SFs become less interconnected after FlnA KD. (A) Representative fluorescence images of scr and FlnA shRNA NIH3T3 cells showing FlnA staining (red), actin (green), nuclei (blue), and merged images. Scale bars, 30 µm. (B) Representative WB for FlnA, vinculin, and tubulin in scr and FlnA shRNA cells. (C) Representative inverted tagRFP intensity images of talin-TS–transfected scr and FlnA KD cells. Scale bars, 30 µm. (D–G) Representative PREM images of scr (D and E) and FlnA KD (F and G) cells. Respective zoomed-in images (E and G) of the boxed regions in D and F. Scale bars, 5 µm in D and F; and 1 µm in E and G. (H) Quantification of overlapping, partially merging and merging actin fibers in scr (gray) and FlnA KD (light red) cells. n = 14 and 20 for scr and FlnA KD cells, respectively. P < 0.05, obtained from Student’s t test. (I) Number of peaks (SFs count) per micron line scan length in scr and FlnA KD cells. n = 14 cells each. Error bars represent SD. (J) Representative FRET index maps of talin-TS–transfected NIH3T3 fibroblasts in scr (left) and FlnA KD (right) cells. Pseudocolor scale indicates FRET index values. Scale bars, 30 µm. (K) Average FRET index normalized to scr in each experiment. Error bars represent SD. n = 86 and 78 cells for scr and FlnA cells, respectively. (L) Histogram of pixelwise FRET index for the same data.