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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. 5 — a Confocal images showing the localization of CD13-mCherry (magenta) and GFP-Rab11 (green) at 1-cell, and adhesion, AMIS, and PAP stage in 2-cell 3D Caco-2 structures. White arrows show the proximity of CD13 puncta with Rab11-positive vesicles near the plasma membrane. b Confocal images of Caco-2 cells showing CD13-mCh (magenta) and GFP-Rab11 (green) vesicles during AMIS formation. Object-based colocalization was performed for CD13 and Rab11, and produced a correlation efficient ρ = -0.08. Arrows show local sites of CD13 accumulation. c Confocal images of GFP-Rab11 (green) and Par6 (magenta) showing their localization in shScr and shCD13 Caco-2 cells. Yellow arrows show regions of concentrated Rab11-positive vesicles. d Quantification of the percentage of Rab11 accumulation at the middle of shScr (n = 92) and shCD13 (n = 24) at 2 cell and >2 cell 3D Caco-2 structures. Dots represent means from each of two independent experiments. e Confocal images of 3D Caco-2 structures at the AMIS stage showing localization of GFP-Rab11 (green) and Par6 (magenta). f, g Quantification of Par6 localization to the periphery (f) or cytoplasm (g) in shScr (n = 20) and shCD13 (n = 22) Caco-2 cells at the AMIS stage. Dots represent individual Caco-2 structures from a single experiment. h Confocal images of control (shScr) and Rab-down (sh1-Rab11, sh4-Rab11) 3D Caco-2 cells immunostained for Par6 (magenta) and laminin (green). Right – quantification of structures with internal Par6 staining as a measure of normal or inverted polarity. The indicated number of structures were measured from (n = 11 shScr, n = 11 sh1Rab11, and n = 9 sh4Rab11) experiments. p-values were determined by ANOVA with Tukey’s posthoc test for multiple comparisons (d, h) or unpaired two-tailed student’s t-test (f, g). Images are representative from two (c) or three (a,b, e, h) independent experiments. Bars: 10 μm.