kim et al- supplemental material onc-2009-00693r · the filters were coated with 10 µg/ml of ......
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Supplementary Information
Supplementary Methods
Semiquantitative RT‐PCR
Total cellular RNA was extracted from cells using Easy BlueTM (Intron Company), dissolved in
diethylpyrocarbonate‐treated water, and quantified by UV scanning. RNA (5 μg) was reverse‐
transcribed, and BLT1, BLT2, NOX1, and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH)
were amplified using an RT‐PCR PreMix Kit (Intron), as previously described (Choi et al., 2008).
Luciferase reporter gene assays for NF‐κB and MMP‐9 promoter activities
Cells were transiently co‐transfected with pcDNA3, pcDNA3‐H‐RasV12, pcDNA3‐BLT1, or pcDNA3‐
BLT2 and the reporter plasmid containing the NF‐κB, AP‐1, or MMP‐9 promoter fused to a
luciferase coding sequence or stable cells (Rat‐2, Rat2‐HO6, or Rat2‐BLT2) were transiently
transfected with the pNF‐κB, AP‐1, or MMP‐9‐Luc reporter plasmid, cultured for 24 h and then
serum‐starved for 12 h. The cells were then harvested and the luciferase activity measured as
described previously (Woo et al., 2000).
Invasion assays
To monitor the invasion potential of Rat2‐HO6, Rat2‐BLT2, or NIH3T3‐HRasV12 cells, BD BioCoatTM
MatrigelTM Invasion Chambers (BD Biosciences, San Jose, CA) were used according to the
manufacturer’s instructions. Cells (5 x 104) were removed from culture plates with trypsin‐EDTA,
washed in DMEM/0.5% FBS, and seeded on top of the rehydrated Matrigel inserts. DMEM
containing 0.5% FBS and 300 nM of LTB4 was added to the lower chamber. Cells were allowed to
migrate for 24 h. The filters were fixed in methanol and stained with hematoxylin and eosin (H&E).
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Invasive ells were counted microscopically in 10 random high‐power fields per filter; each sample
was assayed in duplicate, and 5 independent assays were performed.
Chemotactic transmigration assay
Transmigration assays were performed using Transwell chambers (Corning Costar, Inc.) with 6.5‐
mm diameter polycarbonate filters (8‐μm pore size). Briefly, confluent Rat2, Rat2‐HO6, NIH3T3, or
NIH3T3‐HRasV12 cells were incubated for 12 h in DMEM containing 0.5% FBS. The lower surfaces of
the filters were coated with 10 µg/ml of collagen type I (Sigma‐Aldrich, St. Louis, MO) for 1 h at
37 °C. Cells were trypsinized and suspended in DMEM containing 0.5% FBS before being loaded
into the upper chamber at a final concentration of 5 x 104 cells/well. The cells were then allowed
to migrate to the lower side of the chamber, which contained LTB4. In some assays, cell
suspensions were exposed to inhibitors or antagonists for 30 min before seeding. After incubation
at 37 °C for 6 h, the filters were disassembled, and the upper surface of each filter was scraped
free of cells by wiping with a cotton swab. Cells that had migrated to the underside of the filters
were fixed for 1 min with methanol and stained for 20 min with H&E. Cell migration was
quantified by counting the number of cells on the lower side of the filter in 10 random high‐power
fields per filter. Each sample was assayed in triplicate, and five independent assays were
conducted. Migration data were analyzed by Student’s t‐test using SPSS 12.0 software to examine
differences among cell lines and groups.
Western blotting for MMP‐9 levels
Levels of MMP‐9 in culture supernatants were measured by western blot assay. Briefly, cultured
cells (4 x 105 cells/60‐mm plate) were incubated for 24 h in DMEM containing 0.5% FBS. The
culture supernatant was collected, solubilized in Laemmli buffer with reducing agent, and heated
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at 95 °C for 5 min prior to subjecting samples to 8% SDS‐PAGE. Samples were transferred to
polyvinylidine difluoride membranes and incubated overnight at 4°C in Tris‐buffered saline
containing nonfat dried milk (5%, w/v) and Tween 20 (0.01%, v/v). Subsequently, the membranes
were incubated with the appropriate antibodies, followed by HRP‐conjugated secondary antibody,
and developed by ECL, according to the manufacturer's instructions (Amersham Biosciences). For
inhibitor experiments, cells were treated with inhibitors or antagonists in DMEM medium
containing 0.5% FBS for 24 h. Anti‐MMP‐9 (Sigma‐Aldrich) was used at a dilution of 1:1000.
Tubulin (Sigma‐Aldrich) was used as a loading control.
FACs analysis for BLT2 expression
The cell surface expression of BLT2 in Rat2, Rat2‐HO6, Rat2‐BLT2, NIH3T3, and NIH3T3‐HRasV12
cells was evaluated by flow cytometry using a FACStarPLUS (BD Biosciences) analyzer and rabbit
plyclonal Ab anti‐BLT2 (Life Span Biosciences, Inc.) (1:200 dilution for 2 h), followed by a
fluorescein isothiocyanate (FITC)‐conjugated anti‐rabbit IgG (1:200 dilution, Sigma‐Aldrich). To
evaluate the expression of cell surface BLT2, cells were fixed with 3% paraformaldehyde without
permeablization before incubation with rabbit polyclonal Ab. Rabbit immunoglobulin (IgG, Sigma‐
Aldrich) was used as an isotype control. Data are expressed as the mean fluorescence intensity.
Electrophoretic mobility shift assay (EMSA)
Nuclear protein extracts for electrophoretic mobility shift assays (EMSA) were obtained by lysing
cells (5 x 106) with 200 μl of ice‐cold buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2,
0.1 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT). Samples were centrifuged at 800 x g for 10 min at
4 °C and the supernatants were collected as the cytoplasmic extract. The pellets were
resuspended in 50 μl of Buffer B (20 mM HEPES (pH 7.9), 400 mM NaCl, 1.5 mM MgCl2, 0.1 mM
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EDTA, 5% glycerol, 0.5 mM PMSF, and 0.5 mM DTT) to extract nuclear proteins. After incubation
for 20 min on ice, the samples were centrifuged at 10,000 x g for 30 min at 4 °C, and the
supernatants were collected as nuclear extracts and stored at ‐80 °C. The amount of protein in the
nuclear extracts was quantified using the Bradford method (BioRad, Hercules, CA). The sequence
of the sense strand of the double‐stranded oligonucleotide probes used in the EMSAs was 5'‐AGT
TGA GGG GAC TTT CCC AGG C‐3' for NF‐κB. Oligonucleotides were end‐labeled with [γ‐32P] ATP
using T4 polynucleotide kinase (MBI Fermentas Inc.), annealed, and purified on a Sephadex G‐50
column (Amersham Biosciences, Corp.). Nuclear extracts (10 μg) were incubated with 0.5‐1 ng of
labeled probe (10,000‐20,000 cpm) for 30 min at room temperature in binding buffer (MBI
Fermentas) with 5 mM DTT. Unlabeled oligonucleotides, which were used as competitors, were
added at a 100‐fold molar excess 10 min before addition of the labeled probe. The samples were
separated in a 5% polyacrylamide Tris‐acetate‐EDTA gel, which was dried and then exposed to X‐
ray film at ‐80 °C.
Experimental metastasis assays and morphological and histological analyses
Rat‐2, Rat2‐HO6, and Rat2‐BLT2 cells (5 x 105 cells) from cultures in the logarithmic growth phase
at the time of harvest were prepared for injection. In certain cases, siBLT2 or siMMP‐9‐transfected
cells were prepared for injection. For delivery of siRNA (siBLT2 or siMMP‐9) into cells,
polyethlyleneimine (PEI; Sigma‐Aldrich), a cationic polymer, was used as the delivery vehicle to
prevent degradation and enhance cell membrane penetration of siRNAs, as described previously
(Tan et al., 2005; Zhang et al., 2007). The cells were briefly treated with 0.025% trypsin and 0.1%
EDTA in Hanks’ balanced salt solution and quickly removed from the culture plates by
centrifugation, resuspended in saline containing 300 nM of LTB4 (to ensure BLT2 activation), and
injected within 1 h in 0.1 ml into the lateral tail vein with a 30‐gauge needle. For inhibitor
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experiments, 0.25 mg/kg of U75302, 2.5 mg/kg of LY255283, or 12.5 mg/kg of SB‐3CT was
injected intraperitoneally 3 and 5 days after injection of cells. For siRNA experiments, control (1 μl
of PEI + siRNA control) or dsRNA (1 μl of PEI + 5 μg of siRNA) was injected into the lateral tail vein
5 and 10 days after injection of cells transfected with siBLT2 or siMMP‐9. The mice were
maintained under aseptic barrier conditions until sacrifice at 21 days after cell injection (n = 8 for
each group) to identify pulmonary metastases. After 21 days, the mice were euthanized, and the
number of lung surface metastasis nodules larger than 0.2 mm in diameter and the metastatic
tumor burden evaluated by measuring whole lung weight were determined. To investigate the
mortality of injected mice, animals were monitored for 7 weeks (n = 8 for each group). The lungs
were dissected and fixed in 4% formalin, processed, and embedded in paraffin. Sections (4 μm)
were stained with H&E, examined, and photographed using a BX51 microscope (Olympus, Tokyo,
Japan) equipped with a DP71 digital camera (Olympus). Survival was analyzed by Kaplan‐Meier
plots.
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Supplementary References
Tan PH, Yang LC, Shih HC, Lan KC, Cheng JT (2005). Gene knockdown with intrathecal siRNA of
NMDA receptor NR2B subunit reduces formalin‐induced nociception in the rat. Gene Ther 12:
59‐66.
Woo CH, Lee ZW, Kim BC, Ha KS, Kim JH (2000). Involvement of cytosolic phospholipase A2, and
the subsequent release of arachidonic acid, in signalling by rac for the generation of
intracellular reactive oxygen species in rat‐2 fibroblasts. Biochem J 348 Pt 3: 525‐30.
Zhang S, Zhao B, Jiang H, Wang B, Ma B (2007). Cationic lipids and polymers mediated vectors for
delivery of siRNA. J Control Release 123: 1‐10.
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Supplementary Figure Legends
Supplementary Figure 1. Up‐regulation of BLT2 by H‐Ras. (a‐c) The elevated BLT2 expression
induced by H‐Ras was analyzed by FACS analysis (a‐b) and semiqauntitative RT‐PCR (c). Rat‐2,
Rat2‐HO6, Rat2‐BLT2, NIH3T3, and NIH3T3‐HRasV12 cells were examined for BLT2 antibodies, as
described under Supplementary information (a‐b). # p < 0.05, * p < 0.01. (c) Upper panel;
Expression of BLT2 in Rat‐2 and Rat2‐HO6 cells or NIH3T3 and NIH3T3‐H‐RasV12 cells (4 x 105
cells/60‐mm plate) was examined by semiquantitative RT‐PCR using BLT2‐specific primers, as
reported previously (Choi et al., 2008). Lower panel; Rat‐2 cells and NIH3T3 cells were transiently
transfected with 0, 0.5, 1, or 2 μg of H‐RasV12 expression plasmid, after which the transfectants
were serum‐starved for 12 h in DMEM containing 0.5% FBS and then analyzed for transcript
levels by semiquantitative RT‐PCR using BLT2‐specific primers. The samples were also assayed by
western blot analysis. Antibodies specific for phospho‐ERK, total ERK (Cell Signaling Technology),
or H‐Ras (Novus Biologicals) were used (1:1000 dilution). GAPDH levels are shown as a control.
The results shown are representative of three independent experiments with similar results.
Supplementary Figure 2. ROS generation occurs via BLT2‐NOX1 signaling in Rat2‐HO6 cells. (a)
Rat2, Rat2‐HO6, or Rat2‐BLT2 cells were pre‐treated with DMSO, 5 or 10 μM of LY255283, 0.5 or 1
μM of U75302, and a combination of LY255283 (10 μM) and U75302 (1 μM) and then analyzed for
the level of ROS. (b) ROS levels are regulated by NOX1 in Rat2‐HO6 or Rat2‐BLT2 cells. pSIREN
(control vector) and pSIREN‐siNox1 were transiently transfected into Rat‐2, Rat2‐HO6, or Rat2‐
BLT2 cells using LipofectAMINE‐plus. After 36 h, the cells were assayed for ROS levels, as described
previously (Choi et al., 2008). All data are expressed as the average fold increase relative to
controls ± s.d. * p < 0.001.
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Supplementary Figure 3. The BLT2 signal axis is independent of AP‐1 transcriptional activity. (a‐b)
Rat‐2 cells were transiently co‐transfected with pcDNA3, pcDNA3‐H‐RasV12, and the pAP‐1‐Luc
reporter plasmid (a) or pcDNA3, pcDNA3‐H‐RasV12, pcDNA3‐BLT1, pcDNA3‐BLT2, and the pAP‐1‐
Luc reporter plasmid (b), cultured for 24 h, and then serum‐starved for 12 h. For inhibition tests,
cells were treated with 10 μM LY255283, 1 μM U75302, 20 μM NS398, and 100 μM PDTC for 6 h
before cell harvest, followed by determination of luciferase activity. Data are expressed as the
average fold increase over controls ± s.d. in three independent experiments.
Supplementary Figure 4. Modulation of MMP‐9 via the BLT2 axis in Ras‐activated cells. Rat‐2
cells were transiently co‐transfected with pcDNA3, pcDNA3‐H‐RasV12, and the pMMP‐9‐Luci
reporter plasmid (a) or Rat‐2, Rat2‐HO6, and Rat2‐BLT2 cells were transfected with the pMMP‐9‐
Luc reporter plasmid (b), cultured for 24 h, and then serum‐starved for 12 h. For inhibition tests,
cells were treated with buffer, 100 μM PDTC, 100 μM SN‐50M, or 100 μM SN‐50 for 6 h before
cell harvest to determine luciferase activities. * p < 0.01, ** p < 0.001.
Supplementary Figure 5. In vivo metastatic potential evoked by H‐RasV12 is suppressed by BLT2
inhibition with LY255283. (a‐b) Mice were injected with Rat2, Rat2‐HO6, or Rat2‐BLT2 cells, and
then administered DMSO (control), 2.5 mg/kg LY255283, or 0.25 mg/kg U75302 twice (3 and 5
days after the cell injections) intraperitoneally. Mice were sacrificed 21 days after cell injections
and the lungs were removed and photographed, and then lung tissues were stained with H&E.
The arrowheads indicate metastatic nodules. (c) In vivo metastatic potential evoked by H‐RasV12
in NIH3T3 cells. Mice were injected with NIH3T3‐HRasV12 cells and then administered DMSO
(control) or 2.5 mg/kg LY255283 twice (3 and 5 days after the cell injections) intraperitoneally.
Mice were sacrificed 21 days after cell injections and the lungs were removed and photographed.
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The number of lung surface metastatic nodules and the metastatic tumor burden were then
determined. n = 7 mice. *** p < 0.0001.
Supplementary Figure 6. Up‐regulated BLT2 expression in invasive or metastatic human cancer
tissues. BLT1 and BLT2 expression was detected in 6 aggressive human tumor tissues (brain, skin,
lung, breast, bladder, and thyroid gland) by immunohistochemical staining, as described under
Materials and Methods. Expression of BLT2, but not BLT1, was strongly detected as a dense
purple color in the cytoplasmic area. The results shown are representative of three independent
experiments. Bar, 100 μm.
a
100 101 102 103 104
ControlNIH3T3NIH3T3-HRas V12
0
30
60
90
1
20
150
Cou
nts
FITC BLT2
100 101 102 103 104
ControlRat2Rat2-HO6Rat2-BLT2
0
20
40
6
0
80 1
20 1
40C
ount
s
FITC BLT2
H-Ras
pERK
ERK
BLT2
GAPDH
- 0.5 1 2 DNA(μg)H-RasV12
BLT2
GAPDH
Rat-2
Rat2
-HO6
NIH3
T3-H
-Ras
V12
BLT2
GAPDH
NIH-
3T3
- 0.5 1 2 DNA(μg) H-RasV12
H-Ras
pERK
ERK
BLT2
GAPDH
b
Kim et al, Supplementary Figure 1
c
Rat
-2
Rat
2-H
O6
Rat
2-B
LT2
Rel
ativ
e flu
ores
cenc
e in
tens
ity **
NIH
3T3
NIH
3T3-
HR
asV1
2
#
Rel
ativ
e flu
ores
cenc
e in
tens
ity
Rat2-HO6
*
Rat
2-B
LT2
Rat
-2
Rel
ativ
e In
tens
ity (D
CF)
DM
SO
+ LY
2552
83_U
7530
2
+ LY
2552
83
+ U
7530
2
a b
Rat-2
*
Rel
ativ
e In
tens
ity (D
CF)
- + - - + - pSIREN
- - + - - + siNOX1
Rat2-HO6
*
Rel
ativ
e In
tens
ity (D
CF)
Rat-2
- + - - + - pSIREN
- - + - - + siNOX1
Rat2-BLT2
**
Kim et al, Supplementary Figure 2
a
Rel
ativ
e A
P-1
Luc
Act
ivity
pcD
NA3
+ H
Ras
V12
+ B
LT1
+ B
LT2
b
Rel
ativ
e A
P-1
Luc
Act
ivity
LY25
5283
+ H-RasV12D
MSO
NS3
98
PDTC
+ pcDNA3
U75
302
LY25
5283
DM
SO
NS3
98
PDTC
U75
302
Kim et al, Supplementary Figure 3
a
Rel
ativ
e M
MP-
9 Lu
c A
ctiv
ity
Rat
-2
Rat
2-H
O6
Rat
2-B
LT2
*
b
Rel
ativ
e M
MP-
9 Lu
c A
ctiv
ity
** **
-
Buf
fer
SN-5
0M
SN-5
0
PDTC
-
Buf
fer
SN-5
0M
SN-5
0
PDTC
+ pcDNA3 + H-RasV12
**
Kim et al, Supplementary Figure 4
c
NIH3T3-HRasV12 cells injected
+ DMSO + LY255283
Num
ber o
f Lun
g N
odul
es
DM
SO
LY25
5283
***
Lung
wei
ght (
tum
or b
urde
n:g)
LY25
5283
DM
SO
Pare
nt g
roup
***
a
+ DMSO + LY255283 + U75302Rat2-BLT2 cells injected
+ U75302Rat2-HO6 cells injected
+ LY255283+ DMSO
b
Rat-2 cell injected+ DMSO + LY255283 + U75302
200 μm
Kim et al, Supplementary Figure 5
BrainNon-neoplastic tissue Glioblastoma
BLT
1
100 μm
BLT
2
SkinNon-neoplastic tissue Invasive squamous cell carcinoma
Papillary carcinoma
LungNon-neoplastic tissue Squamous cell carcinoma
BLT
1B
LT2
BreastNon-neoplastic tissue Infiltrating ductal carcinoma
BladderNon-neoplastic tissue Invasive urothelial carcinoma
BLT
1B
LT2
Thyroid glandNon-neoplastic tissue Metastatic papillary carcinoma
Kim et al, Supplementary Figure 6