Fibronectin coated

VSMCs at density of 10⁵ cells/well were cultured and treated with 0.5 mg/ml of CC in fibronectin-coated (10 ug/ml, 2 h; R and D systems, UK) eight-chamber culture slides for 24 h. The cells were then fixed using ice-cold 4% paraformaldehyde for 30 min at room temperature, followed by incubation with ice-cold PBS containing 0.1% Triton-X100 for 10 min. The cells were washed with ice-cold PBS and blocked in 1% BSA in PBS containing 0.1% Triton-X100 for 30 min. F-actin was visualized by staining with Rhodamine phalloidin, 400X (Invitrogen, Stockholm, Sweden) for 20 min in dark followed by staining the nucleus using 4ʹ,6-Diamidino-2-Phenylindole hydrochloride, (DAPI, Sigma-Aldrich, Germany) for 5 min in dark and washed twice with PBS. Slides were airdried and mounted using antifade reagent and were stored in the dark at 4 °C until viewed under microscope. CC were visualized under polarized light and Images were acquired at 20X and 40X magnification and 1024 × 1024 resolution using confocal laser scanning microscope SP8 (Leica, Germany). The images were further processed with LAS X software (Leica, Wetzlar, Germany).

The human NSCLC cell line A549 (bronchioalveolar carcinoma, American Type Culture Collection, ATCC-CCL-185, [49] (link)) was cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and Pen/Strep; a second human lung adenocarcinoma cell line NCI-H441 (ATCC-HTB-174, [50] (link)) was propagated in RPMI 1640 medium with 10% FCS and Pen/Strep. Cells were cultured at 37°C, 5% CO₂. Both cell lines were subjected to DNA typing of short tandem repeats and genotyped to ensure their identity (Leibniz-Institute DSMZ). DMEM, RPMI-1640, FCS, Glutamax and Pen/Strep were purchased from Life Technologies. All cells were cultured on tissue culture-treated plastic and rat tail collagen I (50 µg/ml, BD Biosciences) or fibronectin-coated (50 µg/ml, R&D Systems) glass, respectively at 37°C and 5% CO₂ atmosphere. For migration assay, cells were made quiescent by culture in fresh medium containing 0.5% serum for 1 day.