Fig. 2

Substrate modulus affects actin stress fiber orientation in A2058 cells. A Representative images of the cells analyzed in E for control and FMNL2 depleted cells plated on the indicated substrates. Cells were fixed and stained with phalloidin to visualize actin filaments. Scale bar = 20 μm. B Windrose plots showing the orientation of the actin fibers with respect to the cells’ Feret diameter for control and FMNL2 depleted cells plated on 0.2 kPa and 64 kPa substrates. Increases in substrate modulus and FMNL2 depletion both yield a tighter distribution around 0, indicating increased filament alignment. C An annotated image of a cell with a white bar showing the Feret diameter (FD), with the angle θ being the angle between the FD and an actin fiber shown with the dashed line. θ is then used to calculate the 2D orientational order parameter (S). D Anti-FMNL2 immunoblot confirming the extent of FMNL2 knockdown by transient transfection of siRNA. Tubulin was used as a loading control. E 2D orientational order parameter S quantifying average orientation of F-actin with respect to the cell Feret diameter. Increasing substrate stiffness increases the 2D order parameter. N = 3, with approximately 100 cells analyzed per cell type and substrate. *p < 0.0001