Lu et al
15-PGDH inhibits HCC via 15-keto-PGE2/PPARγ/p21
SUPPLEMENTARY MATERIALS AND METHODS
Materials.
The
plasmids
pCMV6-AC-GFP,
pCMV6-AV-GFP-15PGDH,
pGFP-V-RS, pGFP-V-RS-15PGDH, pGFP-V-RS-WAF1/Cip1/p21, pCMV5-PPARγ,
pGFP-V-RS-PPARγ and pCMV6-entry-PGR2 were purchased from Origene,
Rockville, MD.
The p21 promoter luciferase reporter construct was purchased from
Addgene.
The
pcDNA3-WAF1/Cip1/p21
was
subcloned
from
pCMV6-entry/Myc/DDK-WAF1/Cip1/p21 (Origene). The p53 shRNA vector
(pMKO.1 puro p53 shRNA 1) and pRL-Tk were purchased from Addgene (Plasmid
10671). The mouse monoclonal antibodies against 15-PGDH, p53, CDK2, CDK4,
Cyclin E, cyclin D1, p21, phosphorylated p21, PCNA, COX-2, BrdU, biotin, β-actin
were from Santa Cruz Biotech.
The rabbit polyclonal anti-PPARγ and anti-RB
antibodies were purchased from Cell Signaling Technology (Danvers, MA). The
antibody against histone was purchased from Abcam.
purchased from Evrogen.
The anti-TurboGFP was
The anti-rabbit IgG (Horseradish peroxidase linked
F(ab’)2 fragment from [donkey]) and the anti-mouse IgG (Horseradish peroxidase
linked whole antibody [from sheep]) were purchased from GE Healthcare Limited.
The IRDye 680LT/IRDye 800CW secondary antibodies were purchased from LI-COR
Biosciences.
The PGE2 and 15-keto-PGE2 metabolite biotrak enzyme immunoassay
(EIA) systems and 15-keto-PGE2 were purchased from Cayman Chemicals.
other reagents and compounds were analytical grades (from Sigma and Promega).
1
All
Lu et al
15-PGDH inhibits HCC via 15-keto-PGE2/PPARγ/p21
Isolation of nuclear extract.
Approximately 109 cells were homogenized in 3 ml of
cell lysis buffer (250 mM sucrose, 30 mM KCl, 6 mM MgCl2, 20 mM HEPES pH 7.9,
0.5 mM EDTA, 0.2 mM NaF, 2 mM Na3VO4 and 0.1% Triton X-100 with protease
inhibitors). Nuclei were liberated from the cells by 15 gentle strokes of a tight fitting
pestle in a dounce homogenizer.
The extract was centrifuged at 1500 g for 10
minutes at 4°C. The supernatant was used as the cytosolic fraction. The pellet was
resuspended in nuclear lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 0.1% NP-40
and 0.5% Triton X 100 with protease inhibitors) and the samples were rotated slowly
for at least 40 min at 4°C. The samples were then sonicated to lyse the nuclei and
thoroughly sheer the genomic DNA. The resulting extract was centrifuged at 13,000
g for 15 minutes at 4°C and the supernatant was used as the nuclear fraction.
Electrophoretic mobility shift assay (EMSA).
EMSA was performed using the gel
shift assay system obtained from Promega (Madison, WI).
Oligonucleotides
corresponding to the PPRE consensus site at the p21 promoter were synthesized,
which
include
biotin-labeled
5’-biotin-TGACCAGTGACC-3’)
probes
and
(5’-biotin-AGGTCACTGGTCA-3’;
non-biotin-labeled
(5’-AGGTCACTGGTCA-3’; 5’-TGACCAGTGACC-3’).
probes
Each binding reaction
was carried out with 100 fmol of biotinylated dsDNA probe and 10 µg of purified
nuclear protein (5µg/µl) in 20 μl of binding buffer containing 0.5mg/ml poly(dI:dC)
(Sigma) (25 mM HEPES at PH 8.0 with 50 mM KCl, 0.1%TritonX100, 2mM MgCl2,
3 mM DTT, and 5% glycerol). Twenty-five pmol of unlabeled cold DNA motifs
2
Lu et al
15-PGDH inhibits HCC via 15-keto-PGE2/PPARγ/p21
(250-fold excess) were added in the competition assays. Reactions were carried out
for 30 min incubation at room temperature, followed by overnight incubation at 4°C.
The reaction mixtures were loaded onto 6% TBE polyacrylamide gels and separated
in 0.5%×TBE at 100v on ice. Western blotting for biotin was then performed.
Western blotting.
The logarithmically growing cells were washed twice with
ice-cold phosphate-buffered saline (PBS, Hyclone Lab. INC) and lysed in a lysis
buffer (50 mmol/L Tris-HCl PH8.0, 150mmol/L NaCl, 1%Nonidet P-40, 5mmol/L
ethylenediaminetetraacetic acid [pH8.0], 1mmol/L phenylmethyl sulfonyl fluoride, 2
µg/mL aprotenine, and 2 µg/mL leupeptine, 2 µg/mL pepstatine; for phosphorylation
assay adding 50 mmol/l sodium fluoride, 25 mmol/l glycerophosphate, 1 mmol/L
Na3VO4). The cells lysates were centrifuged at 12,000 x g for 20 minutes at 4°C
after sonication on ice, and the supernatants were obtained. Protein concentration was
measured by using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc). After
boiling for 10 minutes in the presence of 2-mercaptoethanol, the samples were
subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and the separated proteins were transferred onto a nitrocellulose membranes
(Invitrogen, Carlsbad, CA, USA) using the Minipprotein Tetra system (Bio-Rad),
Mighty Transphor (Hoefer Inc.), and Trans-Blot Turbo Transfer system (Bio-Rad).
To visualize the transfer efficiency, membranes were stained with Ponceau S (Sigma),
destained with Milli-Q water, and then blocked in 10% dry milk-TBST (20mM
Tris-HCl [PH 7.6], 127mM NaCl, 0.1% Tween 20) for 1 h at 37°C.
3
Following three
Lu et al
15-PGDH inhibits HCC via 15-keto-PGE2/PPARγ/p21
washes in Tris-HCl pH 7.5 with 0.1% Tween 20, the blots were incubated overnight
with 0.2 µg/ml of antibody at appropriate dilution at 4°C.
Following three washes,
membranes were then incubated with the secondary antibody (horseradish
peroxidase-conjugated immunoglobulin G (GE Healthcare Limited) or IRDye
680LT/IRDye 800CW secondary antibodies (LI-COR Biosciences) at 37°C for 60
min or at 4 ° C overnight in TBST.
Signals were visualized by enhanced
chemiluminescence plus kit (GE Healthcare) or ODYSSEY infrared imaging system
(LI-COR).
Standard western immunoblotting procedures were used with the
following antibodies: the mouse monoclonal antibodies against 15-PGDH (1:500),
p53 (1:1000), CDK2 (1:500), CDK4 (1:1000), cyclin E (1:1000), cyclin D1 (1:1000),
p21 (1:500), phosphorylated p21 (1:500), PCNA (1:1000), COX-2 (1:500), BrdU
(1:1000), biotin (1:1000), β-actin (1:5000); the rabbit polyclonal anti-PPARγ (1:1000),
anti-RB (1:500) antibodies; the antibody against histone (1:1000); the anti-TurboGFP
(1:200). The second antibodies included the anti-rabbit IgG (horseradish peroxidase
linked F(ab’)2 fragment (from donkey) (1:5000), anti-mouse IgG (horseradish
peroxidase linked whole antibody (from sheep) (1:5000), or the IRDye 680LT/IRDye
800CW (1:10000).
Co-immunoprecipitation (IP).
The cells in 100-mm dishes were lysed in 1 ml of
the whole-cell extract buffer A containing 50 mM Tris-HCl pH7.6, 150 mM NaCl, 0.1
mM EDTA, 1% NP-40, 1.0 mM DTT and protease inhibitors. 500 l cell lysates were
used for immunoprecipitation with specific antibodies.
4
In brief, the cell lysate was
Lu et al
15-PGDH inhibits HCC via 15-keto-PGE2/PPARγ/p21
pre-cleared with 30 μl protein A/G-plus agarose beads (Santa Cruz) by rotation at 4°C
for 1 hour. The supernatant was obtained after centrifugation 5000 rpm at 4°C for 5
minutes. The precleared supernatants were incubated with 2 µg antibody by rotation
at 4°C for 4 hours, then incubated with 30 μl protein A/G-plus agarose beads by
rotation at 4°C overnight. The samples were collected by centrifugation at 5000 rpm
at 4°C for 5 min. The precipitates were washed five times with beads wash solution
(50 mM Tris-HCl pH7.6, 150 mM NaCl, 1 mM EDTA and 0.1% NP-40) and then
suspended in 40µl 2×SDS-PAGE sample loading buffer. After boiling for 5 min, the
samples were subjected to SDS-PAGE and Western blotting with specific antibodies.
Repeat IP (rIP) was conducted by cleansing the first precipitates with elution
buffer (0.1% Triton X-100, 0.1% SDS, 0.5% BSA in PBS). 40μl aliquot of
immunoprecipitates was eluted with 750μl elution buffer (50 min incubation at room
temperature). The samples were then subjected to an additional round of IP as
described above.
Chromatin immunoprecipitation.
Formaldehyde cross-linking and chromatin
immunoprecipitation assays are performed according to the protocol provided by
Upstate Biotechnology with modifications.
In brief, approximately 109 cells grown
on 100-mm dishes were cross-linked by formaldehyde which was directly added into
the culture media to a final concentration of 1% (0.68 ml 37%/25 ml media) and the
dishes were rocked for 10 min. The cross-linking was stopped by adding glycine to
a final concentration of 125 mM (3.75 ml of 1 M/25 ml media) with rocking dishes at
5
Lu et al
15-PGDH inhibits HCC via 15-keto-PGE2/PPARγ/p21
room temperature for 5 min. The cells were washed three times with ice-cold PBS
and scraped into 1 ml PBS containing protease inhibitors and collected by
centrifugation (700 x g for 4 min). Cell pellets were suspended in cell lysis buffer
containing 5 mM Pipes (KOH), pH 8.0, 85 mM KCl and 0.5% NP-40 with protease
inhibitors on ice for 10 min. The nuclei were collected by centrifugation (5000 rpm
for 5 min) and suspended in nuclear lysis buffer containing 50 mM Tris-HCl, pH 8.0,
10 mM EDTA, 0.1 % SDS with protease inhibitors. The solution was sonicated on
ice to achieve average chromatin length of about 500 bp. The debris was cleared by
centrifugation at 13,200 rpm for 10 min at 4oC.
Equal amount of the supernatant
was diluted 5-fold in ChIP dilution buffer containing 0.01% SDS, 1% Triton X-100,
1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0, 167 mM NaCl plus protease inhibitors.
The supernatants were pre-cleared with 80μl recombinant protein A agarose beads
(Santa Cruz) coated with BSA, salmon-sperm DNA, and tRNA at 4°C for 30 min
with agitation and collected by a brief centrifugation (5000 rpm for 5 min). 20% of
the collected supernatant was used as total input control and eluted in CHIP elution
buffer for the cross-linking reversal step. The remaining supernatant is divided into
three fractions as following: the first one for negative control without antibody, the
second one incubated with 5 μg antibody; the third one incubated with 5 μg normal
mouse or rabbit IgG for mock IP. After incubation at 4oC overnight with rotation,
the samples were added 60 μl recombinant protein A agarose beads coated with BSA,
salmon-sperm DNA, and tRNA and incubated at 4°C for 4 hrs with rotation. The
agarose beads were washed consecutively for 3-5 minutes on a rotating platform with
6
Lu et al
15-PGDH inhibits HCC via 15-keto-PGE2/PPARγ/p21
1 ml of each solution: (a) low salt wash buffer containing 0.1% SDS, 1% Triton
X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl; (b) high salt wash
buffer containing 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl
pH
8.0, 500 mM NaCl; and (c) LiCl wash buffer containing 0.25 M LiCl, 1% NP40, 1%
deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0; and (d) TE buffer pH8.0 twice.
After washing, immunoprecipitates were eluted in 250 µl CHIP elution buffer
containing 1% SDS in 0.1M NaHCO3 for 1 hr at RT twice.
Reverse formaldehyde
crosslink was performed by adding 1μl 10 mg/ml RNase and 5 M NaCl to a final
concentration of 0.3 M NaCl in the elutant.
After incubation at 65°C for 4-5 hours,
the elutant was added 2.5 volumes 100% ethanol for precipitation at –20°C overnight.
Pellet DNA and debris were dissolved in 100 μl H2O containing 2 μl 0.5 M EDTA, 4
μl 1 M Tris-HCl, pH 6.5 and 1 μl of 20 mg/ml Proteinase K and incubated at 45°C for
1-2 hours. DNA was purified using QiaQuick spin columns and eluted in 50 μl 10
mM Tris-HCl, pH 8.0. 2 μl DNA was used as PCR template.
The GoTaq DNA
polymerase PCR amplification kit (Promega) was utilized and the conditions were
3min at 94°C, followed by 35 cycles of 30s at 94°C, 30s at 55°C and 30s at 72°C,
followed by 10 min at 72°C.
PCR products (10 µl) were analyzed with 1.5%
agarose gel electrophoresis and visualized by ethidium bromide (Sigma) staining
using ImageMaster@VDS (Thermal imaging system FTI-500, pharmacia Biotech).
Real-time PCR was performed with Power SYBR Green PCR Master Mix (Applied
Biosystems) in accordance with the manufacturers’ protocols. Real time PCR
conditions were 10 min at 94°C, followed by 40 cycles of 30s at 94°C, 30s at 55°C
7
Lu et al
15-PGDH inhibits HCC via 15-keto-PGE2/PPARγ/p21
and 30s at 72°C.
Average cycle threshold (Ct) values were calculated from triplicate
reactions of three biological replicates. The WAF1/Cip1/p21 promoter PCR primer
sequences
are:
P1:
5’-GTGGCTCTGATTGGCTTTCTG-3’;
P2:
5’-CTGAAAACAGGCAGCCCAAG-3’. The same primer sets were used for both
the PCR assay and real-time PCR assay.
Immunohistochemistry.
Immunohistochemical stain for PCNA was performed in
the formalin-fixed and paraffin-embedded tissue specimens from the SCID mice
tumor xenograft.
The tissues were fixed with 4% paraformaldehyde, dehydrated,
embedded in paraffin and sectioned at 4μm.
The deparaffinized sections were
treated with 0.3% H2O2 in methanol for 30 minutes to abolish endogenous peroxidase
activity and then microwaved in antigen unmasking solution for antigen retrieval.
Sections were blocked with 10% goat serum in PBS for 1 hour at 37℃ and then
incubated with 4 µg/ml of anti-PCNA antibody (1:100, Santa Cruz Biotech) at 4℃
overnight.
Then the sections were incubated with anti-mouse IgG (Horseradish
peroxidase linked whole antibody (1:200, GE Healthcare Limited) at 37℃ for 1 hour.
Staining was performed using DAB substrate kit for peroxidase according to the
manufacturer's instructions (Vector Laboratories Inc) and counterstained with
hematoxylin (Vector Laboratories Inc).
As negative control, duplicated sections
were immunostained without exposure to the primary antibodies. The percentages
of positive cells were determined by counting the number of positively stained cells
versus total cells in randomly selected 200 × magnification field which included at
8
Lu et al
15-PGDH inhibits HCC via 15-keto-PGE2/PPARγ/p21
least 1000 cells.
Immunofluorescence localization.
Cells with 70% confluence grown on coverslips
were washed twice with cold PBS and fixed in 4% paraformaldehyde for 10 min.
Then the cells were incubated with 0.3% H2O2 in methanol for 30 minutes to abolish
endogenous peroxidase activity.
Following three washings with PBS, the cells were
incubated with PBS containing 0.1% Triton X-100 and 5% DMSO and blocked with
5% BSA in PBS at 37℃ for 1 hour. Subsequently, the cells were incubated with
mouse anti-15-PGDH (1:100,Santa Cruz) at 4℃ overnight.
After three washings
with PBS, the cells were incubated with anti-mouse TRITC-linked IgG (1:200, Santa
Cruz). The cells were then counterstained with DAPI (Invitrogen, diluted 1:1000 in
ddH2O) for 15 min. Coverslips were applied with mounting medium for fluorescent
and images were captured using Olympus FV1000II Laser Scanning Confocal
Microscope (Olympus) by FV10-ASW1.7 software or fluorescence microscope
(Olympus).
9