Fig. 2: Reduced glycocalyx density in cell-cell contacts.

a Post-fixation THS-based glycocalyx labeling in cell-cell-contact. Colored line, quantification line. Pseudocolor, Fire Lookup Table. Scale bars, 5 µm. b Glycocalyx enrichment, fluorescence intensity compared to average single-cell membrane intensity per quantification line post-fixation and in live cells. Thin lines, single-membrane section enrichment, 45 (live) and 26 (fixed) cells; smoothened means (loess smoothening, black line) ± 95% CI (gray ribbon), 3 independent experiments. One-sample Wilcoxon signed rank sum test comparing measured with expected values (µ=2.0; ε² = 0.44 (fixed), ε² = 0.70 (live), large effect sizes). c Intensity (values from b) in live (left) and fixed (right) cells. Superviolins, single-cell fluorescence; color shades, intraexperiment distribution; dots, median per experiment; line, median ± 95% CI. Kruskall-Wallis rank sum test with Bonferonni multiple correction (ε² = 0.25 (fixed), ε² = 0.11 (live), large/moderate effect sizes, respectively). MFI, mean fluorescence intensity. d Glycocalyx enrichment in short (top) and long (bottom) transition zones. Left, representative micrographs. Right, glycocalyx enrichment in cell-cell contacts compared to single-cell membrane (green solid line). Annotations, transition zone lengths. Scale bars, 2 µm. e Transition zone length vs. glycocalyx enrichment. Lines, fits of linear regression analysis; ribbons, 95% CI. Datapoints (dots), mean per cell; 34/26 cells from 2/3 independent experiments for live/fixed cells, respectively. R², adjusted coefficient of determination of linear regression, (P.adj, adjusted p-value). f Glycocalyx fluorescence recovery in membrane zones in live cells. Distance 0, denotes transition zone center (definition: Supplementary Fig. 4b) or the bleached area center in single membrane and cell-cell-contacts. Half-maximum fluorescence recovery time was calculated according to Supplementary Fig. 7a–c. Black thick lines, rolling median (rolling window: 75 iterations) from 25 (single-cell-membrane), 39 (transition zone) and 21 (cell-cell-contact) cells, 3 independent experiments. g Half-maximum recovery per bleached membrane region (experiment from (f)), 25 contact-free membranes, 21 cell-cell-contacts and 67 transition zones from 85 cells pooled from 3 independent experiments. Kruskall-Wallis rank sum test with Bonferonni multiple correction (ε² = 0.04, small effect size). Data from (a–e) and (f–g) derive from the same experiments. Cells (all panels): B16F10. Boxplots: middle-line, median; outlines, 1^st-3^rd quantiles; whiskers, quantiles ±1.5x interquantile range. CI, confidence interval. Source data are provided as a Source Data file.