Rat tail collagen 1

Collagen gels for contraction assays were prepared by using of Rat tail collagen I (Serva, Germany), 10× DMEM, 1 M NaOH (final pH 7.4) and mixed with cells (1 × 10⁶/ml) in 8:1:1:1 ratio previously described in Kamel et al (2014), with final concentration 2 mg/ml. Gel–cells suspension (triplicates) was pipetted into 96 wells pre‐coated with BSA and allowed polymerized in 5% CO₂ at 37°C for at least 30 min. The collagen lattices containing cells were replaced with completed medium with or without inhibitor and allowed to contraction at least 4 days. After day 4, the images were taken and contraction was calculated as a decreased area of original diameters area of 96 wells. Comparison of collagen gel contraction was performed by using Student's unpaired one‐tail t‐test, and P < 0.05 were considered statistically significant.

All tissue culture reagents were from Euroclone SpA except ECGS and heparin (Sigma Aldrich, cat #E2759 and #H3149, respectively). N^G-Nitro-L-arginine methyl ester (L-NAME, cat #N5751), methylcellulose (cat #M0512), FITC-labelled phalloidin (cat #P5282), and DAPI (cat #D9542) were from Sigma Aldrich; collagenase (cat #17454) and rat tail collagen I (cat #47254) from Serva; recombinant human VEGF₁₆₅ (cat #100) from Peprotech; mitomycin (cat #BIA-M1183) from Tebu-bio. Primary antibodies used were: mouse monoclonals anti-eNOS (BD Transduction Laboratories, cat #610296) and anti-β-actin (Sigma Chemicals, cat #A2228), and rabbit polyclonal anti phospho-eNOS (Ser1177) (Cell Signalling Technology, cat #9571). HRP-conjugated secondary antibodies were from Dako (cat #P0260 and #P0399 for rabbit anti-mouse and swine anti-rabbit antibodies, respectively). A goat anti-mouse CY3 (cat #29-0382-75, GE Healthcare) was used in immunofluorescence experiments to detect eNOS.

All tissue culture reagents were from Euroclone SpA except ECGS and heparin (Sigma Aldrich). Charcoal, dextran T-70, MTT, trypan blue, methylcellulose (cat #M0512), E2, T3, DHT, sodium acetate, palmitic acid, and free fatty acid-bovine serum albumin (FFA-BSA) were from Sigma Aldrich; rat tail collagen I from Serva; recombinant human VEGF-165 from Peprotech. palmitic acid was used as a conjugate to FFA-BSA. Briefly, a 75 mM stock solution of palmitic acid dissolved in ethanol was diluted 1:10 with a 10% FFA-BSA solution, and incubated overnight at 37°C before every experiment.