Wz4003

The human cancer cell lines A549, H1299, NCI-H460, MNK45, U251, SW480, SW620, HCT116, Colo320 and HT-29, PC-3, U266, and the mouse breast cancer cell line 4T1 were cryopreserved in the Hematological Laboratory of Zhujiang Hospital (Guangzhou, China). All cell lines were incubated in DMEM medium supplemented with 10% fetal bovine serum at 37 °C with 5% CO₂. WZ4003, SBI-0206965, Chloroquine, and MRT68921 were purchased from Selleckchem (Houston, TX, USA), dissolved in DMSO or water, and stored at −20 °C. CCK-8 was purchased from Dojindo Laboratories (Japan). Mitotracker, DAPI, and TritonX-100 were purchased from Solarbio (Beijing, China).

We treated breast cancer cell lines with NUAK inhibitors WZ4003 (S7317) and HTH-01-015 (S7318), which were purchased from Selleck Chemicals and diluted in dimethyl sulfoxide (DMSO). For determination of cell line drug sensitivity, 1000–3000 cells were seeded in each well of 96-well plates and treated with 1–30 μM of drug. Cell viability was measured using a MTS cell proliferation assay (Promega CellTiter AQueous One) when the cells reached 80–90% confluence 3–5 days after treatment. The surviving fraction of cells in each well was determined relative to DMSO-treated controls. Drug sensitivity experiments were carried out in quadruplicate. Drug sensitivity was determined by calculating the drug concentration required to reduce cell viability by 50% relative to DMSO-treated controls (IC₅₀). IC₅₀ values were determined from dose-response data using GraphPad Prism version 7.

The h‐JBMMSCs were cultured as described in our previous study, and the protocol was approved by the Medical Ethical Commission of the Peking University School of Stomatology (PKUSSIRB‐201520026). The informed consent was obtained from all the patients. The h‐JBMMSCs were obtained from patients undergoing orthognathic surgery and were identified based on positivity for CD73, CD90 and CD105 and negativity for CD34, CD11b, CD19, CD45 and HLA‐DR using flow cytometry 23, 24. The osteoblast‐inducing conditional medium consisted of α‐MEM medium with 10% FBS, 10 nM dexamethasone, 10 mM β‐glycerophosphate and 50 μg/ml L‐ascorbic acid (Sigma‐Aldrich, St. Louis, MO, USA). The complete medium consisted of α‐MEM medium with only 10% FBS. The cells were cultured with or without 1 μg/ml APN (dissolved in PBS, Z03072; GenScipt, Piscataway, NJ, USA) produced using HEK 293 cells with endotoxin <0.01 EU/μg (determined using the LAL method; data not shown). For the CXCR2 inhibitor (10 μM working concentration dissolved in DMSO, SB225002; Selleck, Boston, MA, USA), p38 inhibitor (10 μM working concentration dissolved in DMSO, SB203580; Selleck, USA) and AMPK inhibitor (5 μM working concentration dissolved in DMSO, WZ4003; Selleck, USA) assay, the cells were treated with the inhibitors for 2 hrs before the experiments.