Integrin αvβ5 is necessary in the formation of FCL. (A) U2Os-AP2-GFP/halo, HeLa-AP2-GFP/halo, and MCF7-AP2-halo were plated on FN-coated or non-coated dishes, allowed to settle overnight, stained for integrin αvβ5 and p-Pax, imaged using TIRF, and analyzed for FA coverage. N (images): U2Os glass/FN = 37, HeLa glass = 32, Hela FN = 29, MCF7 glass = 28, and MCF7 FN = 32. (B) U2Os-AP2-GFP cells silenced for integrin β5 (ITGB5) with two shRNAs (shITGB5 #1, #2) or control were imaged using TIRF microscopy at 1-s intervals for 5 min. Representative 5-min kymographs. (C) Analysis of FCL proportions from time-lapse videos in A. N (videos): control = 8, shITGB5 #1 = 11, shITGB5 #2 = 6, from three independent experiments. One-way ANOVA with Tukey’s multiple comparison, F(2, 22) = 44.46, P < 0.0001. (D) U2Os-AP2-GFP cells silenced for integrin β5 (ITGB5) with two shRNAs (shITGB5 #1, #2) or control, were stained for integrin αvβ5 and p-Pax. Representative TIRF images. (E) Analysis of integrin β5 silencing efficiency, n = 10, from one representative experiment. Similar results were observed from three independent experiments. One-way ANOVA with Tukey’s multiple comparison F(2, 27) = 63.29, P < 0.0001. (F) U2Os-AP2-GFP cells plated on non-coated dishes for 20 h were treated with the integrin αvβ5 inhibitor Cilengitide (10 µM) for 15 or 45 min and stained for integrin αvβ5 and p-Pax. Representative TIRF images. (G) Analysis of AP2 signal colocalizing with p-Pax or integrin αvβ5 over time from samples in E. N (images): control = 14, Cil 15 min = 13, Cil 45 min = 10, from one representative experiment. Similar results were observed in four independent experiments. One-way ANOVA and Tukey’s multiple comparison F(2, 99) = 63.38, P < 0.0001. Data are the mean ± SD, ns. non-significant P-value; *** P-value < 0.001. Scale bars, 10 µm.