Jc 1 dye

JC‐1 method was used to measure mitochondrial membrane potential as described previously.32 In brief, following treatment, H9C2 cells were incubated with 2.5 mmol/L JC‐1 dye (Solarbio, Beijing, China) for 30 min in the dark at 37°C. Red JC‐1 aggregates represented a normal hyperpolarized membrane potential, while Green JC‐1 monomers indicated a loss of mitochondrial membrane potential. Images were obtained by the fluorescence microscope at 400× amplification.

The standards of ginsenoside Rb3 and cisplatin was obtained from Aladdin (China). Hematoxylin and eosin were purchased from Nanjing Jiancheng (China). The primary antibody of TGF-β, Smad2, Smad3, p-Smad2, p-Smad3, Bcl-2, Bax, cleaved PARP, cleaved Caspase 3, cleaved caspase 8, cleaved caspase 9, and β-actin were supplied from Abcam (USA). Annexin V-FITC apoptosis kit was purchased from BD (USA). JC-1 dye was obtained from Solarbio (China). Caspase 3, caspase 8, and caspase 9 colorimetric assay kits were purchased from BioVision (USA).

Following incubation and treatment of cells on 12-well plates, 661w cells were loaded with JC-1 dye (Solarbio, Cat#M8650) for 25 min. Next, the cells were washed three times with PBS and photographed immediately under a fluorescence microscope. For each well, JC-1 aggregates fluorescence (red) and monomer JC-1 fluorescence (green) were detected at the excitation/emission spectra of 560/595 nm and 485/535 nm, respectively. The red/green ratio was calculated as an indicator of MMP.

The Mitochondrial membrane potential of MGC-803 cells treated with PEO was measured by JC-1 dye (Solarbio, Beijing, China). The MGC-803 cells in the logarithmic phase were cultured in a 6-well plate at 37 °C, 5% CO₂ (v/v) at a density of 3 ~ 5 × 10⁵ cells per well. After 24 h of treatment with different concentrations PEO (0, 25, 50, and 100 µg/mL), cells were centrifuged and collected. 1 mL Jc-1 reagent (the concentration of Jc-1 was 10 µg/mL) was added to each centrifuge tube and incubated for 20 min at 37 °C. Then, the samples were rinsed twice with PBS and analyzed by flow cytometer.

Sodium hyaluronate was purchased from Yuanye Bio-Technology Co. Ltd (Shanghai, China). Ethylene glycol, sodium periodate (100–150 kDa), rhodamine B, and polyvinyl pyrrolidone were purchased from Aladdin (Shanghai, China). 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA), 4′, 6-diamidino-2-phenylindole (DAPI), potassium ferricyanide, and gelatin were obtained from Sigma. Cy5 was bought from Ruixibio (Xi’an, China). Cell Count Kit-8 (CCK-8) and Live/dead kit were acquired from Dojindo Molecular Technologies (Kumamoto, Japan). The JC-1 dye and Annexin V-FITC Apoptosis Detection Kit was bought from Solarbio (Solarbio, China). Antibodies of MMP3 (ab52915) and were purchased from Abcam (Abcam, UK). Collagen II (sc-52658) was purchased from Santa cruz (Santa cruz, USA). Aggrecan (13,880-1-AP), MMP13 (18,165-1-AP) and SOX9 (67,439-1-Ig) was purchased from Proteintech (Proteintech, China).

H9C2 cells were incubated with 2.5 mmol/L JC‐1 dye (Solarbio, Beijing, China) in the dark at 37 °C for 30 min. Red JC‐1 aggregates represent normal hyperpolarized membrane potential, whereas green JC‐1 monomers represent mitochondrial membrane potential loss. Images were obtained using a fluorescence microscope at 400 × magnification.