Substantial tension is built up at focal adhesions despite impaired RSF assembly. (A) Box plots of focal adhesion (FA) lengths of WT, Dia Inh, and Atn-1 KD U2OS cells (open circle = mean; box = 25th, 50th, and 75th percentile; whiskers = 5th and 95th percentile; n > 1,000 from 10 cells for each condition; ***, P < 0.001 with respect to WT). (B) Time series of GFP-Pxn and traction stress heat map images for representative WT, Dia-inhibited, and Atn-1 KD cells, showing the assembly and disassembly of a representative single focal adhesion and changes in traction stress. Bars, 3 µm. Focal adhesion length and normalized traction stress at the focal adhesion indicated by the red ovals are shown on the right. (C) Bar graph of the mean rates of focal adhesion assembly and disassembly for each of three conditions (error bars indicate SEM; n = 10 adhesions from two cells for each condition). (D) Bar graph of the mean focal adhesion lifetime (error bars indicate SEM; n = 26, 24, and 22 from two WT, Dia Inh, and Atn-1 KD cells, respectively; ***, P < 0.001 with respect to WT). (E) Plot of the mean traction stress exerted at individual focal adhesions (error bars indicate SEM; n = 51, 69, and 29 focal adhesions from three cells for WT, Dia-inhibited, and Atn-1 KD U2OS cells, respectively; ***, P < 0.001 with respect to WT).