FN mediates LTBP-1 fibril formation and incorporation. (A and B) Substrates were coated with purified LTBP-1–EGFP and used to culture hDMfs transfected with siRNA against FN (siFN) or nonrelated control RNA (siNT; A) and FN^−/− MEFs or wild-type (WT) MEFs (B). (C) hDfs and hDMfs were seeded onto substrates coated with LTBP-1–EGFP alone or LTBP-1–EGFP mixed with an excess of FN (100 µg/cm²) and grown for 4 h. (D) hDMfs were pregrown for 7 d before purified LTBP-1–EGFP was added to the culture medium for an additional 24 h in the presence or absence of 20 µM blebbistatin (blebbi). Experiments were performed in FN-depleted culture medium. All samples were stained for EGFP (green), FN (red), and nuclei (blue). (A–E) LTBP-1–EGFP fibrils (A–D) and staining intensity (E) were quantified by image analysis from at least five images per three independent experiments to calculate mean values and SDs (*, P ≤ 0.05; and ***, P ≤ 0.005 using ANOVA followed by a post-hoc Tukey’s multiple comparison test). Inset in E shows FN organization after 7 d of hDMfs without addition of LTBP-1–EGFP. Bars, 25 µm. a.u., arbitrary unit.