RSF formation is suppressed through formin inhibition or reduced expression of Dia1 or Atn-1. (A) Images of F-actin visualized by fluorescent phalloidin (top row) and Pxn immunofluorescence (middle row) are shown for WT U2OS cells, cells treated with 15 µM of the formin inhibitor SMIFH2 (Dia Inh), cells with reduced expression of Dia1 via siRNA (Dia1 siRNA), and cells with reduced expression of Atn-1 by shRNA (Atn-1 KD). Yellow arrows indicate RSFs, and red arrows indicate transverse arcs in the WT cells. Bar, 20 µm. (B) Percentages of cells containing both RSFs and transverse arcs (RSF + TA), transverse arcs only (TA Only), or no actin bundles (None) were determined for the WT, Dia Inh, Dia1 siRNA, and Atn-1 KD cells, as described above (n = 49, 50, 22, and 50 for the four conditions, respectively). Representative images from each classification of cells can be seen in Fig. S3. (C) Mean linear density of RSFs, measured as the number of RSFs per micrometer along the cell edge for each of the four conditions (error bars indicate SEM; n = at least 10 cells containing five to six RSFs; **, P < 0.01 with respect to WT). (D) Mean background-corrected fluorescence (Fluor.) intensity of fluorescent phalloidin staining of F-actin in transverse arcs and RSFs for each condition, normalized to the transverse arcs in WT cells (error bars indicate SEM; n = 20 regions from 10 cells for each condition; **, P < 0.01; ***, P < 0.001 with respect to WT). A.U., arbitrary unit.