The cDNAs encoding BCR/ABL and BCR/ABL-T315I have been previously described (3). All retroviral expression vectors used in this study were based on the bi-cistronic PINCO vector.
Cell lines and patient-derived long-term cultures
The Ba/F3 and Rat-1 cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and were maintained as previously described (3). Ph + ALL patient derived long term cultures (PDLTCs) expressing BCR-ABL-T315I (KÖ) were obtained from a patient enrolled in the German Multi-Center Study Group for acute lymphatic leukemia of the adult (GMALL 07/2003) upon informed and written consent  and were maintained in a serum–free medium consisting of IMDM supplemented with 1 mg/mL of bovine insulin, 5x10-5 M β–mercaptoethanol (Sigma, Steinheim, Germany), 200 mg/mL Fe –saturated human apo–transferrin (Invitrogen, Karlsruhe, Germany), 0.6% human serum albumin (Sanquin, Amsterdam, The Netherlands), 2.0 mM L–glutamine and 20 mg/mL cholesterol (Sigma) . Proliferation was assessed with the XTT proliferation kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions.
Isolation of Sca1+/lin- hematopoietic stem and progenitor cells (HSPCs)
Sca1+/lin- HSPCs were isolated from 8- to 12-week-old female C57BL/6 N mice (Janvier, St. Berthevin, France) after euthanization by CO2 asphyxiation. Bone marrow (BM) was harvested from the femur and tibia by flushing the bones with a syringe and a 26-gauge needle. Sca1+ cells were purified by immunomagnetic beads using MACS cell separation columns according to the manufacturer’s instructions (Miltenyi, Bergisch-Gladbach, Germany). Prior to subsequent use, the purified cells were pre-stimulated for 2 days in DMEM supplemented with 10% FCS (Hyclone/Perbio Science, Bonn Germany), 1% L-Glutamine, 1% Penicillin/Streptomycin, mIL-3 (20 ng/mL), mIL-6 (20 ng/mL) and mSCF (100 ng/mL) (Cell Concepts, Umkirch, Germany).
Transfection and retroviral infection
Ecotropic retroviral supernatants were obtained after transfection of Phoenix packaging cells as described earlier . For infection of target cells, Retronectin® (Takara Bio Inc., Otsu, Japan) was used to enhance infection efficiency according to the manufacturer’s instructions. Then, 2x105 target cells were seeded per well. Infection efficiency was measured after 48 h by determining the percentage of GFP positive cells using flow cytometry.
Soft agar and focus formation assays were performed using untransformed Rat-1 fibroblasts retro virally transduced with PINCO vectors harboring unmutated BCR/ABL or BCR/ABL-T315I. Six-well plates were filled with DMEM supplemented with 10% FCS and 0.5% bacto-agar (DIFCO Laboratories, Detroit, MI, USA) (2 ml per well). Then, 5x103 transduced Rat-1 cells were suspended in “top-agar” (DMEM supplemented with 10% FCS and 0.25% bacto-agar) (1 ml per well) and stacked in the wells. Colonies were counted after 15 days of incubation at 37°C and 5% CO2. For focus formation assays, 4x104 transduced Rat-1 cells were plated per well of a 24-well plate. Foci were stained after 15 days using 1% crystal violet (Sigma).
Colony assays on HSPCs
At day 5 post-infection, Sca1+ cells were plated at 5x103 cells/mL in methyl-cellulose either with mIL-3 (20 ng/mL), mIL-6 (20 ng/mL) and mSCF (100 ng/mL) or without cytokines (Stem Cells Inc., Cambridge, UK). The number of colony forming units (CFUs) was determined 10 days after plating and normalized according to the transduction efficiency.
Western blot analysis was performed according to widely accepted protocols. The following antibodies were used: anti-ABL (α-ABL) (St. Cruz Biotechnology, Santa Cruz, CA, USA), anti-phosphorylated ABL specific for the phosphorylated tyrosine residue 245 (α-p-ABL-Y245) (Cell Signaling, Boston, MA, USA), anti-BCR (α-BCR) (St. Cruz Biotechnology), anti-phosphorylated BCR specific for the phosphorylated tyrosine residue 177 (α-p-BCR-Y177), anti-Crkl, and anti-phosphorylated Crkl (Cell Signaling).
Differences in response rates towards different concentrations of a single inhibitor or inhibitors in combination were analyzed by Student′s t-tests. Statistical analyses were performed using the GraphPad Prism (GraphPad Software, San Diego, CA) software package. Evaluation of the character of the combined effects was performed according to the three dimensional model of Prichard and Shipman using MacSynergy software .