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Supplementary Figure Legends
Figure S1: Phosphorylation status of FES and FER in AML cell lines.
(A) Tyrosine phosphorylation status of FER following FER immunoprecipitation in AML cell
lines. The phosphorylation of FER is shown on the top panel (anti-PY). The fraction of
immunoprecipitated FER is shown below. The expression of FER and of a control protein,
AKT, in the cell lysates are shown in the two lower panels.
(B) Tyrosine phosphorylation status of FES following FES immunoprecipitation in AML cell
lines. The phosphorylation of FES is shown on the top panel (anti-PY). The expression of
FES and of a control protein, AKT, in the cell lysates are shown in the two lower panels.
Figure S2: Reduced FES and FER phosphorylation in MOLM-14 upon inhibition of FLT3
kinase activity.
MOLM-14 cells were treated either with DMSO or with FLT3 inhibitor SU11248 (2 µM) for
90 minutes. FES and FER phosphorylation status was determined by immunoprecipitation
with specific monoclonal antibodies (IP anti-FES and IP anti-FER) and revelation using 4G10
antiphosphotyrosine antibody.
Figure S3: Activation of FES kinases in transfected FLT3-ITD TF-1 cells.
FES or FER proteins were immunoprecipitated from parental or FLT3-ITD transfected TF-1
cell lysates. The phosphorylated forms of the proteins were revealed using 4G10
phosphotyrosine antibody.
Figure S4: Slower rate of cell division in FER-depleted cells.
MV4-11 cells transfected either with control-, fes- or fer-siRNAs were labeled with CFSE.
Left panels: Histogram plots represent the profiles for the 3 cell populations, control-siRNA
(black line), fes-siRNA (grey dotted line) and fer-siRNA (dark spotted line) on day 0 and 3
days following labelling.
Right panels: Profiles of the individual cell populations at day 3. The estimated number of cell
divisions is indicated above the histograms. The analyses were done with FlowJo software.
The profiles presented are from 1 of 3 independent experiments that gave similar results.
Figure S5: Cell cycle is impaired in FER depleted cells.
MV4-11 cells transfected with either control, fer- or fes-siRNAs were stained with anti-BrdUFITC antibody and 7-AAD. The percentage of cells in G0/G1, S, or G2/M phases was
quantified using FlowJo software. The values are from 1 of 3 independent experiments that
gave similar results.
Figure S6: Detection of senescence-associated ß-galactosidase in fes-siRNA MV4-11 treated
cells.
(A) Illustration of ß-gal positive cells in the fes-siRNA cells. Cells transfected with the
indicated siRNA were stained using a Senescence Beta-galactosidase Detection Kit.
(B) Percentage of ß-gal positive cells in the 3 cell populations. The histogram represents the
results of three independent experiments.
(C) Overview of the ß-gal staining in the 3 cell populations. Stained cell populations were
resuspended in a small volume of PBS to visualize the staining of the whole population.
Figure S7: Wild-type FLT3 signaling in OCI-AML5 cells.
OCI-AML5 cells, treated with the indicated siRNAs, were stimulated for 5 min with FLT3
ligand (FL). The activation of ERK1/2, AKT and GSK3beta was detected using activationstate phospho-specific antibodies on cell lysates. The level of expression of the respective
proteins is shown.
Figure S8: FLT3-ligand dependent cell survival in OCI-AML5.
Cells treated with either control-, fes- or fer-siRNAs were deprived of serum to induce cell
death (-FL) and reveal the survival effect of FLT3-ligand (+FL).