Figure 4

Assessment of cytoskeletal integrity via quantification of F-actin structures and density in liver endothelial cells. (A) Representative liver z-stacks stained for F-actin (green) and DAPI (blue), with a focus on the endothelial cell lining of a small vessel for WT and MK2^-/- mice, 2.5 h after i.v. injection of PBS or 450μg/kg TNF; (n^PBS (WT and MK2^-/-)=10 × 3 vessels/animal, n^TNF (WT)=5 × 3, n^TNF (MK2^-/-)=5 × 3). For every vessel, part of the endothelial cell sheet was defined as region of interest (ROI). (B) Actin density, expressed as the percentage of F-actin positive voxels per ROI. (C) The number of segmented F-actin positive structures per 100 000 voxels. (D) The average size of the actin structures, expressed as the total number of F-actin positive voxels per actin structure, normalized over the number of discrete actin structures. Comparisons were made between baseline (PBS) and TNF challenged animals (#), and between genotypes (*) via one-way ANOVA with Sidak’s multiple comparisons test. Error bars indicate SD. *, p≤0.05.