Icrt 14

After surgery, all mice were allowed to rest for 4 weeks and then treated with the β-catenin inhibitor iCRT14 for 6 weeks. iCRT-14 (S8704) was purchased from Selleck (TX, USA) and was first dissolved in DMSO and then further dissolved in corn oil (50 mg·kg⁻¹ body weight) and administered every 3 days for 6 weeks by i.p. injection. Injection of corn oil (S6701, Selleck, USA) was used as a negative control, and celecoxib (S1261, Selleck, USA) was used as a positive control. celecoxib was also first dissolved in DMSO and then dissolved in corn oil (10 mg·kg⁻¹ body weight) and delivered by i.p. injection on the same schedule as iCRT-14.

The human chondrocyte cell Line C28/I2 (BLUEFBIO, China) and the human embryonic kidney 293 cell line (HEK293) (ATCC, USA) were cultured according to the vendor’s instructions. C28/I2 cells were cultured in DMEM/F-12 medium (Gibco, USA), and HEK293 cells were cultured in DMEM (Gibco, USA). All media were supplemented with 10% fetal bovine serum at 37 °C under 5% CO₂. IL-1β (20 ng·mL⁻¹, P00019, Solarbio, China) was used to induce the expression of inflammation-related genes in C28/I2 cells for 12 h. BIO (1 μmol·L⁻¹, S7198, Selleck, USA) was used to treat C28/I2 cells for 12 h to activate β-catenin signaling and induce β-catenin target genes. 50 μmol·L⁻¹ iCRT14 (S8704, Selleck, USA) was used to treat C28/I2 cells for 12 h to inhibit β-catenin signaling. The TCF7 plasmid (40620, Addgene, USA) was transfected into C28/I2 cells or 293 cells using Lipofectamine 3000 to overexpress TCF7. qPCR, Western blotting or ChIP assays were performed 48 h after transfection. The cell lines were tested for mycoplasma-free status before they were used.

Human GBC cell lines NOZ (K-ras mutant) and GBC-SD were provided by Dr. Yingbin Liu (Department of General Surgery, Xinhua Hospital affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, P.R. China) and Kunming Cell Bank of The Chinese Academy of Science (Kunming, P.R. China), respectively. These cells lines were not passaged for more than 6 months after authentication by the cell bank. NOZ and GBC-SD cells were cultured at 37°C in William's E medium or DMEM with 10% fetal bovine serum (FBS) (Invitrogen Technologies, Inc, CA), respectively.
In some experiments, cells were treated with MEK inhibitor trametinib (GSK1120212) and Wnt pathway inhibitor iCRT-14 as the indicated concentrations and times, and the medium and agent were replenished every day. GSK1120212 and iCRT-14 were purchased from Selleck Chemicals and Santa Cruz Biotechnology, respectively. Both of them were dissolved in dimethylsulfoxide (DMSO), aliquoted and stored at −80°C until use. The same volume of vehicle was used as control.