α5β1 integrin in CAFs transduce mechanical forces to Fn. (A) IF staining of NFs and CAFs for active α5 integrin (green, left) and total α5 integrin (red, middle). Bar, 20 µm. Zoomed images have been pseudocolored to show differences in fluorescence intensity, with warmer colors indicating higher intensity. Bar, 5 µm. (B and C) Quantification of mean fluorescent intensity of active (B) and total (C) α5 integrin. 80–95 cells per condition from five independent experiments were analyzed. Error bars indicate SEM for five experiments. ***, P < 0.001; n.s., not significant as determined by Student’s t test. (D) Representative traction-force vector maps of CAFs treated with 5 µg/ml control IgG (left) or α5 integrin function blocking antibody JBS5 (right). Warmer colors indicate areas with high traction forces. (E) The dot plot shows mean traction forces in control and JBS5-treated CAFs. Line indicates mean; error bars indicate SEM. 22 control CAFs and 25 JBS5-treated CAFs were analyzed in four independent experiments. ***, P < 0.001 as determined by Mann Whitney U test. (F) Fn staining of CAFs after 48 h treatment with 5 µg/ml control IgG or α5 integrin function-blocking antibody P1D6. Bar, 20 µm. (G) Measurements of angles between Fn fibers in CAFs treated with IgG or JBS5. Greater than160 angles measured per condition from ≥16 images from three independent experiments. ***, P < 0.001 as determined by Mann-Whitney U test. (H) NHS–ester-488 staining of CAF CDMs generated during 10 μM RGE or RGD treatment. Bar, 50 µm. (I) FFT analysis of CAF CDMs shown in Fig. 4 H.