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. 2018 Nov 2;9:4582. doi: 10.1038/s41467-018-06941-4

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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. 2 — ARTS regulates expansion of the intestinal stem cell niche. a Control and Sept4/ARTS-deleted (S/A^−/−) small intestinal wholemounts, viewed from the base of the crypt. Note the high presence of granularity in S/A^−/− crypts, indicative of Paneth cells. Dashed white circles demarcate the crypt base circumference. b Quantification for wild-type (WT) and S/A^−/− crypt base diameter measured from wholemount tissues. c Images of the crypt base in reporter mice shows enhanced numbers of Sept4/ARTS-deficient Lgr5-EGFP⁺ (Lgr5^EGFP; S/A^−/−) intestinal stem cells (ISCs). Dashed white circles demarcate the crypt base circumference. d Longitudinal sections of small intestinal crypts show that the Lgr5^EGFP; S/A^−/− crypt houses increased numbers of Lgr5-EGFP⁺ ISCs. Dashed white line demarcates the crypt base to the highest point of Lgr5-EGFP signal. e Relative number of Lgr5-EGFP⁺ ISCs per intestinal crypt in control and Lgr5^EGFP; S/A^−/− mice. f Real-time (RT)-PCR shows increased relative Lgr5 mRNA in S/A^−/− intestinal tissue. g WT and S/A^−/− intestinal crypts stained for lysozyme, marking Paneth cells. Dashed white line demarcates the crypt base to the highest point of lysozyme signal. h Relative number of Paneth cells per WT and S/A^−/− crypt. i Brightfield images of organoids grown from isolated WT and S/A^−/− crypts after 8 days in culture. j Quantification for maximum organoid size after 20 days in culture. k, l S/A^−/− intestinal organoids display greater numbers of k Lgr5-EGFP⁺ ISCs and l lysozyme⁺ Paneth cells in organoid crypts. Dashed white lines demarcate organoid crypts. Images and quantitations are representative of n = 3 mice per genotype. All organoid experiments were performed on well triplicates generated from 3 pooled mice per genotype. RT-PCR experiments were performed on 3 pooled mice per genotype, analyzed in triplicates. P values were determined using two-tailed unpaired Student’s t test where *P < 0.05 and ***P < 0.001. Error bars represent ±s.e.m. All experiments were repeated at least twice. Scale bars: 10 µm (c, d, g), 20 µm (k, l), 50 µm (a), 500 µm (i)