Hadscs

The hADSCs were purchased from Cyagen Biosciences Inc. (Guangzhou, China). Briefly, the hADSCs were isolated and purified from fresh human adipose tissues donated from healthy adults less than 45 years of age after liposuction. The cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, United States) containing 10% fetal bovine serum and cultured at 37°C in a humidified atmosphere of 5% CO₂. Osteogenic induction medium was purchased from SALILA (SALILA, Guangzhou, China). The medium was changed every 2 days. To inhibit DNA methylation, hADSCs were treated with 5 μmol/L 5-aza-2′-deoxycytidine (DAC, Sigma Aldrich, Munich, Germany). To inhibit NOTCH signaling, cells were treated with 2 μM DAPT (Selleck). Protein deacetylation was inhibited using 20 μM OSS_128167 (Selleck). For the cycloheximide (CHX)-chase assay, cells were treated with 100 g/mL CHX (Sigma-Aldrich, St. Louis, MO, United States) for the indicated hours in the absence or presence of 5 g/mL actinomycin D (Sigma-Aldrich, St. Louis, MO, United States) and western blot analysis was performed.

Human adipose-derived stromal cells (HADSCs) were purchased from Cyagen Biosciences Co., Ltd. (Suzhou, China). The cells were cultured in a complete culture medium (Cyagen Biosciences Co., Ltd., Suzhou, China).

Human adipose-derived mesenchymal stem cells (hADSCs) isolated from fat tissue were obtained from Cyagen Biosciences (Guangzhou, China) as previously described50 (link). hADSCs were cultured in DMEM/F12 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen) and 100 units/mL penicillin and streptomycin (Invitrogen). Passage 3 hADSCs were used for all experiments. hADSCs were cultivated in serum-free conditions for 24 h prior to stimulation with 10 mM lithium chloride (LiCl) from Amresco (Solon, OH, USA) for 24 h51 (link). 293T cells were cultured in DMEM/F12 (Invitrogen) supplemented with 10% FBS (Invitrogen) and 100 units/mL each of penicillin and streptomycin (Invitrogen). All cells were incubated at 37 °C in a 5% CO₂ humid atmosphere, and the cell medium was changed every 2–3 days.