Contractile forces exerted by PASMC on different stiffness gels were assessed by traction force microscopy essentially as described (39 (link), 46 (link), 47 (link)). Briefly, polyacrylamide substrates with shear moduli of 0.4, 1.6, and 6.4 kPa were prepared (14 (link), 45 (link)) and fluorescent sulfate–modified latex microspheres (0.2 μm, 505/515 ex/em, FluoSpheres, Invitrogen) were conjugated to the gel surfaces as described (47 (link)). PASMC were plated on fluorescent bead–conjugated discrete stiffness gels and grown for 24 hours, at which time they were treated with iloprost (10 μmol/l) or vehicle for 30 minutes before traction force measurements. Images of gel surface–conjugated fluorescent beads were acquired for each cell before and after trypsinization using a Pathway HT fluorescence imaging system (BD Biosciences) and a ×20 magnification objective. Cell area was visualized using CellLight Plasma Membrane-RFP (Invitrogen) as described (47 (link)) to delineate the cell perimeter. Tractions exerted by PASMC were estimated by measuring bead displacement fields, computing corresponding traction fields using Fourier transform traction microscopy (46 (link)), and calculating root-mean-square traction (RMST) with MATLAB (Math-Works) (46 (link)).