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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. 8 — ARTS mediates its functions via interaction with XIAP in the intestinal crypt. a Confocal microscopy image of mouse small intestinal crypt base shows co-localization of ARTS and XIAP within the same cell [n = 4 mice]. b Western blot of XIAP in isolated wild-type (WT) and Sept4/ARTS^−/− (S/A^−/−) crypts. Signal intensity is normalized to GADPH. c Super-resolution stimulated emission depletion (STED) microscopy shows co-localization of ARTS and XIAP in normal and apoptotic human colonic crypt base cells. d Pearson’s coefficient values demonstrate higher co-localization of ARTS and XIAP in both mouse and human apoptotic crypt cells [n = 3 human colons and n = 4 mice. Error bars represent s.e.m.]. e Co-immunoprecipitation (co-IP) of ARTS and XIAP in isolated XIAP^ΔRING small intestinal crypts. XIAP^ΔRING only interacts mildly with ARTS in the absence of apoptotic stimulation. After staurosporine (STS) treatment, efficient binding between ARTS and XIAP^ΔRING is detected. f Western blot and signal intensity of active caspase-3 (CP3) in STS-treated crypts show that deletion of XIAP or RING domain increases cleaved CP3 levels. g Organoids derived from XIAP^−/−, XIAP^ΔRING and S;X^−/− crypts display hindered growth and development after 11 days post seeding. h XIAP^−/−, XIAP^ΔRING and S;X^−/− organoids stained for Ki67. i, j Fold difference in i organoid formation capacity and j number of Ki67⁺ cells in WT, S/A^−/−, XIAP^−/−, XIAP^ΔRING and S;X^−/− organoids. k XIAP^−/−, XIAP^ΔRING and S;X^−/− mice display greater body weight loss than both WT and S/A^−/− mice at 10 days following a 5-day DSS (2.5% w/v) regime [n = 3 mice per genotype]. All blots are representative of n = 3 pooled mice per genotype. Organoid data are representative of n = 3 pooled mice per genotype analyzed in well triplicates. P values were determined between each group or for each genotype compared to the WT control using unpaired two-tailed Student’s t test, where *P < 0.05, **P < 0.01, and ***P < 0.005. Error bars represent ±s.e.m., unless otherwise stated. All experiments were repeated at least twice. Scale bars: 1 µm (c right), 3 µm (c left), 5 µm (a), 100 µm (g, h)