Dimethyloxaloylglycine increases bone repair capacity of adipose-derived stem cells in the treatment of osteonecrosis of the femoral head

Zhu, Zhen-Hong; Song, Wen-Qi; Zhang, Chang-Qing; Yin, Ji-Min

Western blot analysis. To evaluate the influence of DMOG (Sigma-Aldrich) on the expression of HIF-1α protein in ASCs, the cells were seeded on six-well plates at 3x10 5 cells/well and regular medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum) was added with different concentrations of DMOG (0, 200, 500 and 1,000 µM). After 1, 3 and 7 days, total protein was harvested from the cultured cells according to standard protocols (24) . Briefly, cells were washed three times with (PBS; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), and then solubilized in lysis buffer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 4˚C for 10 min. Lysates were centrifuged at 14,000 x g for 15 min. The supernatants were collected and stored at -80°C. The protein concentration was measured using a bicinchoninic protein assay kit (Thermo Fisher Scientific, Inc.). Proteins (10 µg) from each sample were then separated using 12% SDS-PAGE (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred to nitrocellulose membranes. They were then blocked in 5% non-fat milk at room temperature for 1 h. Primary antibodies against HIF-1α (1:800; ab1; Abcam, Cambridge, MA, USA) were added to incubate with the membranes at 4˚C overnight. Then, infrared-conjugated secondary antibodies (1:10,000; ab97040; Abcam) were added to incubate with the membranes for 1 h at room temperature. The resulting membranes were scanned in an Odyssey Scanner (Li-COR Biosciences, Lincoln, NE, USA), and quantified using Odyssey software version 3.0. The protein levels were normalized against those of β-actin (1:500; ab6276; Abcam).