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. 2016 Dec 5;127(1):365–374. doi: 10.1172/JCI88555

Figure 2. Chymase impairs smooth muscle adhesion to fibronectin and focal adhesion complex phosphorylation.

Figure 2

(A) Schematic of alternate pathways important in the transmission of tension. Vh, vinculin head domain; Vt, vinculin tail domain. (B) Adhesion of human ASM cells to fibronectin measured by absorbance of crystal violet at 595 nm. Fibronectin (0.1 μg/ml) or cells were treated with the indicated doses of rhChy for 20 minutes and then chymostatin (10 μg/ml) prior to assessment of adhesion. (C) Adhesion of human ASM cells to collagen I (0.1 μg/ml), vitronectin (0.3 μg/ml), or laminin I (10 μg/ml), as measured by absorbance of crystal violet at 595 nm. Ligands were treated with the indicated doses of rhChy for 20 minutes and then chymostatin (10 μg/ml) prior to assessment of adhesion. (B and C) Data represent the mean ± SEM from triplicate experiments. (D) Representative Western blots and quantitative densitometry for phosphorylated and total vinculin and FAK in human ASM cells plated on poly-l-lysine (300 μg/ml) alone or poly-l-lysine with either fibronectin (1 μg/ml) or collagen I (1 μg/ml), with the addition of rhChy (30 nM) or vehicle for 20 minutes, followed by chymostatin (10 μg/ml). GAPDH was used as a loading control. Poly, poly-l-lysine; FN, fibronectin; C, collagen I. *P < 0.05 and **P < 0.01 versus Poly; #P < 0.05 and ##P < 0.01 versus Poly/FN by 2-way ANOVA. n = 3 distinct experiments.