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. 2021 Aug 4;12:4697. doi: 10.1038/s41467-021-24993-x

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Phase contrast images showing the phenotype of shScr, sh4- and sh5-CD13 knock-down in 3D Caco-2 cysts after 5, 7, and 9 days in culture. b Quantification of the size of 3D Caco-2 structures for the indicated conditions after 5, 7, and 9 days in culture. Data are reported as mean ± SEM for the indicated conditions (n = 129 shScr, n = 126 sh4-CD13, and n = 160 sh5-CD13 cysts) from a single representative experiment. Comparisons of multiple means were performed by ANOVA using Tukey’s post-hoc test on data from day 9. c Quantification of cysts with single prominent lumen of shScr (n = 806), sh4-CD13 (n = 961) and sh5-CD13 (n = 1312) knock-down in 3D Caco-2 cysts on day 10. Individual dots represent mean values from each of 4 independent experiments. d The schematic diagram showing different phenotypes (internal, peripheral, mixed) of apical Par6 observed. e Confocal images of Par6 (green) and F-actin (magenta) showing different Par6 localization (internal, peripheral, mixed) in 3D Caco-2 cysts. Yellow arrow heads indicate peripheral Par6 localization. f Quantification of Par6 localization (internal, peripheral, mixed) in Caco-2 structures for the indicated conditions (n = 358 shScr, n = 336 sh4-CD13, and n = 187 sh5-CD13 cysts) after 10 days in culture. Dots represent the mean from each of 4 independent experiments. g Confocal images of shCD13-expressing Caco-2 cells immunostained for endogenous CD13 (magenta) and Par6 (green) to visualize residual/depleted CD13 in individual cysts with different phenotypes. h Confocal images CD13 knock-down Caco-2 cysts immunostained for Ezrin (green) and laminin (magenta) showing cells with inverted apical-basal polarity. i Quantification of internal Ezrin in shScr (n = 168) and sh4CD13 (n = 150) 3D Caco-2 cysts after 10 days in culture. Dots represent means from each of 8 independent experiments. j Quantification of intact basal laminin in shScr (n = 71) and sh4CD13 (n = 35) Caco-2 cysts after 10 days in culture. Dots represent means from each of two independent experiments. k Images for E-cad (green) and β1-integrin (magenta) showing cell polarity are disrupted in CD13 knock-down 3D Caco-2 cysts. Data are represented as mean ± SEM (b, f) or mean ± SD (c, i, j). p-values were calculated by ANOVA with Tukey’s posthoc test for multiple comparisons (b, c, f) or unpaired two-tailed student’s t-test (i). Images are representative of two (k) three (a), or four (e, g, h) independent experiments. Bars: a, 200 μm; e, 50 μm; g, 20 μm; h and k, 30 μm.