Supplementary Figure Legends
Supplementary Figure 1.
(A) Western analysis of IGF-1 expression. -Actin was used as a loading control. Cells
were treated for 18 hrs with 5 µM imatinib for K562, 1 µM imatinib for KU812 and
JURL-MK1 or PBS (-).
Supplementary Figure 2.
(A) Expression of Stat5b in K562 and JURL-MKI transfected with Stat5b shRNA or
control shRNA after 48 hrs. Western analysis was carried out using Stat5b antibody. βactin was used as a loading control. (B) RT-PCR analysis of Stat5b, Stat5a, Stat3, and actin mRNA expression in K562 and JURL-MK1 cells transfected with Stat5b shRNA or
control shRNA.
Supplementary Figure 3.
(A) Trypan blue exclusion analysis of the effects of Stat5b shRNA on K562 and JURLMK1 viability. (*) represents P-value < 0.01 between viable cells in Stat5b shRNA and
control shRNA transfected cells. (B) MTT analysis of the effects of Stat5b shRNA on
K562 and JURL-MK1 proliferation. (**) represents P-value < 0.05 between Stat5b
shRNA and control shRNA transfected cells. (C) Annexin V analysis of the effects of
Stat5b shRNA on K562 and JURL-MK1 apoptosis. The percentage of cells undergoing
apoptosis was detected using Annexin V fluorescein isothiocyanate (FITC) and
propidium iodide (PI) staining. (*) represents P-value < 0.01 between Stat5b shRNA and
control shRNA transfected cells. (D) Effect of Stat5b shRNA on the cell cycle of K562
and JURL-MK1. Histograms represent the PI staining of DNA content in different phases
of the cell cycle. Percentages of cells in each phase of the cell cycle are shown on the
histograms. For panels A and B, cells were cultured for 72 hrs after transfection. For
panels C and D, cells were cultured for 48 hrs after transfection. For panels A, B, and C,
error bars represent standard deviation from three independent measurements.
Supplementary Figure 4.
(A) Trypan blue exclusion assay of K562, KU812, and JURL-MK1 viability after
treatment with AG490 or DMSO (Con) for 72 hrs. (*) represents P-value < 0.01 between
viable cells in AG490 treated and untreated cells. (B) MTT analysis of K562, KU812,
and JURL-MK1 proliferation after treatment with AG490 or DMSO (Con) for 72 hrs.
(**) represents P-value < 0.05 between AG490 treated and untreated cells. (C) Annexin
V assay of K562, KU812, JURL-MK1 apoptosis after treatment with AG490 or DMSO
(Con) for 72 hrs. (**) represents P-value < 0.05 between AG490 treated and untreated
cells. The percentage of cells undergoing apoptosis was detected using Annexin V
fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining. (*) represents Pvalue < 0.01 and (**) represents P-value < 0.05 between PP2 treated and untreated cells.
(D) Cell cycle analysis of K562, KU812, and JURL-MK1 after treatment with AG490 or
DMSO (Control) for 48 hrs. Histograms represent the PI staining of DNA content in
different phases of the cell cycle. Percentages of cells in each phase of the cell cycle are
shown on the histograms. For panels A-C, error bars represent standard deviation from
three independent measurements.
Supplementary Figure 5.
(A) Expression of Hck in K562 and JURL-MK1 transfected with Hck shRNA or control
shRNA after 48 hrs. Western analysis was carried out using Hck antibody. β-actin was
used as a loading control. (B) Expression of IGF-1 in K562 and JURL-MK1 transfected
with Hck shRNA or control shRNA after 48 hrs. Western analysis was carried out using
IGF-1 antibody. β-actin was used as a loading control.
Supplementary Figure 6.
(A) Expression of Bcr-Abl in K562, KU-812, and JURL-MK1. Western analysis was
carried out using c-Abl antibody. β-actin was used as a loading control.
Supplementary Materials and Methods
Reagents and Antibodies - Imatinib was obtained from the Royal University Hospital
(Saskatoon, SK, Canada). AG490, PP2, and AG1024 were purchased from Calbiochem
(San Diego, CA, USA). IGF-1 polyclonal rabbit antibody used in Western analysis is
from Abcam Inc. (Cambridge, MA, USA) and IGF-1 polyclonal rabbit antibody for
immunohistochemistry is from Biogenesis (Kingston NH, USA). -Actin mouse antibody
is from Sigma-Aldrich (Oakville, ON, Canada). Polyclonal rabbit phospho-p44/42 MAP
Kinase (Thr202/Tyr204) antibody, polyclonal rabbit phospho-AKT (Ser473) antibody,
polyclonal rabbit total AKT antibody polyclonal, polyclonal rabbit c-Abl antibody, and
phospho-Stat5b antibody (Tyr694) are from Cell Signaling Technology (Danvers, MA,
USA). Hck rabbit polyclonal antibody used for flow cytometry and Western analysis,
phospho-Hck rabbit antibody (Tyr411), monoclonal Stat5b antibody used for chromatin
immunoprecipitation assay and Western analysis, and polyclonal mouse Stat5a antibody
are from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal IGF-1 mouse
antibody used for flow cytometry and recombinant human IGF-1 are from R & D
Systems (Minneapolis, MN, USA). Monoclonal mouse ERK1/2 is from BD Biosciences.
Stat5b and control shRNA plasmids are from Panomics (Fremont, CA, USA). Hck and
control shRNA plasmids are from Origene (Rockville, MD, USA).
Immunoprecipitation – Immunoprecipitations were performed as previously described
(Pastural et al., 2007). Briefly, 5x106 cells/ml were treated with or without imatinib (1
µM) for 1 hr. Cells were lysed and supernatants were precleared with 50 µl protein A
Sepharose (anti-Stat5a Ab) and protein G Sepharose (anti-Stat5b Ab). Sepharose was
removed and anti-Stat5a or anti-Stat5b (4 µg) antibody was added and incubated for 1 hr
at 4 0C. 50 µl protein A Sepharose (anti-Stat5a Ab) and protein G Sepharose beads (antiStat5b Ab) were added and incubate for 1 hr at 4 0C. Immunprecipitated proteins were
characterized by Western analysis with anti-Stat5b, anti-Stat5a, and anti-phoshoTyr694Stat5 antibodies.
PCR - cDNA was amplified in a 25 μl reaction containing 12.5 μl SybrGreen PCR
Master Mix (Bio-Rad), 100 pmol primers, and 0.25 μg cDNA on a Mini-Opticon RealTime PCR machine (Bio-Rad) using the following program: 1 cycle at 95oC for 5 min; 45
cycles at 95oC for 45 s, 60.6oC 45 s, 72oC for 45 s. Primer sequences for IGF-1,
hypoxanthine phosphoribosyltransferase (HPRT), Stat5b, and Stat3 amplification are
listed in Supplementary Table 1. HPRT expression was used to normalize mRNA
expression.
Flow Cytometry - 106 cells were fixed and permeabilized with IntraPrep reagents
(Beckman Coulter, Marseille, France). Proteins were stained using an unconjugated
specific primary antibody at a 1:20 dilution in Intraprep Reagent 2 for 15 min. Cells were
washed in PBS and labeled for 20 min using a FITC-conjugated secondary anti-mouse
antibody for IGF-1 and Stat5b and a FITC-conjugated secondary anti-rabbit antibody for
Hck staining. Labeled cells were washed in PBS, resuspended in 500 l of PBS
containing 0.5% formaldehyde, and analyzed using an Epics XL flow cytometer
(Beckman Coulter). All labeling reactions were performed in the absence of specific
primary antibody as a control for background staining. For flow cytometry experiments a
minimum of 20,000 events were collected. Cells were gated for side scatter and forward
scatter populations to eliminate data from cell debris, dead cells, and clumps of two or
more cells. Gated populations were then analyzed for fluorescence using FLOWJO
software (Ashland, OR, USA). The histograms represent the results from three separate
experiments.