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. 2020 Jul 14;11:3516. doi: 10.1038/s41467-020-17091-x

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© The Author(s) 2020

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. 4 — a Left: MDCK cells, expressing GFP-IRSp53, were mock transfected or transfected with Apple-RAB11a or RFP-RAB35, and seeded as single cells on Matrigel and fixed after 5 h of growth. Cysts were processed for epifluorescence to visualize GFP-IRSp53 (green) and Apple-RAB11a or RFP-RAB35, or stained with rhodamine-phalloidin (red), anti-PODXL (red) or anti-7 (red), and DAPI (blue). Arrowheads, IRSp53 colocalization with trafficking markers. Scale bar, 10 µm. Right: Intensity profiles of the green and red channels over the vesicular structures (dashed lines in the merge channels). b Top: MDCK cell control (Ctr) and IRSp53-KO were seeded as single cells on Matrigel and grown for 24/36 h. The cysts were stained with anti-β-catenin (green) and anti-PODXL (red) antibodies, and DAPI (blue). Asterisks, PODXL multi-foci in IRSp53-KO cyst. Arrowheads, PODXL staining at the basal membrane in IRSp53-KO cyst. Scale bar, 10 µm. Bottom: Quantification of multi-foci cysts (left) and inverted polarity cysts (right). Data are means ± SD. Four-cell stage cysts were analysed, as at least 20 cysts/experiment in n = 7 (Ctr) and n = 5 (IRSp53-KO) independent experiments. P value, student’s t-test two-tailed (multi-foci) or one-tailed (inverted polarity). *p < 0.05; ***p < 0.001. Source data are provided as a Source Data file. c Top: MDCK control (Ctr), IRSp53-KD or RAB35-KD cells were seeded as single cells on a Matrigel and grown for 24/36 h. The cysts were fixed and stained with anti-β-catenin (green) and anti-PODXL (red) antibodies, and DAPI (blue). Asterisks, PODXL multi-foci in IRSp53-KD and RAB35-KD cysts. Arrowheads, PODXL staining at the basal membrane in IRSp53-KD and RAB35-KD cysts. Scale bar, 10 µm. Bottom left: Quantification of multi-foci cysts (left) and inverted polarity cysts (right). Data are means ± SD. Four-cell stage cysts were analysed, as at least 20 cysts/experiment in n = 7 (Ctr; IRSp53-KD) and n = 3 (RAB35-KD) (multi-foci) or n = 7 (Ctr), n = 4 (IRSp53-KD) and n = 2 (RAB35-KD) (inverted polarity) independent experiments. P value, student’s t-test two-tailed (multi-foci) or one-tailed (inverted polarity). *p < 0.05; ***p < 0.001. Source data are provided as a Source Data file. Bottom right: immunoblotting of IRSp53, RAB35 and vinculin levels.