Fig. 4. BECLIN1-deficient intestinal organoids display increased apoptosis and morphological defects.

a, b Deletion of BECLIN1, but not ATG7, by addition of 4-hydroxytamoxifen (4-HT) led to significantly smaller intestinal organoids. a, c There was also a significant reduction in the number of “buds” per organoid formed, indicative of reduced stem cell-containing crypt formation. d BECLIN1- and ATG7-deficient organoids were analyzed for propidium iodide (PI) and Annexin V staining by flow cytometry, to detect apoptotic cell death, over days 4 to 7 post the first 4-HT dose. Consistent with our in vivo observations, we saw enhanced apoptosis in the Becn1^ΔIEC but not Atg7^ΔIEC or wild-type organoids. Data are representative of at least n = 3 independent experiments. Graphs indicate the mean ± 95% confidence interval, except for the FACS plot in d which indicate mean ± S.D. Significance determined by Welch’s unpaired t test for b and c, and by 2way ANOVA Tukey’s multiple comparisons test for d. The ^{+/+ IEC} and ^{fl/fl IEC} refer to intestinal organoids derived from Becn1^+/+;Vil1-CreERT2^Cre/+ or Atg7^+/+;Vil1-CreERT2^Cre/+ and Becn1^fl/fl;Vil1-CreERT2^Cre/+ or Atg7^fl/fl;Vil1-CreERT2^Cre/+ mice.