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. 2019 Apr 2;10:1492. doi: 10.1038/s41467-019-09525-y

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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. 6 — IEC of Rnf5^−/− mice activate immune responses and changes AMPs expression by IRE1α/sXBP1 signaling. a Western blot analysis of the indicated proteins in lysates of MODE-K-shCon and MODE-K-shRNF5 cells treated with thapsigargin (TG, 1 μM) for indicated time. b qRT-PCR analysis of mRNA levels of sXBP1, XBP1 and target genes in small intestine-derived IECs from naive WT or Rnf5^−/− mice (n = 4). c Representative micrographs of immunohistochemical (IHC) staining of BiP in the jejunum, ileum, and colon of YUMM1.5 tumor-bearing WT or Rnf5^−/− mice (scale bar = 25 μm). Lower graphs show quantification of IHC staining (n = 12 fields per group). Staining was scored semi-quantitatively based on staining intensity (0, 1, 2, or 3) multiplied by the percentage of positively stained cells (0–100), to give a maximum IHC score of 300. d Quantification of BiP and CHOP IHC staining in ileum sections from WT and Rnf5^−/− mice co-housed for 4 weeks prior to tumor inoculation (n = 12). e qRT-PCR analysis of sXBP1 mRNA levels in MODE-K-shCon and MODE-K-shRNF5 cells treated with the indicated concentration of MKC-4485 for 24 h (n = 3). f MFI of MHC class I and II on bone marrow-derived dendritic cells (BMDCs) incubated with media alone (no stimulation), conditioned media (CM) from shControl or shRNF5 MODE-K cells treated with MKC-4485 (n = 4). g qRT-PCR analysis of Defa1 mRNA levels in MODE-K-shCon and MODE-K-shRNF5 cells (n = 3). h qRT-PCR analysis of AMPs mRNA levels in IECs in MODE-K-shCon and MODE-K-shXBP1 cells (n = 3). i, qRT-PCR analysis of Defa1 mRNA levels in MODE-K cells treated with 2 μM MKC-4485 for 24 h (n = 3). Data are representative of three independent experiments (b, e–i) and two independent experiments (a, c, d) ≥3 mice per group. Graphs show the mean ± s.e.m. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001 by two-tailed t test or Mann–Whitney U test (b, c, g–i) or one-way ANOVA with Tukey’s correction for multiple comparisons (d–f)