ATG16L1 protects intestinal organoids from necroptosis by maintaining mitochondrial homeostasis. (A) Representative TEM images of mitochondria classified into type 1 (cristae are maintained), type 2 (mildly swollen and a moderate decrease in the number of visible cristae), and type 3 (severely swollen and >70% of cristae are missing, or highly dysmorphic and electron dense). At least 20 cells in each organoid were analyzed per group from three mice each. (B) Representative fluorescent images of organoids on day 3 treated ± TNFα and NAC and stained with MitoSOX Red. Quantification of MitoSOX mean fluorescence intensity (MFI) in the epithelium shown. Staining in the lumen was excluded. At least 20 organoids were quantified from three mice each. (C–E) Viability of organoids from Atg16L1^f/f and Atg16L1^ΔIEC mice treated ± TNFα and NAC (C), B6 and Rip3^−/− mice treated ± TNFα and CCCP (D), and B6 and Park2^−/− mice treated ± TNFα and Nec-1 (E). n = 3 mice each. (A and B) Bars: 0.5 µm (A); 50 µm (B). Data points in B represent individual organoids, and data points in C–E are mean of three technical replicates. Bars represent mean ± SEM, and at least two independent experiments were performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by one-way ANOVA and Tukey’s test in B and unpaired t test in C–E.