Imatinib

Renal cell carcinoma cell line, A498, was purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and penicillin–streptomycin (Gibco-BRL, Carlsbad, CA, USA) in 37°C incubator. For the establishment of imatinib-resistant A498 (A498/R), A498 cells were incubated with 1 mM imatinib (Sigma-Aldrich, Shanghai, China), and the concentration of imatinib was increased by 1 mM per week. The cells were treated with an increased concentration of imatinib for 5 months. Five months later, A498 cells were incubated with 20 mM imatinib for another 1 month to establish A498/R cell line. A498/R was transfected with shRNA (short hairpin RNA) targeting PDIA6 (shPDIA6) or the negative control (shNC) (Genepharma, Shanghai, China) by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). A498/R was also cotransfected with shPDIA6 and pcDNA-FZD1 by Lipofectamine 2000. The transfection efficiency was confirmed by western blot. Two days later, the cells were treated with 10 mM imatinib for 24 hours before functional assays [15 (link)].

The human CML cell line (K562) was generously provided by Dr. Kai-Wen Hsu from the Research Center for Cancer Biology at China Medical University, Taichung, Taiwan. The K562-IR cells, a derivative of the K562 cell line, were developed through a two-month exposure to 0.05 μM of Imatinib (Sigma-Aldrich, St. Louis, MO, USA), followed by one month of exposure to increasing Imatinib concentrations of 0.1, 0.5, 1, and 5 μM [21 (link)]. During the K562-IR cell line establishment, the culture medium was refreshed weekly. The cells were cultured in Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and supplemented with 10% (v/v) fetal bovine serum (Biological. Industries, Kibbutz Beit Haemek, Israel), along with 100 units/mL of penicillin and 100 mg/mL of streptomycin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cell cultures were maintained at 37 °C in an incubator with 5.0% CO₂. Lentiviral transfection was conducted using a multiplicity of infection (MOI) of 5 for 72 h, followed by antibiotic selection with either neomycin (Sigma-Aldrich, St. Louis, MO, USA) or puromycin (Sigma-Aldrich, St. Louis, MO, USA) for 48 h. The genomic Indel (insertions or deletions) profiling was assessed using Sanger sequencing and immunoblotting.