Figure 2.
Age-dependent propensity of IECs for efferocytosis. (A and B) Immunostaining of intestinal epithelial stem cell organoid–derived cell monolayers generated from adult (A) or neonatal (B) small intestinal tissue 2 h after coculture with growth factor–starved tdTomato expressing IECs (starved IECs, red) derived from adult stem cell organoids. White squares indicate the area that is magnified; white arrowheads in the magnified image highlight intracellular tdTomato (red)-positive cell material. Counterstaining with phalloidin (green) and DAPI (blue). Bar, 100 μm. (C) Three-dimensional image reconstruction to illustrate the dtTomato-positive starved IEC (red, highlighted with a red line) engulfed by a neonatal stem cell organoid–derived IEC (highlighted with a white line). Counterstaining with phalloidin (green) and DAPI (blue). (D) Quantitative analysis of the number of internalization events of tdTomato-positive growth factor–starved cells/cell material (TR+ cells) by intestinal epithelial stem cell organoid–derived cell monolayers generated from neonatal (N) or adult (AD) small intestines per mm2. 30–49 image fields with the size of 1.25 × 1.00 mm obtained from two to three independent experiments were analyzed. Mann–Whitney U test. ****, P < 0.0001. (E and F) TEM images of a neonatal intestinal epithelial stem cell organoid–derived cell monolayer co-cultured with growth factor–starved intestinal epithelial stem cell organoid cells (starved IECs) for 2 h. (E) White arrowheads illustrate the membrane extensions of monolayer cells directed toward the starved IECs. Bar, 5 µm. (F) The white squares indicate the areas that are magnified in panels i and ii. Three stages of efferocytosis: uptake of cell debris (magnified in i), endosomal compartments with loosely packed cargo suggestive of an EEf, and with electron-dense cargo suggestive of an LEf (magnified in ii). Bar, 5 µm (panels i and ii, 1 µm). (G) TNF-α (pg/ml) in the cell culture supernatant of neonatal intestinal epithelial stem cell organoid–derived cell monolayers left untreated, stimulated with S. Typhimurium (MOI 10:1) or exposed to growth factor–starved intestinal epithelial stem cell organoid cells (starved IECs) for 2 h. Two independent experiments with three replicates were analyzed, the graph shows one representative experiment. One-way ANOVA Kruskal–Wallis test with Dunn’s post-test. ****, P < 0.0001; ns, non-significant.

Age-dependent propensity of IECs for efferocytosis. (A and B) Immunostaining of intestinal epithelial stem cell organoid–derived cell monolayers generated from adult (A) or neonatal (B) small intestinal tissue 2 h after coculture with growth factor–starved tdTomato expressing IECs (starved IECs, red) derived from adult stem cell organoids. White squares indicate the area that is magnified; white arrowheads in the magnified image highlight intracellular tdTomato (red)-positive cell material. Counterstaining with phalloidin (green) and DAPI (blue). Bar, 100 μm. (C) Three-dimensional image reconstruction to illustrate the dtTomato-positive starved IEC (red, highlighted with a red line) engulfed by a neonatal stem cell organoid–derived IEC (highlighted with a white line). Counterstaining with phalloidin (green) and DAPI (blue). (D) Quantitative analysis of the number of internalization events of tdTomato-positive growth factor–starved cells/cell material (TR+ cells) by intestinal epithelial stem cell organoid–derived cell monolayers generated from neonatal (N) or adult (AD) small intestines per mm2. 30–49 image fields with the size of 1.25 × 1.00 mm obtained from two to three independent experiments were analyzed. Mann–Whitney U test. ****, P < 0.0001. (E and F) TEM images of a neonatal intestinal epithelial stem cell organoid–derived cell monolayer co-cultured with growth factor–starved intestinal epithelial stem cell organoid cells (starved IECs) for 2 h. (E) White arrowheads illustrate the membrane extensions of monolayer cells directed toward the starved IECs. Bar, 5 µm. (F) The white squares indicate the areas that are magnified in panels i and ii. Three stages of efferocytosis: uptake of cell debris (magnified in i), endosomal compartments with loosely packed cargo suggestive of an EEf, and with electron-dense cargo suggestive of an LEf (magnified in ii). Bar, 5 µm (panels i and ii, 1 µm). (G) TNF-α (pg/ml) in the cell culture supernatant of neonatal intestinal epithelial stem cell organoid–derived cell monolayers left untreated, stimulated with S. Typhimurium (MOI 10:1) or exposed to growth factor–starved intestinal epithelial stem cell organoid cells (starved IECs) for 2 h. Two independent experiments with three replicates were analyzed, the graph shows one representative experiment. One-way ANOVA Kruskal–Wallis test with Dunn’s post-test. ****, P < 0.0001; ns, non-significant.

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