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. 2020 Mar 19;11:1457. doi: 10.1038/s41467-020-15303-y

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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. 1 — HeLa cells treated with negative control (siCtrl, a–c), or µ2-adaptin (siAP2M1, a), dynamin-2 (siDNM2, b–c), or clathrin heavy chain (siCLTC, c) siRNAs. a, b Measure of anti-CD166 and transferrin (Tf) endocytosis by loss of surface assay in flow cytometry. a Number of independent experiments: anti-CD166, n = 3; Tf, n = 4. b Three independent experiments. NS, not significant. ****P < 0.0001 (RM two-way ANOVA with Bonferroni’s multiple comparison test). c Continuous uptake of anti-CD166 antibody for 10 and 30 min. Internal fluorescence quantified from confocal images and plotted as the relative percentage of siCtrl condition at 10 min. n cells, two independent experiments. NS not significant. ****P < 0.0001 (Kruskal–Wallis test with Dunn’s multiple comparison post hoc test, two-sided). The Western blots against α-adaptin (a), dynamin (b, c), and clathrin heavy chain (c) document the efficiency of siRNAs. Loading control, α-tubulin. d Continuous co-uptake of anti-CD166 antibody (red) and fluorescent Tf (green) for 5 min in HeLa cells. No co-localization between the two markers (see insets). Representative of three independent experiments. e–h Endocytosis imaged by lattice light-sheet microscopy on live cells. Full 3D volume of 60 planes per U2OS cell acquired within 3 s. Average endocytic intensity traces per lifetime, and classification of endocytic events for anti-CD166 (e) and Tf (g) uptake. Green, endogenous genome-edited CLTA-mRFP; Orange, cargo. Lifetime cohorts of endocytic trajectories show a “dome”-shaped intensity profile of cargoes and co-tracking or not with clathrin. f, h 2D projections of acquired 3D stacks of cargo (red), clathrin (green), and overlay (yellow). Representative of ten experiments. All ten experiments were used for quantifications (e, g). Scale bars, 5 µm (d), 10 µm (f, h). Data are mean ± s.e.m. Source data are provided as a Source Data file and in Supplementary Fig. 11 (blots).