Supplementary Figure 1: Overexpression of radial fiber components, Zyxin or VASP, is not sufficient to reverse chirality.

RFP-Zyxin (a) and GFP-VASP (c) localized to radial fibers and focal adhesions. Actin was visualized either by co-transfection with LifeAct-GFP (green in a) or phalloidin staining (red in c). Scale bars, 10 μm. RFP-Zyxin (b) or GFP-VASP (d) expressing cells were filmed to follow their actin dynamics. The expression level in an individual cell was assessed by measuring either total RFP or total GFP fluorescence intensity respectively. The y-axis represents fluorescence intensity in a log scale; the cells were sorted in ascending order of their respective protein expression level along the x-axis. Each bar on the histogram represents an individual cell. Colour-coding indicates the type of actin cytoskeleton dynamics for each cell. Green – cells that do not demonstrate swirling during the entire period of observation (∼10 hours). Blue – cells demonstrating positive tilt of radial fibers and counter-clockwise swirling similar to control cells. No clockwise swirling events were observed in these experiments. 58 cells assessed for RFP-Zyxin, and 72 cells assessed for GFP-VASP.