Extended Data Figure 6: ER stress-induced enteritis of Xbp1 deficiency exacerbated by autophagy impairment correlates with age and cell death in intestinal epithelial cells and is dependent on IRE1α-mediated NF-κB activation.
From: Paneth cells as a site of origin for intestinal inflammation
a, Representative images of TUNEL-labelled intestinal epithelium (brown) (n = 7). Scale bars, 50 μm. b, Correlation of TUNEL+ cells with the severity of inflammation. Linear regression analysis of Atg16l1/Xbp1ΔIEC (left) and Atg7/Xbp1ΔIEC (right) with significant R2 and P value for deviation from zero shown (left panel, n = 5/5/7/7; right panel, n = 5). c, Linear least square regression analysis for the correlation of enteritis histology score with cell death and age of animals by genotype. Each dot represents a single animal (grey, wild type; yellow, Atg16l1ΔIEC; blue, Xbp1ΔIEC; red, Atg16l1/Xbp1ΔIEC mice) and the plane represents the linear regression for the enteritis histology score as a function of age and TUNEL labelling for Atg16l1/Xbp1ΔIEC mice (n = 6/13/12/12). Note that the severity of inflammation significantly correlates with numbers of TUNEL+ intestinal epithelial cells and age only in Atg16l1/Xbp1ΔIEC mice (R2 = 0.602, P = 0.016). The three-dimensional (3D) plot is also available online in video format (Supplementary Video 1). Regression analysis was performed using the R package lessR (http://cran.r-project.org/web/packages/lessR/index.html), last accessed May 2013. d, shCtrl or shXbp1 MODE-K cells were co-silenced for Atg16l1 (siAtg16l1) or with scrambled siRNA (siCtrl), and analysed by flow cytometry for annexin V and propidium iodide (PI) uptake (one-way ANOVA with post-hoc Bonferroni; mean ± s.e.m.). e, Densitometry of the immunoblot shown in Fig. 3b (n = 3; one-way ANOVA with post-hoc Holm’s corrected unpaired Student’s t-test; mean ± s.e.m.). f, Immunohistochemical staining for p-IκBα in the small intestinal epithelium (n = 3). Scale bars, 20 μm. g, NF-κB consensus sequence binding assay after stimulation of shCtrl and shXbp1 MODE-K cells for 5 and 20 min with indicated concentrations of TNF. h, Nfkbia expression, a prototypic NF-κB-transactivated gene, after TNF stimulation of shCtrl and shXbp1 MODE-K cells (mean ± s.e.m.). i, Representative images of TUNEL-labelled sections after administration of BAY11-7082 or vehicle (n = 3/4/4). Scale bars, 50 μm. j, Enteritis histology score of mice treated with the NF-κB inhibitor BAY11-7082 or vehicle (n = 7/9/8; median shown; Kruskal–Wallis with post-hoc Holm’s-corrected Mann–Whitney U-test). k, Expression of Nfkbia in Ern1- and control-silenced shXbp1 and control MODE-K cells after TNF stimulation (mean ± s.e.m.). Results represent three (h, k) or two (d, g) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
