Fig. 2

Retrieval of CD93 is regulated through its cytoplasmic domain. a: Time course image analysis of CD93 localization during cell spreading. CD93-YFP was transiently transfected into HUVECs. Cells were detached from the plate, resuspended in complete growth medium, plated on the substrate, and fixed at different spreading times as indicated. Exogenous CD93 was imaged as yellow. Scale bars, 10 μm. b: Quantification of exogenous CD93 levels in the apical bud and cytoplasm of ECs treated as in a. Bars represent the percentage of total fluorescence intensity/area. Data are presented as the mean/cell ± SD of three independent experiments (n = 3 cells per group). **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. 5 min; 2-way ANOVA with Dunnett’s post-test. c: Periodic z-series of cells treated as in a were acquired at 10 min after plating to generate a vertical section (lateral xz, 90 z-sections of 115 nm each). The corresponding cell boundary is indicated by a dotted line. An arrowhead indicates the point of view of the lateral xz image. Scale bar, 15 μm. d: The schematic diagram illustrates CD93 wild type (CD93-YFP) and the deletion mutant (CD93∆C-YFP) lacking the cytoplasmic domain fused with YFP. The deleted amino acid residues are indicated. CTLD, C-type lectin-like domain; EGF-like, Epidermal Growth Factor repeats; Mucin, Mucin-like domain; TM, transmembrane domain; Cy, cytoplasmic domain. e: CD93∆C-YFP deletion mutant was transiently transfected into HUVECs. Cells were detached from the plate, resuspended in complete growth medium, plated on the substrate, and fixed at early phases of spreading (from 5 to 10 min). Exogenous CD93∆C-YFP was imaged as yellow. Scale bars, 8 μm. Merged and DIC images are shown. f: Quantification of exogenous wild type and mutant CD93 levels in the apical bud and cytoplasm of transfected cells as in a and e. Bars represent the percentage of total fluorescence intensity/area. Data are presented as the mean/cell ± SD of three independent experiments (n = 15 cells per group). ****P < 0.0001; unpaired t-test