Gsmtx4

ADSCs were pretreated with PD98059 (HY-12028, 80 μM, MedChemExpress), GsMTx4 (HY-P1410A, 5 μM, MedChemExpress), and Yoda1 (HY-18723, 30 μM, MedChemExpress) for 1 hour as requested, and the cells were harvested 2 hours after LIPUS stimulation without specification. ADSCs were lysed on ice with RIPA buffer (Beyotime, Shanghai, China) containing the protease inhibitor PMSF (Beyotime, Shanghai, China) and Phosphatase Inhibitor Cocktail (YEASEN, Shanghai, China). The protein concentrations of cell lysates were measured using a BCA protein assay kit (Beyotime, Shanghai, China). Protein samples (15 μg) were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. The membranes were then blocked with 10% bovine serum albumin (BSA) for 1 hour at room temperature and incubated with primary antibodies at 4°C overnight. The primary antibodies used in this study were ERK (ab184699, 1 : 10000, Abcam, USA), p-ERK (ab76299, 1 : 5000, Abcam, USA), VEGFA (ab214424, 1 : 1000, Abcam, USA), and GAPDH (ab181602, 1 : 10000, Abcam, USA). After being rinsed with TBST, the membranes were hybridized with secondary antibodies and reacted with ECL solution (NCM Biotech Co., Ltd, Suzhou, China). The chemiluminescent images were taken, and the density of each protein band was determined using ImageJ software.

CFs were transfected with siRNAs by using Lipofectamine™ 2000 (Thermo Fisher Scientific, USA). The siRNA was used to knockdown Piezo1 in CFs. The targeted sequence of the siRNA directly against rat Piezo1 RNA was 5′-CGGCCAACAUAAAGAACAUTT. The targeted sequences of siRNAs directly against rat integrin β1 RNA was 5′-GCCAGAUGGAGUAACAAUATT. The negative control was performed with a scrambled siRNA (5′-UUCUCCGAACGUGUCACGUTT).
CFs were transfected with a plasmid to overexpress YAP. The CFs were seeded on PEG-RGD hydrogel disc with a diameter of 35 mm. Then, a preincubated mixture (including Opti-MEM, DNA, and Lipofectamine™ 2000) was quickly dropped into each culture plate. Approximately 6–8 h after transfection, the medium was removed and replaced with new medium (including FBS and penicillin/streptomycin), and the CFs were allowed to recover for 3 days on the matrices prior to analysis.
For the inhibition treatments, cells were respectively treated with 5 μg/mL integrin β1-blocking antibody (BD Pharmingen™, USA), 2.5 μM GsMTx4 (MedChemExpress, USA), 1 μM PF-562271 (MedChemExpress, USA) and 2 μM PLX-4720 (MedChemExpress, USA), and the control groups were added into the corresponding amount of DMSO diluted in culture media.