Figure 6. Images were also taken using a 4× objective with BD Pathway.

3 × 3 montages were captured for cells and nuclei stained with Alexa 488 Phalloidin and DAPI respectively. The montages captured complete pillars and trenches. The images were cropped to the regions above the pillars and these cropped images were used for segmenting cells using CellProfiler. For analysis, only the cells with centers within the ROIs were considered. Cellular orientation for cells in the ROIs was plotted as beeswarm plots for each mechanical stimulation condition (replicates pooled together but depicted in separate colors). In the beeswarm plots, the orientation of individual cells are plotted as a scatter with added spread where points overlap, making the plots appear thicker for angles along which a large number of cells are oriented. The beeswarm plots display the distributions of cell orientation angles with respect to the trench major axis. Thus, preferential alignment perpendicular to the maximum strain direction results in a bulge in the plot at 90° while preferential alignment along the flow produces a bulge in the plot at the angle that the trench major axis makes with the flow. Median values are marked with a solid blue line and a central ‘o’. Dotted red lines plus ‘|’ in the left corner and dotted green lines plus ‘*’ in the left corner mark the expected medians if the alignment was perfectly along the flow or perpendicular to the maximum principal strain respectively. Medians were calculated for orientation angles with respect to the reference line that would put the perfect alignment case at 90°, and were shifted appropriately if the reference line was not along the trench major axis.