FN-rich ECM inhibits the formation of FCL and RA. (A) U2Os-AP2-GFP cells plated on PLL-, BSA-, FN-, VTN-, Col I-, Col IV-, LN111-coated, or non-coated dishes overnight. Samples were imaged with TIRF microscopy at 1-s intervals for 5 min. Representative 15-s time projections and 5-min kymographs of time-lapse videos from samples in Fig. 1 B. (B) Left: U2Os-AP2-GFP cells plated on non-coated dishes overnight were stained for FN. Representative TIRF images. Right: Graph showing the reduced brightness of AP2 in regions with higher FN signal (measured from a 1.5 µm × 1.5 µm region around each AP2 spot, n = 32988 AP2 spots, 27 images from one representative sample). (C) U2OS cells plated on non-coated (NC) or FN-coated (10 µg/ml) dishes were analyzed for clathrin exon 31 density by RT-PCR; n = 3 biological replicates. (D) U2Os-AP2-GFP were plated to non-coated dishes and stained for integrin αvβ5 and VTN. Representative TIRF images. (E) Analysis of integrin αvβ5 fluorescent intensity of samples from Fig. 2 A. N (images): FN = 15, VTN/Col I/LN111/non-coated = 21, Col IV = 17. Results were obtained from one representative experiment; similar results were observed in four individual experiments. F(5, 120) = 85.49, P < 0.0001. (F) U2Os-AP2-GFP cells plated on 10 µg/ml FN, 5 µg/ml FN, 1 µg/ml FN, FBS, or complete MEM medium-coated dishes were stained for FN and integrin αvβ5. Representative TIRF images. (G) FN integrated fluorescent density of dishes coated as in G, and plated or not plated with U2Os cells. N = 16-10/sample, from two independent experiments. F(9, 129) = 184.8, P < 0.0001. Data are the mean ± SD, ns. non-significant P value; *** P value < 0.001. Scale bars, 10 and 5 µm; insets, except in D, are 2 µm. Source data are available for this figure: SourceData FS1.