Supplementary methods:
Cell lines: Akt WT, Akt1
−/−
(Akt1 KO), Akt1/2
−/−
(Akt DKO) MEF, pTEN
+/−
, pTEN
−/−
MEF
were the kind gifts of Dr. N. Hay (Department of Biochemistry and Molecular Genetics,
University of Illinois, Chicago, IL) and cultured in DMEM with 10% FBS.
Experimental agents:
Primary antibodies for Notch-1, Notch-2, Notch-3, Notch-4, Jagged-1, Jagged-2, Dll-1,
Dll-4, FoxM1, VEGF and MMP-9 were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). Antibody against Akt and phospho-Akt (Ser473) were purchased from Cell Signaling
Technology. The monoclonal antibody to β-actin and PI3K inhibitor, LY294002, were purchased
from Sigma-Aldrich. All secondary antibodies were obtained from Pierce (Rockford, IL).
Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA). Chemiluminescence
detection of proteins was done with the use of a kit from Amersham Biosciences (Amersham
Pharmacia Biotech, Piscataway, NJ). Protease inhibitor cocktail, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), and all other chemicals were obtained from Sigma (St.
Louis, MO).
Preparation of Nuclear extract:
The ICN transfected PC-3 cells were washed with cold phosphate-buffered saline and
suspended in 0.15 ml of lysis buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1
mM EGTA, 1 mM DTT, 1 mM PMSF, 2 µg /ml leupeptin, 2 µg /ml aprotinin, and 0.5 mg/ml
benzamidine]. The cells were allowed to swell on ice for 20 min and then 4.8 µl of 10% Nonidet
P-40 was added. The tubes were then vigorously mixed on a vortex mixer for a few seconds and
centrifuged for 120 sec in a microfuge. The nuclear pellet was re-suspended in 30 µl of ice - cold
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nuclear extraction buffer [20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1
mM DTT, 0.5 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 0.5 mg/ml benzamidine]
and incubated on ice with intermittent mixing. The tubes were then centrifuged for 5 min in a
microfuge at 4ºC, and the supernatant (nuclear extract) was collected in a cold eppendorf tube
and stored at –70ºC for later use. The protein content was measured by BCA protein quantitation
method.
Electrophoretic mobility shift assay (EMSA) for measuring NF-B activity:
EMSA was performed by incubating 10µg of nuclear protein extract with IRDye TM 700labeled NF-B oligonucleotide (LI-COR, Lincoln, NE). The incubation mixture included 2 µg of
poly (dI-dC) in a binding buffer. The DNA-protein complex formed was separated from free
oligonucleotide on 8.0% native polyacralyamide gel using buffer containing 50 mM Tris, 200
mM glycine, pH 8.5, and 1 mM EDTA and then visualized by Odyssey Infrared Imaging System
using Odyssey Software Release 1.1 (LI-COR, Lincoln, NE). For loading control, 10 g of
nuclear proteins from each sample were subjected to Western blot analysis for retinoblastoma
protein, which showed no alternation after transfection.
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Supplementary Figure legends:
Supplementary Figure-1: Over-expression of Notch-1 in PC-3 cell line promoted cell
growth, inhibited cell apoptosis, increased S-phase fraction, and increased cell invasion.
A, Left panel: the high expression of Notch-1 was confirmed by Western blotting analysis in PC3 ICN cells. Middle panel: MTT assay showing that PC-3 ICN promoted cell growth. Right
panel: ELISA showing that over-expression of Notch-1 inhibited cell apoptosis.
B, Cell survival of PC-3 and PC-3 ICN. PC-3 and PC-3 ICN cells were evaluated by the
clonogenic assay. Photomicrograph showing differences in colony formation of cells (Left
panel). There was a significant increase in the colony formation by PC-3 ICN cells compared
with PC-3 cells (Right panel). P values represent comparisons between PC-3 and PC-3 ICN
using the paired t test.
C, The PC-3 and PC-3 ICN cells were harvested for cell cycle analysis using propidium iodide
staining. X axis, DNA content; Y axis, the number of nuclei. The S phase fraction increased from
35.51% in PC-3 cells to 43.43% in PC-3 ICN cells.
D, Left panel: Invasion assay showing that PC-3 ICN treated with genistein resulted in high
penetration through the Matrigel-coated membrane, compared with control cells. Right panel:
value of fluorescence from the invaded cells. The value indicated the comparative number of
invaded cells.
Supplementary Figure-2: Over-expression of Notch-1 up-regulated pAkt, NF-B and its
target gene VEGF and MMP-9.
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A, Left panel: Western blot analysis showing that over-expression of Notch-1 increased the
expression of pAkt, MMP-9 and VEGF. Middle and right panel: Real-time RT-PCR showing
that over-expression of Notch-1 increased the MMP-9 and VEGF mRNA levels.
B, Over-expression of Notch-1 increased the activity of MMP-9 and VEGF. MMP-9 and VEGF
activity was assayed by ELISA showing that MMP-9 and VEGF levels in the culture medium
were increased in PC-3 ICN cells.
C, Left panel: NF-B DNA-binding activity was measured by EMSA in PC-3 and PC-3 ICN
cells. Right panel: NF-B DNA-binding activity was quantified.
Supplementary Figure-3: FoxM1 was decreased in Akt knock-out MEF cell lines. CS:
control siRNA; NS: Notch-1 siRNA;
A, MEF cell growth was measured by MTT assay. Knock-out of Akt inhibited MEF cell growth.
B, Left panel: FoxM1 protein was decreased in Akt knock-out cell lines using Western blot
analysis. Right panel: FoxM1 mRNA was decreased significantly in Akt 1KO and Akt DKO cell
lines using Real-time RT-PCR.
C, Left panel: Notch-1 siRNA decreased pAkt and FoxM1 in Akt WT cell line using Western
blot analysis. Right panel: PTEN KO MEF showed higher expression of pAkt and FoxM1 as
assessed by Western blotting analysis.
Supplementary Figure-4: Effect of taxotere on prostate cancer cell growth and apoptosis. P
values represent comparisons between cells treated with taxotere and untreated control using the
paired t test. *P < 0.05, **P < 0.01, compared to untreated control.
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A, Cells were seeded in 96-well plates at 5,000 cells per well and treated with varied
concentrations of taxotere for different time period. After treatment, cell densities were
determined by MTT assay. Each value represents the mean  SD (n = 6) of three independent
experiments.
B, Left panel: cell survival of human PCa cell lines PC-3 and LNCaP. Cells treated with varied
concentrations of taxotere for 72 hours were evaluated by the clonogenic assay.
Photomicrographic difference in colony formation of cells untreated and treated with taxotere.
Right panel: There was a significant reduction in the colony formation by PC-3 and LNCaP cells
treated with taxotere compared with untreated control cells.
C, Cell death assay for measuring apoptosis induced by taxotere. Cells were cultured in RPMI
containing 5% FBS and exposed to different doses of taxotere for 72 hours. Apoptosis was
measured by Histone DNA ELISA.
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