ECM analyses by NHS staining

To promote ECM generation, 0.1% gelatin pre‐coated coverslips were treated with 1% glutaraldehyde in PBS for 30 min and washed before being treated with 1 M ethanolamine (30 min, RT). Fibroblasts were then plated (DMEM/F12 0.5% FBS) and activated in presence of 8 ng/ml TGF‐β. Confluent monolayer was scratched using sterile 200 μl pipette tip, and cells were washed (PBS) and new medium added (DMEM/F12 0.5% FBS + TGF‐β). The medium was changed every 2 days (with fresh TGF‐β) until scratch closing (8 days). At day 8, samples were decellularized using alkaline detergent extraction buffer (0.5% Triton X‐100, 20 mM NH₄OH in PBS). After 2 min, coverslips were washed with PBS, and DNase treated (10 μg/ml) for 30 min at 37°C. To visualize the ECM, the coverslips were labelled with 2 μg/ml NHS‐ester Alexa Fluor 488 dye (A20000; Thermo Fisher Scientific) diluted in 50 mM sodium bicarbonate buffer pH 9 for 15 min in the dark. Coverslips were washed, treated with 200 mM Tris buffer pH 7.5 for 10 min to deactivate the NHS esters and blocked in 1% BSA before being mounted in Fluorescent Mounting Medium. Images were acquired using Cell Observer videomicroscope (Zeiss), Zen 2012 blue edition. Files were cropped and contrast adjusted using Photoshop (Adobe).