Acrylamide monomer

Polyacrylamide gels were prepared as previously described^{55 (link)}. Briefly, 10 kPa and 100 kPa poly(acrylic acid) (PAA) gels were prepared by combining acrylamide monomers (Sigma Aldrich) at 10% and varying the concentration of bis-acrylamide cross-linkers (Sigma Aldrich) from 0.1% to 0.4%, respectively. Polymerization was initiated by the addition of TEMED (Sigma Aldrich) followed by 10% Ammonium persulfate (Sigma Aldrich) at a volume ratio of 1:250 and 1:100, respectively. The gel solution was pipetted between two glass coverslips, one of which having been treated by APTMS 0.5% (Sigma Aldrich) followed by 0.5% glutaraldehyde (Sigma Aldrich) to firmly attach the gel to the coverslip.
PAA functionalization was achieved using the ultraviolet (UV) activated cross-linker Sulfo-SANPAH (Thermo Fischer). Each gel was coated with a 20 mg per mL solution of Sulfo-SANPAH and exposed to 365 nm UV light for 10 min. The gel was then washed to remove any excess cross-linker and then coated with a 500 µg per mL solution of TNP-BSA and incubated at 4 °C for 12 h. Gels were then washed and coated with a 10 µg per mL IgE anti-TNP solution for 1 h at room temperature, followed by a further washing step.

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 functionalised by silanizing with (3-aminopropyl)trimethoxysilane (Sigma). Subsequently 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^{53 (link),54} . To start the polymerisation reaction, 5 μl of 10% ammonium persulphate (Sigma) and 0.75 μl N,N,N′,N′-tetramethylethylenediamine (TEMED, Sigma) were added into 0.5 ml mixtures. To allow cell adhesion, gels were treated with 1 mg/ml N-sulphosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate (sulpho-SANPAH, Sigma) which was activated by UV light. Finally, gels were incubated with 10 μg/ml collagen I from rat tail overnight at 4 °C and UV-sterilised prior to cell seeding.

EVs from the same samples analysed for their cytokine profile were also examined for the content of other proteins. The EVs were lysed in 8 M urea and 100 mM ammonium bicarbonate on ice and sonicated using a Vibra-Cell ultrasonic processor (Sonics & Materials Inc., Newtown, CT, USA). The samples were reduced and alkylated using 5 mM tributylphosphine (Sigma-Aldrich, St. Louis, MO, USA) and 20 mM acrylamide monomers (Sigma-Aldrich, St. Louis, MO, USA) for 90 min at room temperature, and the excess acrylamide was quenched with 20 mM dithiothreitol (Sigma-Aldrich, St. Louis, MO, USA). The urea was then diluted to 1 M with 100 mM ammonium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA). EV lysates, ranging from 10–100 µg of protein, were digested with trypsin from porcine pancreas, proteomics grade (Sigma-Aldrich, St. Louis, MO, USA) at a 1:50 enzyme:protein ratio, and incubated at 37 °C for 16 h. Insoluble membrane components were removed by ultracentrifugation (100,000× g for 3 h). The digested EV proteins were de-salted using Discovery DSC-18 solid phase extraction columns (SUPELCO, Bellefonte, PA, USA). The peptide concentration was determined using the Pierce quantitative colorimetric peptide assay (Thermo Fisher Scientific, Waltham, MA, USA) and prepared for LC-MS/MS analysis.

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.

Sodium deoxycholate (NaDC), acrylamide monomer (AAm), ethylene glycol dimethacrylate cross- linker (EGDMA), benzoyl peroxide (BPO), Human serum albumin (HSA) with a molecular weight (69 kDa) and absolute ethanol were obtained from Sigma-Aldrich (St. Louis, MO, USA). High molecular weight poly (vinyl chloride) (PVC), dioctyl phthalate plasticizer (DOP), aliquate 336S and tetrahydrofuran (THF) were obtained from Fluka AG (Buchs, Switzerland). Several aqueous solutions of NaDC were prepared with freshly de-ionized water (18.2 MΩ cm specific resistance). The appropriate buffer for the potentiometric measurements was prepared from 30 mM NaHCO₃-Na₂CO₃ buffer, pH 9.2, ionic strength 0.1M NaCl.HSA stock solution (100 mg/dL) was prepared by dissolve 0.1 g of bovine serum albumin and 20 mg of sodium azide in water in a 100-mL volumetric flask. The solution was diluted to the volume with water and stored at 4 °C. Working HSA solutions were prepared by appropriate dilutions of the stock albumin standard solution and stored at 4 °C. Albumin solutions containing 20 mg/dL sodium azide show no evidence of bacterial growth after storage for 6 months at 4 °C.