N sulfosuccinimidyl 6 4 azido 2 nitrophenylamino hexanoate

To prepare polyacrylamide gels, glass coverslips were washed twice with 70% ethanol, activated with 0.1 m NaOH, and silanized with (3‐aminopropyl)trimethoxysilane (Sigma). The glass coverslips were then treated with 0.5% glutaraldehyde (Sigma) for 30 min and washed extensively with sterile Milli‐Q water. Mixtures of Milli‐Q water, acrylamide monomers (Sigma), and crosslinker N,N‐methylene‐bis‐acrylamide (Sigma) were prepared according to previously determined formulations to obtain 2 kPa gels.^[49] For the polymerization reaction, 5 µL of 10% ammonium persulfate (Sigma) and 0.75 µL N,N,N′,N′‐tetramethylethylenediamine (Sigma) were added into 0.5 mL mixtures. To functionalize gels with ECM proteins, gels were first treated with 1 mg mL⁻¹N‐sulfosuccinimidyl‐6‐(4′‐azido‐2′‐nitrophenylamino) hexanoate (Sigma), which was activated by ultraviolet (UV) light. Finally, gels or silanized glass coverslips were incubated with 10 µg mL⁻¹ collagen I from rat tail (Sigma) or human fibronectin (Sigma) for 3 h at room temperature (RT) and UV‐sterilized prior to cell seeding.

Round microscopy cover slides were washed with 70% ethanol and 0.1 m NaOH, covered with 3‐aminopropyltrimethoxysilane (3‐APTS, Sigma) for 3 min for activation, incubated for 30 min in 0.5% glutaraldehyde (Sigma), and washed in sterile MilliQ water. Polyacrylamide solutions containing acrylamide monomers (Sigma), crosslinker N,N‐methylene‐bis‐acrylamide (Sigma) and PBS in different concentrations were prepared to create different Young's modulus (0.5, 2, 4.5, 10, 20 and 115 kPa) as previously described [27 (link)]. 5 μL of 10% ammonium persulfate (Sigma) and 0.75 μL N,N,N′,N′‐tetramethylethylenediamine (Sigma) were added into 0.5 mL mixtures, and one drop of the mixture was placed on rain repellent‐treated microscopy slides, and the activated cover slides were placed on top. After polymerisation (3–10 min), the cover slides with polyacrylamide gels were washed with PBS. Next, the cover slides were treated with 1 mg·mL⁻¹N‐sulfosuccinimidyl‐6‐(4′‐azido‐2′‐nitrophenylamino) hexanoate (Sigma) and exposed to ultraviolet (UV) light to allow subsequent collagen‐I binding. The cover slides were incubated at room temperature with 10 μg·mL⁻¹ rat‐tail collagen‐I (Sigma) for 3 h, washed with PBS and placed in UV light for sterilisation. Wells containing cover slides were seeded with SKUT1 cells or primary SMC, uLMS, LM or MM cells.

To prepare polyacrylamide gels, glass coverslips were washed twice with 70% ethanol, activated with 0.1 M NaOH, and silanized with (3-aminopropyl) trimethoxysilane (Sigma). Then the glass coverslips were treated with 0.5% glutaraldehyde (Sigma) for 30 min and washed extensively with MilliQ water. Mixtures of MilliQ water, acrylamide monomers (Sigma), and crosslinker N,N-methylene-bis-acrylamide (Sigma) were prepared according to previously determined formulations^{62 ,63 (link)}. For the polymerization reaction, 5 μl of 10% ammonium persulfate (Sigma) and 0.75 μl N,N,N′,N′-tetramethylethylenediamine (Sigma) were added into 0.5 ml mixtures. To functionalize gels with collagen, gels were first treated with 1 mg/ml N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate (Sigma), which was activated by ultraviolet (UV) light. Finally, gels were incubated with 10 μg/ml collagen I from rat tail (Sigma) or human collagen VI (Rockland) for 3 h at room temperature (RT) and UV-sterilized prior to cell seeding.