Figure 6

Reduced CLIC1 expression affects capillary-like sprouting and branching. A. Microscopy on day 7 of the fibrin bead assay showed that endothelial cells with CLIC1 knockdown had altered sprouting morphology. CLIC1 knockdown sprouts tended to be shorter, less branched, and less robust. Black arrows indicate long, robust, lumen-containing sprouts while red arrows indicate stunted tube structures. Scale bar, 10 μm. B. Microscopy on day 11 of the fibrin bead assay showed that endothelial cells with reduced CLIC1 expression formed tube-like structures; however these structures were stunted and possessed a branching defect when compared to structures formed by control cells. Black arrows indicate anastomosis or long tube-like structures. Red arrows indicate lack of branching or stunted tube-like structures. Scale bar, 30 μm. C. The average number of sprouts formed per bead was tabulated and quantified, shown here with standard deviation. Reduced CLIC1 expression was found to be accompanied by a significant reduction in the average number of sprouts per bead. D. Quantification of the average number of branches per sprout indicated that CLIC1 knockdown endothelial cells formed significantly fewer branches per sprout. The average number of branches per sprout is shown with standard deviation. E. Quantification of the number of lumen-containing sprouts revealed no significant difference between control cells and cells with CLIC1 knockdown. The average percentage of lumen-containing sprouts relative to total number of sprouts per cell line is shown with standard deviation. (**p < 0.001).