Ggti 286

The antibodies, anti-human IgG (Fc) (BioConcept AG, Switzerland), AIIB2 (integrin β₁-subunit blocking antibody), P5H9 (α_vβ₅-integrin blocking antibody) and P1B5 (integrin α₃-subunit blocking antibody; all 10 μg/mL supernatants; DSHB, Iowa) were incubated with cells on ice for 30 minutes prior to SCFS. To inhibit cell signaling, wortmannin (100 nM; Sigma-Aldrich)47 (link), LY294002 (50 μM; Cell Signaling Technology)48 (link), NSC23766 (50 μM; Merck Millipore)49 (link), EHT 1864 (100 μM; R&D Systems)50 (link), GGTI 2147 or GGTI286 (10 μM; Merck Millipore)51 (link), Akt inhibitor IV (1 μM; Merck Millipore)52 (link), Akt inhibitor VIII (20 μM; Merck Millipore)53 (link), SecinH3 (20 μM; Merck Millipore)54 (link), blebbistatin (10 μM; Merck Millipore)55 (link) or Y-27632 (10 μM; Merck Millipore)57 (link) were added to the measurement media at the concentrations given. Cells were then incubated for 30 minutes at 37°C prior to SCFS. All inhibitors were dissolved in DMSO and stored at −20°C.

Target proteins were perturbed by incubating fibroblasts at 37 °C for 30 min in SCFS media with inhibitors as follows; latrunculin A (LatA, 1 µm, Sigma), SMIFH2 (20 µm, Merck Millipore), CK666 (200 µm, Tocris Bioscience), blebbistatin (20 µm, Sigma), Y27632 (10 µm, Sigma), Y11 (10 µm, Tocris Bioscience), LY249002 (10 µm, Cell Signaling Technology), GGTi286 (10 µm, Merck Millipore), or PP2 (20 µm, Tocris Bioscience) in DMSO. Fibroblasts were incubated with C3 toxin (2 µm, cytoskeleton) in glycerol (50% v/v, PanReac AppliChem) at 37 °C for 3 h in SCFS media. All inhibitors were present in the given concentrations during SCFS experiments. DMSO and glycerol (50% v/v) were tested as carrier controls to confirm no effect on adhesion forces from carrier solvents. For heparin treatment, FN substrates and matrices as well as fibroblasts were incubated with a saturating concentration^[6] of heparin (100 µg mL⁻¹, Sigma) at 37 °C for 1 h in SCFS media before SCFS measurements. heparin was present at the given concentrations during SCFS experiments.