Cell contraction assay kit

Gel contraction assay was performed using a cell contraction assay kit (Cat: CBA-201, Cell Biolabs, Inc, San Diego, CA) according to the manufacturer’s instructions. CAFs were trypsinized, washed twice with PBS, resuspend with complete medium(10ul) mixed with collagen and 5xDMEM, total volume 500 ul. This 500 μl of cell-collagen mixture was plated into 24-well plates and incubated at 37 °C for 2 h (for collagen polymerization). Gel contraction was initiated by releasing the gel from the side of the well, and changes in the collagen gel size were observed. The complete medium was changed every 2 days.

HTM cell contraction was tested by a cell contraction assay kit (Cell Biolabs, Inc.) following the manufacturer’s instructions. In brief, HTM cells at a density of 2 ×10⁶ cells/mL were mixed with collagen gel and added to a 24-well plate. After 1 h of incubation at 37 °C for polymerization, the culture medium was added to each well of a plate. After 48 h of incubation, the edge of the gel was carefully detached using a syringe needle. After 13 h, the gel area did not change, and natural contraction was observed. Then, 1 ml of the TIPARP small-molecule inhibitor RBN-2397 (1 μM) was added to the medium. The images were captured at 24 h. The gel area was calculated using ImageJ.

According to the protocol (Cell Contraction Assay Kit, Cell Biolabs, San Diego), the collagen solution, 5× phosphate-buffered saline (PBS), and neutral solution were mixed and diluted in proportion and then placed on ice. The number of uterine smooth muscle cells was adjusted to 800,000 cells/100 µl cell suspension. The cell suspension and diluted collagen solution were mixed (1:4) to configure the gel, and the gel was added to a 24-well plate at 500 µl/well and then incubated for 1 h in an incubator at 37°C to allow the gel to solidify. The gel was divided into 7 groups: normal glucose group, high glucose group, normal with CoCl₂ group, CoCl₂ with echinomycin group, high glucose with echinomycin group, CoCl₂ with L-methionine group, and high glucose with L-methionine group. After collagen solidification of the first three groups, they were cultured in the corresponding medium. The gels of the latter four groups were first incubated in a CoCl₂ medium or high glucose medium for 4 h, then changed to the corresponding medium containing echinomycin or L-methionine, and incubated for 4 h. KCl and oxytocin were added to every group at 4 h after gel solidification. The gel areas of each group were observed and recorded with a camera at the time of solidification (0 h) and the addition of KCl/Oxytocin at 4 h; the areas were measured using Image J.

Dihydrorotenone was provided by Dr. Kyeong Kyu Kim at Sungkyunkwan University (Suwon, Korea). An annexin-V staining kit was purchased from BD Bioscience (Franklin Lakes, NJ). An EZ-cytox cell viability assay kit was purchased from Daeill Lab (Seoul, Korea). A cell contraction assay kit was purchased from Cell Biolabs, Inc (San Diego, CA). Twist1 (Cat: ab50887, Abcam, Cambridge, MA), FSP1 (Cat: 07-2274, Millipore, Burlington, MA), PDGFRα (Cat: S3164, Cell Signaling, Danvers, MA), PDGFRβ (Cat: ab32570, Abcam, Cambridge, MA), FAPα (Cat: 53066, Abcam, Cambridge, MA), α-SMA (Cat: (E184) 04-1094, Millipore, Burlington, MA) and α-tubulin antibodies (Cat: SC-8035, Santa Cruz, Dallas, TX) were used for immunoblotting.

Cell contraction assay was evaluated using a Cell Contraction Assay kit according to the manufacturer's instructions (CELL BIOLABS CBA‐201). Briefly, HASMCs were harvested and suspended at 5 × 10⁵ cells/ml, and the collagen lattice was prepared by mixing two parts of cell suspension and eight parts of cold collagen gel solution. Subsequently, 500 μl of the cell–collagen mixture was cast into each well of a 24‐well plate and allowed to polymerize at 37°C for 1 h. After collagen polymerization, cells were incubated in SMC growth medium (Medium 231 plus SMGS) for 24 h. During which stress developed. Upon release of the collagen lattice from the culture dish, the embedded cells become free to contract the deformable lattice, thus reducing its surface area. This was quantified 24 h after detachment of the gel from the dish using ImageJ and expressed as the percentage of the area of the entire well.

The 3D collagen gel contraction assay was performed with Cell Contraction Assay Kit (Cell Biolabs Inc., CA, USA) according to the manufacturer’s protocol. The cells (10⁶) treated with CH-SEVs, LW-SEVs, miR-218 mimics, negative control or TGIBL1 were mixed with cold Collagen Gel Working Solution. Then, 0.5 mL of a mixture of cells and collagen was transferred into 24 well plates, which were cultured for 1 h at 37 ºC with 5% CO₂. Then, 0.8 mL of 10% FBS culture medium was added to each well and incubated for 48 h after that. Collagen contraction was initiated by gently releasing the gel from the sides of platelets, after the cells were treated with 10 mM BDM contraction mediators. The collagen contraction gel was obtained, and the areas were measured using ImageJ.