Xav 939

For osteogenic induction, BMSCs (1×10⁶ cells/well) were seeded into 6-well plates and cultured in the complete culture medium supplemented with osteogenic induction medium (OIM) containing 10⁻⁷ M dexamethasone, 10 mM β-glycerophosphate and 50 µM ascorbate-2-phosphate (all purchased from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 14 days (21 (link)). Next, the cells were collected and subjected to ossification assessment.
For 1,25(OH)₂D₃ treatment, BMSCs were incubated with 1, 5, 10, 20 or 50 nM of 1,25(OH)₂D₃ (Sigma-Aldrich; Merck KGaA) dissolved in ethanol for 48 h. Equivalent volume of 100% ethanol was used in the negative control group. Furthermore, XAV-939 (Selleck Chemicals, Shanghai, China) treatment was used to inhibit Wnt/β-catenin signaling in BMSCs at a concentration of 10 µM for 1 h, with equal volume of DMSO (Beyotime Institute of Biotechnology) as a negative control. For co-treatment of 1,25(OH)₂D₃ and XAV-939 (MedChemExpress, Inc.), the cells were first treated with 10 µM XAV-939 for 1 h at 37°C, followed by treatment with 10 nM 1,25(OH)₂D₃ for 48 h at 37°C.

The human myeloid leukemia cell lines, NB4, HL60, KG1, U937, Kasumi-1, THP-1 and K562 were purchased from American Type Culture Collection. All cells were cultured at 37°C in 5% CO₂ atmosphere in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and streptomycin (North China Pharmaceutical Co., Ltd.).
NB4 and HL60 cells were selected to conduct the following experiments. Both cell lines were treated with 10 µg/ml dauno-rubicin (DNR; Shenzhen Main Luck Pharmaceuticals Inc.) for 24 h at 37°C to evaluate the cell sensitivity to DNR. In addition, NB4 and HL60 cells were treated with 10 µM XAV939 (MedChemExpress USA), or 10 µM XAV939 + 10 µg/ml DNR and 20 mM LiCl (Sigma Aldrich; Merck KGaA), or 20 mM LiCl + 10 µg/ml DNR for 24 h at 37°C to perform mechanism-related experiments.