Figure 5.

PD 102807 inhibits TGF-β–induced signaling and function in ASM cells in an M3-dependent manner. (A) SMAD-Luc transfected human hTERT ASM cell lines were preincubated with PD 102807 (1 or 10 μM) for 20 min and then stimulated with TGF-β (1 ng/mL) for 6 hours. Luciferase activity was determined as described in Methods. (B) hTERT ASM cell lines were treated with TGF-β (1 ng/mL) ± PD 102807 (10 μM) for 3 days. Cells were then fixed and permeabilized, and smooth muscle α-actin (α-SMA) (488 nm; green) and phalloidin (594 nm; red) expression was determined by immunofluorescence. Cells were counterstained with DAPI (blue) as described in Methods. Representative images are shown. (C) hTERT ASM cells were stimulated with TGF-β (1 ng/mL) with or without MCh (10 μM) or PD 102807 (10 μM) for 3 days, and α-SMA expression was determined by immunoblotting. Representative immunoblots for α-SMA are shown; loading was corrected for β-actin. (D) ASM cell contraction was after cells were treated with TGF-β (1 ng/mL) with or without PD 102807 (10 μM) for 3 days. Collagen gel contraction data are shown as percent reduction in gel area after histamine stimulation (1 μM; 10 min). Data are means ± SEM from four to five experiments. *P < 0.05 and ****P < 0.0001 versus vehicle-stimulated condition; ^#P < 0.05, ^##P < 0.01, and ^####P < 0.0001 versus vehicle-pretreated TGF-β condition; one-way ANOVA followed by Bonferroni multiple comparison test. Veh = vehicle.