X omat bt film

Total protein was isolated from chondrocytes by incubation in RIPA lysis buffer (Beyotime, Cat P0013K) supplemented with Pierce^™ Protease Inhibitor Mini Tablets (Thermo Fisher Scientific, Cat A32955). Protein concentrations were quantified using a BCA protein assay kit (Beyotime). A 20 μg aliquot of protein was denatured, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto a piece of nitrocellulose membrane (Beyotime). Nonspecific bindings were blocked by incubation in a blocking buffer (Beyotime) for 30 min, and the membrane was incubated in properly diluted primary antibodies against SIRT1, SOX9, and GAPDH at 4°C overnight. Next, the membrane was incubated in horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Blots were detected using SuperSignal West Pico Substrate (Thermo Fisher Scientific) and X-OMAT BT Film (Beyotime). The protein intensity was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Human BM-MSCs were purchased from Cyagen Biosciences Inc. (Guangzhou, China). Fetal bovine serum (FBS), alpha minimum essential medium (α-MEM), penicillin, streptomycin, TRIzol^® reagent, Dulbecco’s modified Eagle medium (DMEM), trypsin-ethylene diamine tetraacetic acid (EDTA), horseradish peroxidase-conjugated secondary antibodies, protease inhibitor tablets, and SuperSignal West Pico Substrate were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Standard cell culture plates and flasks were purchased from Costar (Tewksbury, MA, USA). Fibronectin, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), 4’,6-diamidino-2-phenylindole (DAPI), paraformaldehyde, L-ascorbic acid, dexamethasone, β-glycerol phosphate, Sirtinol, Compound C (CC), and phosphate buffered saline (PBS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against SOD1, SOD2, CAT, GPx1, SIRT1, AMPK, p-AMPK (phospho T183 + T172), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Abcam (Cambridge, MA, USA). Cell lysis buffer, polyacrylamide gels, nitrocellulose membranes, and X-OMAT BT Film were purchased from Beyotime Institute of Biotechnology (Haimen, China). All other materials and reagents were purchased from diverse conventional suppliers.

Human adult BM-MSCs were purchased from Cyagen Biosciences Inc. (HUXMA-01001, Guangzhou, China). Standard cell culture plates and flasks were purchased from Costar (Tewksbury, MA, USA). Fetal bovine serum (FBS), alpha minimum essential medium (α-MEM), Dulbecco’s modified Eagle medium (DMEM), penicillin, streptomycin, TRIzol^® reagent, protease inhibitor tablets, horseradish peroxidase-conjugated secondary antibodies, and SuperSignal West Pico Substrate were purchased from Thermo Fisher Scientific (Waltham, MA, USA). ResV, NAM, phosphate buffered saline (PBS), H₂O₂, dexamethasone, L-ascorbic acid, β-glycerol phosphate, paraformaldehyde, fluorescein diacetate (FDA), and 2′,7′-dichlorofluorescein diacetate (DCF-DA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against SIRT1, SOD1, SOD2, CAT, GPX1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Abcam (Cambridge, MA, USA). Cell lysis buffer, polyacrylamide gels, nitrocellulose membranes, and X-OMAT BT Film were purchased from Beyotime Institute of Biotechnology (Haimen, China).