Establishing Imatinib-Resistant K562 Cell Line

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.