Figure 7

KIF11 deficiency impairs LEC migration and sprouting.

(A) 5 x 10⁴ serum starved cells were plated per fibronectin-coated Transwell chamber and given 10 hours to migrate in the presence of increasing concentrations of ispinesib (0 to 200 nM) before fixation and staining of the nuclei with DAPI. Resulting membranes (2 examples on left) were scored for the movement of nuclei through the Transwell membrane and results represented in a plot (right). Data are shown as SD, n = 3 biological replicates (with 2 technical replicates per biological replicate). Scale bar: 50 μm. (B) Single-cell tracking migration assay follows wound closure ability over 15 hours in DMSO (Ctrl) and 50 nM ispinesib-treated cells. Scale bar: 200 μm. (C) Number of cells in experimental wound-window area for Ctrl and cells treated with 50 nM ispinesib over time (frames). (D) Distance and direction of a subset of cells illustrated as star plots of cell tracks for Ctrl and cells treated with 50 nM ispinesib. (B–D) One representative experiment is shown from n = 2. (E) 3D spheroid sprouting assay. Sprouting was stimulated in LECs by VEGFC 150 ng/mL either in the presence of DMSO vehicle or several doses of ispinesib (25, 50, 100 nM). Average sprout length in μm (28 spheroids analyzed) and total number of sprouts per spheroid (15 spheroids analyzed) were quantified in each condition. One representative image is shown per condition from n = 2 biological repeats. *P < 0.05, **P < 0.01, ***P < 0.001, ns, nonsignificant. Two-tailed unpaired Student’s t test. Scale bar: 100 μm.