Yoda1

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.

4 kDa FITC-dextran was obtained from Maokang Biotechnology (Shanghai, China). Roswell Park Memorial Institute (RPMI) 1640 medium and Dulbecco’s Modified Eagle Medium (DMEM) was procured from Gibco (USA). Antibodies for Piezo1, Occludin and ZO-1 were procured from Proteintech (China). The following chemicals were obtained from Servicebio (China): fluorescein (FITC) Tunel Cell Apoptosis Detection Kit, Phosphate Buffered Saline (PBS) and Hank’s Balanced Salt Solution (HBSS). Fluorescent-conjugated antibodies for flow cytometry were procured from BD Bioscience (USA). Secondary antibodies conjugated with AlexaFluor-488 or Alexa Fluor 594, Annexin V-FITC /PI Apoptosis Detection Kit, JC-1 Mitochondrial Membrane Potential Assay Kit, SYBR qPCR Master Mix, Rhod-2 AM and Fetal Bovine Serum (FBS) were procured from Yeason (China). Yoda1 and MitoSOx Red were procured from MedChemExpress (USA). MitoTEMPO was acquired from Topscience (China). DMEM no Ca²⁺ was obtained from Meiluncell (China).

Human proximal tubular cells (HK2 cells) were obtained from ATCC and grown in DMEM/F12 (Corning) containing 10% FBS (Quacell Biotechnology) and 1% penicillin/streptomycin (Corning) and maintained at 37°C in 5% CO₂ atmosphere. Mouse kidney proximal tubular cells (mPTCs) were prepared as previously described (57 (link)). Briefly, mPTCs were isolated from 10-week-old C57BL/6J mice and cultured in DMEM/F12 (Corning) containing 10% FBS (Quacell Biotechnology) at 37°C in 5% CO₂ atmosphere.
The HK2 cells and mPTCs were seeded on 6-well plates (Thermo Fisher Scientific) at a density of 2 × 10⁵ cells/well for 24 hours and were serum-starved for 12 hours. The medium was changed to fresh serum-free DMEM/F12 before treatment. For morphological assessments, the cells were fixed with 4% paraformaldehyde and photographed using a microscope (Leica) connected to a digital camera with a macroconversion lens. For profibrotic response experiments, HK2 cells and mPTCs were treated with cyclic stretch for 24 hours or TGF-β₁ (5 ng/mL, R&D Systems) for 48 hours, accompanied with either GsMTx4 (1 μM or 5 μM, TAIJIA biotech) or Piezo1 siRNA (RIBOBIO) pretreatments. Yoda1 (0.2 μM, MedChemExpress) was used to stimulate HK2 cells for 1 to 24 hours, with or without Piezo1 siRNA. A total of 1 μM Yoda1 was used to stimulate mPTCs for 24 hours. All the cells were then collected for the protein or RNA analyses.