Extended Data Fig. 3: LSEC control experiments and deep learning workflow for quantification of LSEC porosity.

a, Effect of different SEMA3A-Fc concentrations on LSEC size. Cells were cultured for 4 h, starved for 1 h, and treated with SEMA3A-Fc for 1 h. After fixation, phalloidin was used to stain F-actin fibers, and DAPI was used to stain cell nuclei. Cells were imaged using an Axioscope (Zeiss) and the NIS-Elements imaging software, and 10 images of each condition were obtained and analyzed using the Fiji image processing package. Per image, the cell size of at least 26 cells was measured. b, The CellTiter-Glo® Cell Viability Assay (Promega) was performed after SEMA3A-Fc treatment of isolated LSECs to determine the amount of ATP present (n = 3 independent LSEC isolations). c, Mouse LSEC were isolated and cultured in EBM-2 media for 1, 2 or 24 h, after 4 h pre-culture. Fenestrae were analyzed for their frequency and diameter; LSEC porosity was also determined. For each condition, 10 images (taken from different LSECs) were analyzed per experiment (n = 3 independent LSEC isolations). d, LSECs were isolated and incubated in EBM-2 media for 4 h. An arrow points to a potential magnetic bead located within a fenestra. Scale bars = 400 nm (left) and 100 nm (right, n = 1 LSEC isolation). e, Correlation analyses of 30 images that were analyzed either manually or using a deep learning workflow, for fenestrae frequency, fenestrae diameter and LSEC porosity. Each dot represents one image analyzed. f, Example image of LSEC pre- and post-processing (output file) as received by the deep learning workflow, scale bars = 2 µm. A one-way ANOVA with multiple comparisons was used for statistical analysis, and statistical significance was corrected for multiple comparisons using a Dunnett´s post hoc test in (a) and (b), and a one-way ANOVA with a Tukey´s post hoc test was used to test for statistical significance in (c). In graphs (a-c) individual data points and mean ± SEM are presented.

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