Active integrin α5β1 at fibrillar adhesions inhibit FCL and RA formation. (A) U2Os-AP2-halo cells were plated to FN-coated or non-coated dishes and stained for Tensin1 and active integrin β1 12G10. Representative TIRF images. (B) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for p-Pax. Representative TIRF images. (C) Analysis of RA coverage from samples in B. N (images): shScr control = 35, shTNS1 #1/#2 = 44, from three independent experiments. One-way ANOVA with Tukey’s multiple comparison, F(2, 120) = 56.26, P < 0.0001. (D) U2Os-AP2-GFP-ITGB5-mScarlet cells silenced for Tensin1 with two different shRNAs (shTNS1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for active integrin β1 (12G10 antibody). Representative TIRF images. (E) Analysis of 12G10 fluorescent intensity from samples in D. N (images): shScr = 20, shTNS1 #1 = 15, shTNS1 #2 = 11, from one representative image. Similar results were observed in three individual experiments. One-way ANOVA with Tukey’s multiple comparison, F(2, 43) = 87.81, P < 0.0001. (F) A schematic illustration of the results shown in this figure. Data are the mean ± SD, *** P value < 0.001. Scale bars, 10 µm; insets, 5 µm.