} in WT, rrp-8(kun54), rsks-1(ok1255), and AD-treated worms at 1 day of adulthood. BF, bright field. Representative animals; the anterior is indicated on the left and the posterior is indicated on the right. Scale bar represents 10 μm. g Quantification of the fluorescence intensity of PHA-4::GFP from f. Data are presented as the means ± SD of at least 20 worms for each worm strain. Significant difference between WT and rrp-8(kun54) mutant background, ANOVA, ***P < 0.001. h Immunoblotting of PHA-4::GFP {wgIs37[pha-4::GFP]} with anti-GFP antibody in WT, rrp-8(kun54), rsks-1(ok1255), and AD-treated background worms at 1 day of adulthood. WT worms without GFP was used as a negative control and WT worms with PHA-4a::GFP {kunEx136[Pvha-6::pha-4a::GFP]} as a positive control. The relative protein levels of PHA-4::GFP were labeled