Effect of FlnA KD on tension polarity. (A–D, i) Representative images of talin-TS (tagRFP) images. (A and B, ii) Mean FRET index versus FA angle/orientation for peripheral adhesions in stretched scr (A, ii) and FlnA KD (B, ii) cells. Blue arrowhead indicates the direction of stretch with respect to image axis (n = 1,273 and 695 peripheral FAs for A, ii; and B, ii, respectively). Purple line is smoothened plot obtained by 50-point adjacent averaging. (C–D, ii) Alexa Fluor 647–conjugated SNAP dye to label SNAP-tagged WT FlnA (C, ii) or integrin binding–deficient FlnA (D, ii). (A–D, iii) Histogram of average FRET index per FA for perpendicular and parallel FAs in 10% stretched condition. Inset to A–D, iii: Mean FRET index normalized to the perpendicular FAs within each cell. (A–D) Cells with scr (A), FlnA KD (B), and FlnA KD rescued with either WT full-length human FlnA (C) or integrin binding–deficient (I2092C/I2283C) FlnA (D). n = 441 and 398, 240 and 171, 524 and 335, and 258 and 450 perpendicular and parallel FAs for A, iii; B, iii; C, iii; and D, iii, respectively. Error bars represent SD.