Bm mscs

Human BM-MSCs (Cyagen Biosciences Inc., Guangzhou, China) were initially seeded at a density of 5000 cells/cm² as previously described [13 (link)]. The cells were cultured in alpha minimum essential medium (α-MEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Thermo Fisher Scientific) in a humidified 37°C/5% CO₂ incubator with medium change every three days. After reaching 80% confluence, BM-MSCs were harvested by treating with 0.25% trypsin-EDTA (Thermo Fisher Scientific), counted for cell number, and replated in multiwell culture plates for the next stage of the experiments.
KGN (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) at the stock concentration of 20 mM. To examine the effect of kartogenin on BM-MSCs, cells were treated with 10⁻⁸ M, 10⁻⁷ M, and 10⁻⁶ M KGN. Untreated cells served as the control group. Cells treated with 0.005% DMSO served as the vehicle control group. To investigate the role of SIRT1 in KGN-mediated antioxidant functions, BM-MSCs were treated with 10 mM NAM (Sigma-Aldrich) and 10⁻⁶ M KGN.

Human bone marrow mesenchymal stem cells (BM-MSCs; catalog no. PCS-500-012) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM; D5030, Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; F2442; Sigma-Aldrich, St Louis, MO, USA), 100 U/mL Penicillin (Invitrogen, USA), and 100 μg/mL Streptomycin (Invitrogen, USA).
For osteogenic differentiation, the BM-MSCs were cultured in DMEM (Invitrogen, USA) containing 1% FBS (F2442; Sigma-Aldrich, USA), 200 μM l-glutamine (G7513; Sigma-Aldrich, USA), 10 nM dihydroxyvitamin D3 (D1530; Sigma-Aldrich, USA), 10 mM β-glycerolphosphate (50020; Sigma-Aldrich, USA), 100 nM dexamethasone (D4902; Sigma-Aldrich, USA), and 80 μg/mL ascorbic acid phosphate (A8960; Sigma-Aldrich, USA). And the medium was refreshed every 3–4 days.
To evaluate the effects of Forkhead Box O3 (FOXO3) on the autophagy of BM-MSCs, 100 nM autophagy activator Rapamycin (RAPA; R0395; Sigma-Aldrich, USA) was added into the BM-MSCs transfected with lentivirus carrier of small interfering RNA for FOXO3 (siFOXO3). 5 mmol/L autophagy inhibitor 3-methyladenine (3-MA; M9281; Sigma-Aldrich, USA) was added into BM-MSCs transfected with lentivirus carrier of overexpressed FOXO3 plasmid.

Cell viability was assessed using MTT assays, with each experimental group consisting of multiple wells (6 wells per group) for parallel experiments. Cells in each group were treated with complete medium containing various concentrations (0, 5, 10, 15, 20, 25 µM) of nanoparticles for 2, 12, and 24 h, while the control group received an equal volume of PBS solution. After the designated incubation period, 20 µl of MTT reagent (5 mg/ml) was added to each well of a 96-well plate and incubated for 4 h. The reaction was stopped by adding 100 µl of dimethyl sulfoxide (DMSO) and incubating at 37 °C in the dark for 10 min. Cell proliferation was determined by measuring the absorbance at 490 nm.
Mouse bone marrow-derived MSCs (Invitrogen, USA) were cultured in DMEM-F12 medium (HyClone, Logan, UT, USA) supplemented with 10% FBS (HyClone), 1% gentamicin (GIBCO-BRL Life Technologies, Gaithersburg, MD, USA), and 1 × Glutamax (Invitrogen). Human hepatoma cell lines SMMC.7721 were obtained from the Chinese Center for Type Culture Collection (CCTCC, Wuhan, China) and cultured in Dulbecco's modified Eagle's medium (DMEM, HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY). Both cell types were incubated in a humidified incubator at 37 °C with 5% CO₂. Mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) were purchased from Invitrogen.

The UCB obtained from the umbilical vein after the neonatal delivery of an infant was processed within 24 h for the isolation and separation of mononuclear cells (MNCs) with Ficoll-Hypaque solution (density = 1.077 g/cm³; GE Healthcare, Uppsala, Sweden), followed by previous protocol [20 (link), 21 (link)]. This protocol was approved by the Institutional Review Board of MEDIPOST Co., Ltd. (MP-2014-07-1-1). MNCs were washed with phosphate buffer saline (PBS) and cultured in minimum essential medium α medium (MEM-α, Gibco/Invitrogen, Carlsbad, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (Gibco) at 37°C in a humidified atmosphere containing 5% CO₂. The culture medium was changed twice per week. The basic characterization of UCB-MSCs is summarized in Supplementary Table 1. Primary BM-MSCs and human AT-MSCs were purchased from Promega (Gibco/Invitrogen, Heidelberg, Germany). For growth kinetics, the trypan blue exclusion method was performed to analyze the expansion of live cells. At each passage (P), MSCs were cultured for 5 days, then reseeded at a cell density of 2,000 cells/cm². The PD at each passage was calculated by dividing the logarithm of 2 [20 (link)]. The analysis of PD was continued until the proliferation of cells was stopped.