Figure S1. Effect of transient dasatinib washouts on cell death and Bcr-Abl signaling in CML
cells
KU812 cells were treated with (A) 100 nM dasatinib for 30 min followed by the standard
(STD) wash and recultured for 48 h in drug-free media or (B) continuously with 1 nM
dasatinib for 48h. Protein lysates were analyzed by western blot for Bcr-Abl signalling (left)
and apoptotic signaling (right).
Figure S2: OPT washouts prevent induction of cell death in CML cells.
KU812 (left) and Meg01 (right) cells were incubated either continuously with 1 nM dasatinib,
100 nM dasatinib for 72 h or transiently exposed to 100 nM dasatinib for 30 min followed
either by the STD or optimal (OPT) wash and recultured for 72 h in drug-free media. Cells
were then analyzed by Annexin V/7-AAD staining (Data are mean±SEM, n=3).
Figure S3. Extension of high dose imatinib exposure prior to OPT washouts induce cell
death in CML cells.
Imatinib treatment for >2 h prior to OPT wash induces cell death. (A) KU812 cells were
incubated with 32.5 μM imatinib for 0-8h, followed by OPT wash and then cultured for 72h
in drug-free media. (B) Meg01 cells were incubated with 32.5 μM imatinib for 0-8h, followed
by OPT wash and then cultured for 72 h in drug-free media. Cells were then analysed by
Annexin V/7-AAD staining.
Figure S4. Extension of dasatinib exposure prior to OPT washouts induce cell death in
primary CP-CML CD34+ cells.
Newly diagnosed CP-CML CD34+ cells were treated with (A) 100 nM dasatinib or (B) 32.5 µM
imatinib for 0-8 h, followed by OPT wash. Cells were then analysed by Annexin V/7-AAD
staining (n=3 mean+SEM).
Figure S5. Pimozide and STAT5i induce cell death following in K562 and Meg01 CML cells.
K562 (A) and Meg01 (B) cells were transiently exposed to 100 nM dasatinib followed by
either STD wash or OPT wash in the presence or absence of either 10 µM of the STAT5
inhibitor pimozide (left) or 50 µM of the STAT5 inhibitor N′ -((4-Oxo-4H-chromen-3yl)methylene) nicotinohydrazide (STAT5i) (right). Cells were analysed by Annexin V/7-AAD
staining (n=3, mean±SEM).
Figure S6. Pimozide and STAT5i induce cell death following the imatinib OPT wash.
Newly diagnosed CP-CML CD34+ cells were treated with 32.5 µM imatinib (IM) for 30 min,
followed by OPT wash in the presence or absence of the STAT5 inhibitors (A) pimozide or (B)
STAT5i and then cultured for 72 h in imatinib-free media containing that STAT5 inhibitor.
Cells were analysed for CFCs at 2 weeks (n=3, mean±SEM).
Figure S7. STAT5 inhibition, with 30 min dasatinib exposure on Bcr-Abl signaling in CML
cells.
De novo CP-CML CD34+ cells were transiently exposed to 100 nM dasatinib in the presence
or absence of either 10 µM of the STAT5 inhibitor pimozide or 50 µM of the STAT5 inhibitor
N′ -((4-Oxo-4H-chromen-3-yl)methylene) nicotinohydrazide (STAT5i), followed by the OPT
wash and then cultured for 30 min in dasatinib-free media containing pimozide or STAT5i.
Cells were then analysed for pSTAT5(Y694) by intracellular phospho flow cytometry (n=1)