Loss of viability in ATG16L1-deficient intestinal organoids is associated with an IFN signature. (A) Unsupervised clustering based on expression of most variable genes by genotype and treatment with 20 ng/mL TNF-α for 2 hours. n = 4 replicates per group, each replicate was derived from separate mice. (B) Heat map of genes with a twofold change in Atg16L1^ΔIEC (ΔIEC) over Atg16L1^f/f (f/f) organoids. ISGs are highlighted with red and bold. (C) Pathway analysis of genes differentially expressed between f/f and ΔIEC naive organoids. (D) Quantitative reverse-transcription polymerase chain reaction (RT-PCR) measurement of indicated ISG expression normalized to actb in small intestinal organoids from B6 mice that were treated or not with 100 nM ruxolitinib at day 3. n = 3 mice each. (E) Viability of small intestinal organoids stimulated with 20 ng/mL TNF-α and/or 100 nM ruxolitinib for 48 hours. n = 3 mice each. (F) Western blot analysis of cell death–related proteins at day 3. f/f and ΔIEC organoids cultured with or without 100 nM ruxolitinib were treated with 20 ng/mL TNF-α for 2 hours. Blots are representative of ≥2 independent repeats. (G) Viability of small intestinal organoids stimulated with 20 ng/mL TNF-α and/or 500 μM 2-aminopurine (2-AP) for 48 hours. n = 3 mice each. Representative images (H) and viability (I) of small intestinal organoids from Atg16L1^ΔIEC mice transduced with lentiviruses encoding shRNAs targeting Eif2ak2 or a nonspecific control and treated or not with 20 ng/mL TNF-α for 48 hours; n = 3 mice each. Scale bars, 1 mm. Data points in D, E, G, and I are mean of technical replicates. Bars represent mean ± standard error of the mean, and ≥2 independent experiments were performed. **P < .01, ***P < .001, ****P < .0001.