Extended Data Fig. 3: β-II spectrin-KO cells fail to align with shear despite normal tyrosine phosphorylation of junctional proteins.
From: The spectrin cytoskeleton integrates endothelial mechanoresponses
Endothelial cells were subjected to constant fluid flow or maintained in static culture for 30 min. Cells were then fixed and immunostained for phosphorylated protein tyrosine residues (p-tyrosine). a, Representative polar histograms of F-actin orientations analyzed as described in Methods from one representative experiment as in b. b, Confluent endothelial cells grown in static or shear conditions were fixed and immunostained for VE-cadherin to visualize cell borders. Aspect ratios were calculated as the longest axis divided by the shortest axis of each cell. Here and elsewhere, for each condition, on the left: jittered data points (in shades of blue) represent individual measurements and are color-coded according to biological replicate. Means of individual experiments are presented in black. Overall means ± SE are indicated in red. Histograms of all data points indicating the mean (solid line) as well as the 25th and 75th percentiles (dashed line) are shown to the right of the individual data. n = 4 independent experiments, analyzing 141,172,204,346 wildtype static-; 109,136,195,371 wildtype shear-; 83,142,147,377 KO clone 4 shear-; and 171,352,130,129 KO clone 6 shear-exposed cells. c, Representative micrographs for indicated conditions, scale-bar: 5 μm. Representative of 3 independent experiments. d, Ratio of phospho-tyrosine fluorescence at junctions (VE-cadherin-positive structures) and cytosol for individual cells. n = 3 independent experiments, analyzing 140,175,72 wildtype static-; 168,109,99 wildtype shear-; 73,89,72 KO clone 4 static-; 83,142,81 KO clone 4 shear-; 74,91,92 KO clone 6 static- and 103,143,38 KO clone 6 shear-exposed cells. P values are from one-way ANOVA of experimental means (b,d).
