Y 27632

Commercially available primary HUVEC (C2519A, Lonza, mixed sex) were cultured in EBM-2 (CC-3162, Lonza) supplemented with the Endothelial Growth Medium (EGM-2) bullet kit (CC-3162, Lonza) and 1× antibiotic-antimycotic (Gibco). Commercially available primary normal human lung fibroblasts (CC2512, Lonza, mixed sex) were cultured in DMEM (Gibco) with 10% fetal bovine serum (FBS) and 1× antibiotic-antimycotic (Gibco). All cells were maintained at 37°C and 5% CO₂. For contractility inhibition experiments, HUVEC were treated with 10 µM (-) Blebbistatin (B0560-1MG, Sigma) for 15 min at 37°C or 10 µM Y-27632 (10187-694, VWR) for 30 min at 37°C then immediately fixed in 4% PFA. For induction of contractility, HUVEC were treated with 0.5 U/ml thrombin (T7201-500UN, Sigma) for 10 min at 37°C. For microtubule depolymerization, HUVEC were treated with 10 µM nocodazole (M1404, Sigma) for 20 min at 37°C then immediately fixed in methanol.

For Matrigel assays, Matrigel (BD Biosciences, Bedford, MA, USA) was poured at 4°C in 24 Transwells inserts (8-μm pores Falcon, Colorado Springs CO, USA) and polymerized as described [16 (link)]. Fibrillar collagen I matrices were prepared by mixing bovine collagen (2 mg/ml) (Nutragen, Advance biomatrix San Diego, CA, USA) and rat tail collagen (4 mg/ml) (BD Biosciences, Bedford, MA, USA). The preparation was added to Transwell inserts (8 μm pores, Falcon, Colorado Springs CO, USA) and allowed to polymerize as described [2 (link)]. Lower and upper chambers of a transwell system (Nunc, Thermo Fisher, Waltham, MA, USA) were filled in with RPMI 1640 containing 10% FCS and 20 ng/mL rmM-CSF (eBiosciences, Bedford, MA, USA) or 1% FCS and 20 ng/mL rmM-CSF, respectively. After serum starvation for 4 h with RPMI containing 1% FCS BMDMs (2 x 10⁴) cells were seeded in the upper chamber and allowed to migrate. After 48 h z-series images were acquired at 30-μm intervals with a Zeiss (Jena, Germany) observer microscope. The % of migrating cells was determined from z-stack images of the matrix and normalized to the total number of cells in the field of view. Where indicated, the Rho-associated protein kinase (ROCK) inhibitor Y27632 (10 μM) (VWR international, Randor, Pensilvania, USA) was added to the upper and the lower chambers.