Wnt agonist 1

NSCLC cell lines with mutated EGFR (19del), HCC827 (Keygenbio, Nanjing, China) were cultured at 37°C in a humidified 5% CO₂ atmosphere. The cell lines were cultured in RPMI‐1640 medium. To culture the cell lines, 10% fetal bovine serum was supplemented in the medium. Gefitinib (Selleckchem, Houston, TX, USA), which is a type of EGFR–TKI, was mixed in the medium at increasing concentrations to generate and maintain resistant cell lines for approximately six months. The WNT/β‐catenin inhibitor XAV‐939 and WNT/β‐catenin activator WNT agonist 1 were purchased from Selleckchem (Houston, TX, USA).
The following primary antibodies were used: anti‐GAPDH (Abcam, ab181602), mTOR (CST,mAb #2983), Phospho‐mTOR (Ser2448) (CST, mAb #5536). AKT (Abcam, ab32505), p‐AKT (Abcam, ab192623).

Human breast cancer cell lines T47D, MCF7, MDA-MB-231, MDA-MB-468 and SK-BR-3 were purchased from the Cell Bank of Type Culture Collection of Shanghai Biological Institute, Chinese Academy of Science (Shanghai, China). The breast epithelial cell line MCF10A was purchased from the American Type Culture Collection (Manassas, VA, USA) and used as control cells. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂.
Cell transfection was conducted using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturers' protocols. The transfection was performed 48 h prior to the subsequent analysis. The siRNAs were provided by GenePharm Co. (Shanghai, China; cat. no. 17892). Wnt agonist 1 was purchased from Selleck Chemicals (Shanghai, China) and used at a final concentration of 10 µM at 37°C for 24 h. The culture medium was replaced every two days unless otherwise stated.