Fig. 4

Monolayer dynamics in three-dimensional iBMEC microvessels. A Tight junction (TJ)-labeled iBMECs support tracking of monolayer dynamics in real time. B Apoptotic, mitotic, and quiescent cell populations identified from imaging of n = 1871 cells from n = 6 iBMEC-TJ microvessels. C Comparison of mitosis, apoptosis, and turnover rates between iBMECs in 3D microvessels (n = 7) and 2D monolayers (n = 6). Significance analysis performed using a Mann–Whitney test. D Apoptotic, mitotic, and quiescent cells are identified from changes in tight junction fluorescence and phase contrast imaging over 100 min for quiescent cells and 60 min for apoptotic and mitotic cells, with an interval of 5 min. E Heatmap summarizing morphological changes during imaging. Only squares corresponding to a statistically significance change are shaded by magnitude of change (log₂FC) and direction of change (blue: increasing over time, red: decreasing over time) labeled with asterisks representing significance as calculated using the maximally significant result between a Wilcoxon matched-pairs signed rank test to compare first and last time points and using an F test to determine if linear regression of the morphological time course displayed a statistically non-zero slope. F, G Heatmaps comparing morphological metrics of cell types before (t = − 30 min) and after (t = 30 min) cellular events. Only squares corresponding to a statistically significant comparison are shaded by magnitude of change (log₂FC) and direction of change (blue: higher than quiescent cells, red: lower than quiescent cells) labeled with asterisks representing significance calculated using a Kruskal–Wallis test and post hoc Dunn’s multiple comparisons test relative to quiescent cells. Specific violin plots of interest are also shown. Data are presented as mean ± SD. NN nearest neighbor. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. See also Additional file 1: Figs. S8–S10 and Additional file 3; all analysis conducted on TJ-labeled iBMECs