Optimazition experiments for visualizing FCL and RA co-assembly. (A) U2Os-AP2-ITGB5-mScarlet cells plated on non-coated dishes were treated with Cilengitide (10 µM) or DMSO for 25 min. Samples given Cilengitide were washed twice with fresh complete medium (except no-wash control), fixed at the respective recovery time points, and stained for p-Pax. Representative TIRF images. (B) Analysis of RA coverage for samples in A. N (images): control = 31, no wash = 32, 15-min recovery = 30, 30-min recovery = 30, 1-h recovery = 31, 2-h recovery = 30, 4-h recovery = 30, from one representative experiment. Similar results were observed in three independent experiments. (C and D) Additional examples of individual ITGB5 and AP2 events from experiments shown in Fig. 5. In C, an event where an RA grows from a stabilized FCL/ITGB5 cluster. A kymograph for the line on time zero is shown on the right. In D, three events are shown. One FCL-stabilized ITGB5 cluster (yellow arrowheads) and two non-stabilized ITGB5 clusters (blue arrowheads). Open blue arrows represent frames post-disappearance of ITGB5 clusters. Scale bars, 10 µm.