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. 2019 Nov 14;179(5):1144–1159.e15. doi: 10.1016/j.cell.2019.10.015

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© 2019 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Figure S2 — In Vitro Culture of a Self-Organizing Colonic Epithelial Monolayer, Related to Figure 2

(A-B) β-catenin staining demonstrated the distinct cell shapes on ALId0 and ALId21 (A). Cell height was quantified and plotted to compare ALId21 and ALId0 (B). n = 3 samples/group.

(C-D) Microvilli length measured in the transmission electron microscopy images and compared between ALId28 and ALId0. n = 3 samples/group (10 cells per sample)

(E) Transmission electron microscopy images showed the morphology of goblet cells (left panel) and enteroendocrine cells with secretory granules (right panel). Yellow dashed line outlined an enteroendocrine cell.

(F) Actin (green) and UEA1 (red) double staining described polarization and the presence of a mucus layer in ALId21 monolayers.

(G) Immunostaining of major colonic epithelium lineage markers on ALId0 monolayers.

(H) Principal component analysis of transcriptomic profiles of monolayers harvested on ALId0, ALId4, ALId7, ALId14 and ALId21.

(I) GSEA-based analysis comparing the mRNA profiles of ALId21 cells to published goblet (left panel) and enteroendocrine cells (right panel) (Jadhav et al., 2017). See also Tables S1 and S2.

(J) H&E staining showed a cell extrusion event (arrow and higher power view on the right) on ALId21.

(K) ALId21 monolayer culture was dissociated by trypsin digestion and re-plated onto a new Transwell followed by 7 days of submersion and 14 days of ALI. Cell morphology after the passaging procedure is defined by H&E staining. Goblet cell and enteroendocrine cell differentiation was demonstrated by Muc2 and Chga staining.

(L) EdU and Ki67 co-staining on monolayers that were labeled by EdU incorporation for 3 h and washed out for various times (0, 1 and 6 days).

(M-N) Hopx^GFP+ (left panel in M), Lgr5^GFP+ (right panel in M) or Hopx^CreERT2 (middle panel in M) transiently labeled cells co-stained for Ki67 on ALId21. The proliferation rate of Hopx-labeled cells on ALId21 was plotted in the form of a pie chart (left panel in N). The percentage of Hopx+ cells within the Ki67% loci was plotted as a pie chart (right panel in N). n = 3 samples/group.

Mean values ± SD are shown. Two-tailed Student’s t test: ^∗p < 0.05; ^∗∗p < 0.01. Bars: (A, F, G, J, K, L and M) 25 μm. Histological, in situ hybridization, EM and immuno-fluorescent images are representative of at least three samples examined.