Figure 5: XBP1 regulates regenerative DDR in a DDIT4L - mTOR-dependent manner.

(A) RNA sequencing of n = 3 organoid samples from n=3 individual animals. (B) Venn diagram of uniquely and commonly regulated genes in intestinal organoids of H2b/Xbp1^ΔIEC and H2b/p53^ΔIEC vs. H2b^ΔIEC mice. (C) qRT-PCR analysis of small intestinal crypt cells of 52 weeks old H2b/Xbp1^fl/fl (n = 3, 3 males), Xbp1^∆IEC (n = 6, 1 female, 5 males), H2b^∆IEC (n = 6, 2 females, 4 males) and H2b/Xbp1^∆IEC (n = 5, 2 females, 3 males) mice. qRT-PCR analysis of (D) ModeK cells, treated with PBS, 2.5 μM AraC or 50 μM Nutlin for 24 hours and (E) of ModeK cells transfected with siRNA Ctrl and p53 and treated with PBS or 2.5 μM AraC for 24 hours, both experiments representative of a minimum of 3 individual experiments with n = 3 technical replicates. Relative mRNA expression is normalized to Actb (β-actin) or Gapdh. Representative images (F) and (G) statistical analysis of the p-4E-BP1-stained SI of 52 weeks old H2b/Xbp1^fl/fl (n = 3, 3 males), Xbp1^∆IEC (n = 3, 1 female, 2 males), H2b^∆IEC (n = 3, 1 female, 2 males), H2b/Xbp1^∆IEC (n = 13, 8 females, 5 males) and H2b/p53^∆IEC (n = 3, 3 males) mice. Western blots, representative of each n = 3 individual experiments, of (H) ModeK cells transfected with siRNA against Rnaseh2b for 24 hours, (I) untreated intestinal organoids of the indicated genotypes and (J) small intestinal crypt cells from age-matched H2b/Xbp1^fl/fl (2 females), Xbp1^∆IEC (2 females), H2b^∆IEC (1 female, 1 male) and H2b/Xbp1^∆IEC (2 females) mice after exposure to chronic DSS colitis. ß-Actin and ß-Tubulin served as loading controls. Data are expressed as mean ± standard error of the mean and significance was determined using an unpaired Student’s t-test (D,E) or one-way ANOVA(G) * p < .05, ** p < .01, *** p < .0001, **** p < .00001.