Download Lokeshwar et al: Supplemental Information Antibodies and

Lokeshwar et al: Supplemental Information
Antibodies and constructs used in this study: Antibodies were obtained from the following
companies: Cell signaling (Boston, MA): Akt, phospho (ser473)-Akt, phospho-Bad (ser-136),
Bcl-XL, bid, capse-9, caveolin-1, c-erbB2(p-Tyr1221/1222), cleaved caspase 3, cleaved caspase
8, cleaved PARP, DR5; Santa Cruz Biotechnology (Santa Cruz, CA): CD44, DR4, Fas, FAS-L,
HAS2; Sigma-Aldrich (St Luis MO): FADD; Novocastra (Newcastle upon Tyne, UK): CD168
(RHAMM); Lab-Vision/Thermo Fisher (Fremont, CA): c-erbB-2; Epitomics (Burligame, CA):
MMP-2, MMP-9, IKB-D, EGFR, p-EGFR (tyrosine 1143). BD Pharmingen (San Jose, CA): bax
(6A7), bad, bcl-2.
pNF--luc (p-NF-KB-Luc cis reporter plasmid) was purchased from
Stratagene, La Jolla, CA. Coumerin and 4-hydroxy coumerin were obtained from Indofine
Chemical Co (Hillsborough, MJ).
A detailed description of motility and invasion assays: Matrigel• invasion assay was
carried out as described previously (16,18,19) except that 4-MU was added in both chambers of
the Transwell; for details, please see supplemental information. The cells in the top chamber
were resuspended in RPMI 1640 + ITS (insulin, transfeffrin and selenium, Sigma Aldrich, St
Luis, MO) medium and the bottom chamber contained growth medium as the chemoattractant.
For motility assay, 8-Pm pore Transwell with similar experimental set up was used and the
migration of the cells was assayed after 18 h incubation. In both assays, HA (50 Pg/ml) was
added to the top and the bottom chambers of some wells. The cells in the top and bottom
chambers were determined using MTT assay (16,18,19); the cells adhered to the lower surface
of the filter were included in the bottom chamber fraction. To neutralize the effect of 4-MU on
cell growth, percent invasion or motility were calculated as (O.D. bottom chamber ÷ O.D. (top +
bottom chambers)) x100.
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Transient transfection assays with myr-Akt plasmid: PC3-ML cells were transiently
transfected with pcDNA3-Myr-HA-Akt1 plasmid (myr-Akt; Addgene, Inc, Cambridge, MA) using
Lipofectamin 2000 (Invitrogen, Carlsbad, CA). 24-h following transfection, cells were treated
with 4-MU (0.4 mM). Cell growth, apoptosis, and gene expression (Q-PCR) were evaluated after
48 h. Alternatively, PC3-ML cells were transiently co-transfected with myr-Akt and pNF--luc
and pGL4.74[hRluc/TK] plasmids and, 24 hours later, treated with 4-MU,. The firefly luciferase
and Renilla luciferase activities were assayed 24 hours following 4-MU treatment.
Determination of MVD and the TUNEL assay: Staining of microvessels was performed as
described previously (18,19). Microvessel density (MVD) was determined by 2 readers
independently counting microvessels in 10 fields and expressed as mean ± sd using a Nikon
H550L microscope with a video screen camera (18,19). Terminal deoxynucleotidyl transferase
dUTP nick-end labeling (TUNEL) assay was performed to determine the extent of apoptosis in
tumor tissues, using the In situ Cell Death detection kit (Roche Diagnostics; Indianapolis, IN).
The apoptosis index was calculated by counting the number of TUNEL positive cells in three
high power fields for each specimen in the vehicle and in the 4-MU-treated groups and
expressed as mean ± sd.
Tumor extracts and immunoblotting: Tumor tissues (~ 30 mg) were solubilized in 50 mM
Tris.HCl, pH 7.4, 150 mM NaCl, 1% NP40, 0.1% SDS, 50 mM sodium fluoride, 0.1 mM sodium
vanadate, protease inhibitor cocktail (10 Pg/ml; Sigma-Aldrich, St. Louis, MO). The extracts
were clarified by centrifugation at 15,000 rpm for 15 min and analyzed (~ 20 Pg protein) using
specific antibodies.
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Table 1: Primers for Q-PCR: Sequences for the forward and reverse complementary PCR
primers used in the Q-PCR analyses of the specific genes are indicated along with the GenBank
accession number for the gene.
Gene
CD44s
CD44v
Accession #
NM_001001391.1
NM_001001390.1
MMP-2
MMP-9
E-actin
NM_004530
NM004994
NM_001101
RHAMM
Caveolin1
IL-8
NM_012484.1
NM_000584.2
CXCR1
NM_000634.2
CXCR2
NR_002712.1
CXCR4
NM_003467.2
SDF1-v1
SDF1-v2
HAS1
NM_199168.2
NM_000609.4
NM_001523
HAS2
HAS3
U54804
NM_005329
Forward Primer
5’CTGTACACCCCATCCCAGAC3’
5’CAGGTGGAAGAAGAGACCCAA
3’
5’CCTGATGTCCAGCGAGTG3’
5’CTGCCAGGACCGCTTCTACT3’
5’CAACTGGGACGACATGGA3’
5’CAGCTGGAAGATGAAGAAGGA
3’
5’ACCCACTCTTTGAAGCTGTTG3’
Reverse Primer
5’TGTGTCTTGGTCTCTGGTAGC3’
5’ATGACTTCCAAGCTGGCCGTG
GCT3’
5’-AGCCAGATCACCTTCCACA
CACAA-3’
5’-AGCAGGAAGATGAGGACA
ACAGCA-3’
5’TCATCAAGCAAGGGTGTGAG3’
5’TCTCAGCCCTCTTCAAAAACTTCT
3’
5’GCAAGGAGTTCTTGGCACG
TCATT3’
5’ACAATACAGCAAACTGGCGGATG
C3’
5’GCTGAGGTCACTGGGATGAA3’
5’GGGAAGCCAGGATCCATTTT3’
5’CTCAGGGCACTGCAGGATGT3’
5’GTTGGCCTTGGGGTTCAG3’
5’GCATGTAGTTGTAGCTGAAAAGG
3’
5’GAACTTGAAATTGGCACCAGG3’
5-ggctccaaggaaagcataga-3
5’CACAGAAGGTCCTGGTGG TA3’
5’CATTGAAAAGCTGCAATCACA3’
5’GGTGGGGACGTGCGGAT
C3’
5’TGAACAAAACAGTTGCCCTTT3’
5’CTCTACTCCCTCCTCTATATGT
C3’
5’ATGCAGGATACACAGTGGAA
GTAG3’
5’TTCCCATCTATGACCATGACAA3’
5’AACTGCCACCCAGATGG
A3’
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Table 2 (Supplement): Summary of the effect of 4-MU on gene expression in the absence
or presence of HA. The data in columns 1 (4-MU % untreated) and column 2 (4-MU + HA: %
untreated) were calculated from the normalized mRNA expression levels presented in Figure 4,
for 4-MU (0.4 mM) and 4-MU + HA (50-Pg/ml) treated samples. The results are expressed at
percent fold change in 4-MU or 4-MU + HA samples when compared to untreated control (=
100%) or HA-treated control (= 100%), respectively. *: CD44s is the dominant isoforms
expressed in PC3-ML cells, and therefore, data in the table are presented for CD44s.
Gene
4-MU (% untreated)
Caveolin -1
HAS2
RHAMM
CD44s*
IL-8
MMP-2
MMP-9
CXCR1
CXCR2
CXCR4
CXCR7
12.9 ± 2
33.8 ± 4.1
36.8 ± 5.7
34.1 ± 6.3
10.4 ± 1.2
33.3 ± 3.7
29.6 ± 8.6
52.9 ± 5.9
69.6 ± 10.3
31.3 ± 7.3
51.2 ± 10
4
4-MU+HA (% HA-treated
control)
23.8 ± 3.5
64.9 r 5.4
100.5 ± 3.7
78.4 ± 7.4
48.1 ± 2.4
71.4 ± 3.7
79.6 ± 8.3
105 ± 5.9
98.4 ± 6.3
102.7 ± 5.9
216 ± 20.2
Table 3: Analysis of serum chemistry in vehicle and 4-MU treated animals: Blood from
vehicle and 4-MU treated (450 mg/kg) was analyzed for clotting time, blood urea nitrogen,
creatinine, serum glutamic pyruvic transaminase (SGPT) and alkaline phosphatase. Data are
mean r sd.
Parameter
Vehicle
4-MU treated
clotting time (sec)
173.2 ± 9.3
171.8 ± 7.3
Blood urea nitrogen (mg/dl)
27.8 ± 2.7
28.8 ± 2.8
Creatinine (mg/dl)
0.28
0.3
SGPT (U/L)
59.2 ± 4.9
56.5 ± 9.4
Alk Phosphatase (U/L)
79.0 ± 15.1
74.7 ± 19.6
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Figure S1: Effect of HA receptor siRNA treatment on PC3-ML cell growth and apoptosis:
PC3-ML cells were transfected with CD44, RHAMM or CD44 and RHAMM siRNAs. Cell
proliferation (A) and apoptosis (B) were examined after 72 h, by cell counting and Cell Death
ELISA kit, respectively.
Figure S2: Effect of myr-Akt expression on 4-MU induced effects in PC3-ML cells. PC3-ML
cells were transfected with either vector or myr-Akt plasmid. Twenty-four hour following
transfection, cells were treated with myr-Akt. A: total Akt and phospho-Akt expression in PC3ML transfectants after 24 h of 4-MU treatment (0.4 mM). B: Cell proliferation: PC3-ML
transfectants were treated with 4-MU (0.4 mM) for 48 h and the cells were counted. C:
Apoptosis: PC3-ML transfectants were treated with 4-MU (0.4 mM) for 48 h and apoptosis was
determined using the Cell Death ELISA kit. D: NFkB promoter activity: PC3-ML cells were cotransfected with vector/myr-Akt and NFkB plasmids. Twenty-four hours following transfection,
the cells were exposed to 4-MU and the firefly luciferase and Renilla luciferase activities were
assayed after 24 h. E: PC3-ML transfectants were treated with 4-MU (0.4 mM) for 48 h and the
expression of HAS2, IL-8, CD44, RHAMM and MMP-9 mRNAs was examined by Q-PCR.
Figure S3: Effect of 4-MU treated on body weight and the weights of specific organs. A:
Animals in the vehicle and 4-MU treated (450 mg/kg) groups from the experiment described in
Figure 6 A were weighed twice weekly until the end of the study. Data: Mean r sd. B: At
necropsy (day 35) prostate, testes and seminal vesicles were weighed from animals in the
vehicle and 4-MU treated (450 mg/kg) groups from the experiment described in Figure 6 A.
Figure S4: Effect of TXTR and 4-MU combination on PC3-ML cell growth. PC3-Ml cells
(12,000 cells/well) were exposed to various concentrations of TXTR(0- 5000 pM (0-5 nM)) in the
presence or absence of 4-MU (0-0.6 mM) for 48 hours. Following incubation, MTT (3-(4,56
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well at 250-Pg/ml
concentration. Following a two hour incubation, the insoluble purple formazan product was
dissolved in dimethylsulfoxide and the optical density was measured at 515 nm. The data are an
average of duplicate wells.
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