Figure 4.
Figure 4. ATG16L1 is necessary for the survival of intestinal organoids and Paneth cells. (A–C) Representative images of small intestinal organoids derived from Atg16L1f/f (f/f) and Atg16L1ΔIEC (ΔIEC) mice on the indicated days of culture (A). Area (B) and bud number (C) were quantified on day 7. At least 20 organoids were quantified from three mice each. (D) Viability of organoids on the basis of morphology (see Fig. S2 C) ± 20 ng/ml TNFα. n ≥ 10 mice each. (E) LDH cytotoxicity of organoids on day 2 quantified by LDH release assay. n = 3 mice each. (F) Time-series images corresponding to Video 1 in which organoids were treated with 20 ng/ml TNFα at time 0 in the presence of PI (red). Digits represent hours:minutes. Representative of three independent repeats. (G) Quantification of TNFα in culture supernatants from D on day 3. n = 11 per group. (H) Viability of organoids ± 20 µg/ml anti-TNFα antibody. n = 3 mice each. (I–K) Representative H&E images (I) and immunofluorescence images (J) of lysozyme (red) and DAPI (blue) of organoids on day 5. TNFα was added on day 4. Triangles indicate Paneth cells in I. (K) Total number of Paneth cells (left), total number of IECs (middle), and frequency of Paneth cells normalized to total IECs (right) were quantified from images in I. At least 20 organoids were quantified from three mice each. (L and M) Representative images of goblet cells (triangles) in periodic acid–Schiff (PAS)/Alcian blue staining of organoids from I (L) and quantification of total number of goblet cells (left), total number of IECs (middle), and frequency of Paneth cells normalized to total IECs (right; M). At least 20 organoids were quantified from three mice each. (A, F, I, J, and L) Bars: 100 µm (A); 50 µm (F, I, J, and L). Data points in B, C, G, K, and M represent individual organoids, and data points in D, E, and H 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 unpaired t test in B, C, D, E, G, and H and one-way ANOVA and Tukey’s test in K and M.

ATG16L1 is necessary for the survival of intestinal organoids and Paneth cells. (A–C) Representative images of small intestinal organoids derived from Atg16L1f/f (f/f) and Atg16L1ΔIEC (ΔIEC) mice on the indicated days of culture (A). Area (B) and bud number (C) were quantified on day 7. At least 20 organoids were quantified from three mice each. (D) Viability of organoids on the basis of morphology (see Fig. S2 C) ± 20 ng/ml TNFα. n ≥ 10 mice each. (E) LDH cytotoxicity of organoids on day 2 quantified by LDH release assay. n = 3 mice each. (F) Time-series images corresponding to Video 1 in which organoids were treated with 20 ng/ml TNFα at time 0 in the presence of PI (red). Digits represent hours:minutes. Representative of three independent repeats. (G) Quantification of TNFα in culture supernatants from D on day 3. n = 11 per group. (H) Viability of organoids ± 20 µg/ml anti-TNFα antibody. n = 3 mice each. (I–K) Representative H&E images (I) and immunofluorescence images (J) of lysozyme (red) and DAPI (blue) of organoids on day 5. TNFα was added on day 4. Triangles indicate Paneth cells in I. (K) Total number of Paneth cells (left), total number of IECs (middle), and frequency of Paneth cells normalized to total IECs (right) were quantified from images in I. At least 20 organoids were quantified from three mice each. (L and M) Representative images of goblet cells (triangles) in periodic acid–Schiff (PAS)/Alcian blue staining of organoids from I (L) and quantification of total number of goblet cells (left), total number of IECs (middle), and frequency of Paneth cells normalized to total IECs (right; M). At least 20 organoids were quantified from three mice each. (A, F, I, J, and L) Bars: 100 µm (A); 50 µm (F, I, J, and L). Data points in B, C, G, K, and M represent individual organoids, and data points in D, E, and H 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 unpaired t test in B, C, D, E, G, and H and one-way ANOVA and Tukey’s test in K and M.

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