Cba 201

Gene expression changes in cells were performed by qPCR after total RNA was extracted using Direct-zol RNA MiniPrep Kit (Zymo Research R2052). Quantitative PCR was performed in a ViiA 7 Real-Time PCR System. Protein expression changes was monitored using Western blotting. After blocking, the blots were probed with antibodies against collagen I (COL1, Abcam ab6308), VWF (Novus NBP2-33003) or αSMA (Abcam ab5694). For loading control, the blots were immunoblotted with antibodies against GAPDH (Cell Signaling #2118). Band quantification was performed using ImageJ. The IncuCyte Live-Cell Imaging System was used to monitor cell proliferation or migration. After adding different treatments cells were monitored by IncuCyte. Cell counts were analyzed by the IncuCyte S3 Analysis software. Gel contraction assays was performed using the cell contraction kit from Cell Biolabs (CBA-201). Immunofluorescence on cells was performed using anti-SMA antibodies (Abcam ab5694) or anti-VWF antibodies (Novus NBP2-33003) followed by Alexa Fluor secondary antibodies (Thermo Fisher).

Dermal FBs from HS patients were treated with 10 μM of the LATS kinase inhibitor TRULI/Lats-IN-1 (MedChemExpress HY-138489) or 0.1–10 μM of the YAP/TEAD inhibitor verteporfin (Cayman Chemical 17334) for 48–72 hours. Additional 6-hour cytokine stimulations were performed using IL-1β (10 ng/mL; R&D Systems 201-LB-005) and TNF-α (10 ng/mL; R&D Systems 210-TA-005). Gene expression changes in cells were performed by quantitative PCR after total RNA was extracted using Direct-zol RNA MiniPrep Kit (Zymo Research R2052). Quantitative PCR was performed in a ViiA 7 Real-Time PCR System (Applied Biosystems). Protein expression changes were monitored using Western blotting. After blocking, the blots were probed with antibodies against collagen I (COL1, Abcam ab6308) or α-smooth muscle actin (Abcam ab5694). For loading control, the blots were immunoblotted with antibodies against GAPDH (Cell Signaling 2118). Band quantification was performed using ImageJ (NIH) (50 (link)). The IncuCyte Live-Cell Imaging System was used to monitor cell proliferation or migration. After addition of different treatments, cells were monitored by IncuCyte. Cell counts were analyzed by the IncuCyte S3 Analysis software. Gel contraction assays were performed using the cell contraction kit from Cell Biolabs (CBA-201).

For HCAECs, cells underwent EndMT induction for 5 days with or without MC1568. MPLECs were treated with 4-OH tamoxifen or vehicle and with or without EndMT, as described above. Thereafter, cells were trypsinized and resuspended in complete medium at 1 × 10⁶ cells/mL. Cell suspensions were mixed 1:4 with a collagen gel working solution according to the manufacturer’s protocol (CBA-201, Cell Biolabs) and plated onto 48-well plates using 250 μL/well and allowed to set for 1 hour. After the collagen was set, 500 μL serum-free medium was loaded into each well for 1 hour to serum starve the cells before replacing 400 μL of serum-free medium with complete medium plus lysophosphatidic acid (LPA) (catalog 3854, R&D Systems; contraction initiator, final concentration at 10 μM), and the gels were then released. Gels were imaged after 24 hours and contraction measured by calculating gel area using ImageJ software.

Cell proliferation was tested using the WST cell proliferation kit (BioVision) according to the manufacturer’s instruction. 5 × 10³ cells were plated on 96-well plates and cultured for overnight before the WST assay. In vitro wound healing assay was performed using radius 24-well cell migration assay kit (CBA-125; Cell Biolabs, Inc.) according to the manufacturer’s instruction. Collagen gel contraction assay was conducted using cell contraction assay kit (CBA-201; Cell Biolabs, Inc.) according to the manufacturer’s instruction. All assays were tested in triplicates and assays were repeated three times.

The contractility of hMSMCs was evaluated using a cell contraction kit (CBA-201, Cell Biolabs Inc. San Diego, CA, USA) according to the manufacturer’s instructions. Collagen solution was prepared and mixed with cell suspension, then added to a 24-well plate and cultured for 1 h to form a solid gel. Then, 1 mL of medium was added, and the cells were cultured for an additional 48 h. The hMSMCs were placed in either a hypoxic or normoxic environment and stimulated with oxytocin (10 nM at final concentration) to induce contractions. After 2 h of treatment, the gel was released by gently scraping along the well walls with a tip of a sterile pipette. The area of the gel was measured at 1–4 h using a ChemiDoc XRS+ (Bio-Rad, Hercules, CA, USA), and the value of gel area was calculated by Image Lab.