Af1333

The tibias were minced and homogenized with RIPA lysis buffer and protease inhibitor cocktail (Beyotime) at 4 °C to obtain 1:9 (W/V) whole homogenate. The homogenates were centrifuged at 8000× g for 10 min under 4 °C to obtain the femoral proteins. Aliquots of protein extract were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane, operating on a Bio-rad instrument (Hercules, CA, USA). The membrane was incubated with primary antibodies against GSK-3β (AG751, Beyotime), phospho-GSK-3β (Ser 9, AF1531, Beyotime), β–Catenin (AF0069, Beyotime), Runx2 (D1L7F, CST, Danvers, MA, USA), Nrf2 (AF7623, Beyotime), HO-1 (AF1333, Beyotime), and the loading control β–actin (2148, Abcam) or α/β–tubulin (2148S, CST). Horseradish peroxidase-conjugated secondary antibody (Beyotime) was used to enhance ECL chemiluminescence detection. The protein bands were scanned and analyzed by ImageJ software (NIH, Bethesda, MD, USA). Each protein band was normalized to its β–actin band, and the levels of phosphorylated proteins were normalized to the levels of their corresponding total proteins. The results were calculated as percentage change to the ND group.

The cells were lysed with RIPA lysis buffer (P0013B, Beyotime) on ice for 30 min. The supernatants of cells were harvested after centrifugation at 12,000 g for 10 min at 4°C. A BCA protein assay kit (P0012S, Beyotime) was used to determine the total protein concentration. Protein extracts were mixed with loading buffer and boiled at 95°C for 10 min. The boiled samples were loaded onto SDS-polyacrylamide gels followed by electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with TBS-tween containing 5% skim milk, inculcated with either RUNX2 (1:1,000 dilution, S12556, Cell Signaling Technology), BMP2 (1:1,000 dilution, AF0075, Beyotime), HMOX-1 (1:1,000 dilution, AF1333, Beyotime), BID (1:1,000 dilution, AF6306, Beyotime), IL-6 (1:1,000 dilution, AF7236, Beyotime), HIF-1α (1:1,000 dilution, CSB-PA002906, CUSABIO), PRKAA2 (1:2,000 dilution, CSB-PA805325LAO1HU, CUSABIO), β-actin (1:5,000 dilution, AF5003, Beyotime), or GAPDH (1:10,000 dilution, S2118, Cell Signaling Technology) antibodies overnight at 4°C. The membranes were washed and inculcated with HRP-labeled secondary antibody (1:5,000 dilution, ab205718, Abcam). Detection was done using clarity western ECL (WBKLS0100, Millipore). The images were acquired through the e-Blot Touch Imager and semiquantitative analyses were performed by the FIJI software.

Sal B was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Potassium chloride (KCl) was obtained from Sigma (St. Louis, MO, USA). Dihydroethidium (DHE) was from Life Technologies (Carlsbad, CA, USA). Protease inhibitor cocktail was from Sigma Chemicals (St. Louis, MO, USA). Antibodies against gp91^phox (1 : 1000, sc-130543), Keap1 (1 : 500, sc-515432), and NQO1 (1 : 500, sc-32793) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Cleaved caspases-3 (1 : 500, ab49822) and Nrf2 (1 : 1000, ab92946) were from Abcam (Cambridge, UK); HO-1(1 : 1000, AF1333) and Histone H3 (1 : 500, AF7101) were from Beyotime Institute of Biotechnology (Shanghai, China); GAPDH (1 : 2000; #2118), Bax (1 : 1000, #14796), and Bcl-2 (1 : 1000, #3498) were from Cell Signaling Technology (Beverly, MA, USA).