Propidium iodide solution

HCCLM3, HepG2, Huh7, and SMMC-7721 cells were treated with DMSO or ML-323 for 24 h. The cells were collected and fixed with 70% absolute ethanol at 4 °C overnight, and then propidium iodide solution (50 μg/mL; Solarbio) containing RNase A (30 μg/ml; Solarbio) was added at 37 °C for 30 min and detected by flow cytometry (NOVOCYte3130; ACEA Biosciences, Hangzhou, China).

MC3T3-E1 cells originated from Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China). Minimum essential medium-alpha modification (α-MEM) and foetal bovine serum (FES) were provided by Gibco (Gaithersburg, USA). MC3T3-E1 cells were cultured with α-MEM supplemented with 10% FBS at 37 °C in a 5% CO2 atmosphere.
High-glucose (25.0 mmol/L) medium was produced by adding extra D-glucose purchased from Sigma–Aldrich Co., Ltd. (Shanghai, China) to the ordinary medium. β-glycerophosphate and ascorbic acid were purchased from Aladdin Co., Ltd. (Shanghai, China), and β-glycerophosphate (10 mM) and ascorbic acid (50 mg/mL) dissolved in α-MEM supplemented with 10% FBS acted as the osteogenesis medium. Anti-GSK3β, anti-p-GSK3β (S9), anti-β-catenin, anti-Cyclin D1, anti-Cmyc, and anti-Runx2 were purchased from Abcam (Cambridge, UK). Alizarin Red S staining solution, propidium iodide solution and DAPI solution were purchased from Solarbio Co., Ltd. (Beijing, China). Furthermore, Beyotime Biotechnology (Suzhou, China) provided an EdU Cell Proliferation Kit with Alexa Fluor 555, BCIP/NBT Alkaline Phosphatase Colour Development Kit, Alkaline Phosphatase Assay Kit, anti-β-actin, HRP-labelled secondary antibody and Enhanced Chemiluminescence Kit. XAV939 was purchased from MedChemExpress Company (New Jersey, USA).