Ficoll paque density gradient centrifugation

K562 cells were maintained in RPMI 1640 medium (Corning) supplemented with 10% fetal bovine serum (FBS) (Invitrogen). HaCaT cells were maintained in MEM-EBSS medium (Corning) with 10% FBS. HEK293T cells were cultured with DMEM medium (Corning) supplemented with 10% FBS. HaCaT cells were transiently transfected with X‐tremeGENE (Roche Applied Science) and HEK293T cells with PEI (Polyscience). K562 or HaCaT stable cell lines expressing ABL or Smad4 variants were generated by lentivirus infection and selected with puromycin/G418 at appropriate concentrations. Bone marrow samples were collected from CML patients. PBMCs were separated from bone marrow samples by Ficoll-Paque density gradient centrifugation (Miltenyi Biotec, Bergisch Gladbach, Germany). PBMCs were maintained in a-MEM medium (Corning) supplemented with 10% fetal bovine serum (FBS) (Invitrogen).

This study was authorized by the Ethics Committee II of the Medical Faculty Mannheim of the Heidelberg University (2018-566N-MA). Written informed consent was given by all participants. Blood samples were collected from 8 healthy donors (3 males, 5 females, mean age: 50 years), 26 CP-CML patients in DMR or MMR (9 males, 17 females, mean age: 63 years), 5 CP-CML patients with loss of MMR (2 males, 3 females, mean age: 69 years), 5 de novo untreated CP-CML patients (1 male, 4 females, mean age: 47 years), and 3 BP-CML patients (1 male, 2 female, mean age: 59 years) (Table 1). Treatment with TKI included imatinib, nilotinib, dasatinib, and bosutinib, respectively. PBMCs were separated from whole-blood samples by Ficoll-Paque density gradient centrifugation (Miltenyi Biotec, Bergisch Gladbach, Germany).