Depletion of integrin β1 or Integrin α5 promotes FCL and RA formation. (A) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB1 #1, #2) or control shRNA were plated on FN-coated dishes and stained for p-Pax and imaged using TIRF microscopy at 1-s intervals for 5 min. Representative 5-min kymographs. (B) Analysis of FCL proportions from samples in A. N (videos): shScr = 12, shITGB1 #1 = 11, shITGB1 #2 = 10, from three individual experiments. Tukey’s multiple comparison, F(2, 30) = 27.81, P < 0.001. (C) U2Os-AP2-GFP cells silenced for integrin β1 with two different shRNAs (shITGB #1, #2), or integrin α5 with two different shRNAs (shITGA5 #1, #2), or control shRNA, were plated on FN-coated dishes and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (D) Analysis of RA coverage from integrin β1 silenced samples in C. N (images): control shRNA = 30, shITGB1 #1 = 27, shITGB1 #2 = 18, from two independent experiments; similar results were observed in four independent experiments. One-way ANOVA with Tukey’s multiple comparison, F(2, 72) = 276.2, P < 0.0001. (E) Analysis of RA coverage from integrin α5 silenced samples in E. N (images): control shRNA = 33, shITGA5 #1 = 40, shITGA5 #2 = 37, from three individual experiments. One-way ANOVA with Tukey’s multiple comparison, F(2, 107) = 44.46, 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.