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Epigenetic silencing of TIMP-2 in prostate cancer
Pulukuri et al.
SUPPLEMENTARY INFORMATION
“Epigenetic Inactivation of the Tissue Inhibitor of Metalloproteinase-2
(TIMP-2) Gene in Human Prostate Tumors”
Sai MuraliKrishna Pulukuri, Sruthi Patibandla, Jitendra Patel, Norman Estes, and Jasti
S. Rao
Evaluation of TIMP-2 immunostaining. TIMP-2 immunostaining positivity was
determined by pathologist according to the percentage of positive cells and staining
intensity. An average value of two independent scores was presented in the present study.
The percentage of TIMP-2 positive cells was divided into five grades (percentage scores)
: <10% (0), 10-25% (1), 25-50% (2), 50-75% (3), and >75% (4). The intensity of staining
was divided into four grades (intensity scores) : no staining (0), light brown (1), brown
(2), and dark brown (3). TIMP-2 staining positivity was determined by the formula:
overall scores = percentage score × intensity score. The overall score of ≤ 3 was defined
as negative, of > 3 - ≤ 6 as weak positive and of > 6 as strong positive.
Immunoblot analysis. Cells were lysed in RIPA buffer and proteins quantified using a
BCA assay (Pierce, Rockford, IL). Equal amounts were separated on SDS-PAGE gels.
Membranes were probed with antibodies against: TIMP-1 (Oncogene, San Diego, CA),
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TIMP-2 (Oncogene, San Diego, CA), TIMP-4 (Biomeda, Foster City, CA) and GAPDH
(Abcam, Cambridge, MA). Antibodies against MMP-9 and MMP-2 were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA).
Chromatin immunoprecipitation assay. Chromoatin immunoprecipitation analysis was
used to determine the binding activity of MeCP2 in the TIMP-2 promoter in prostate
cancer cell lines. Chromoatin immunoprecipitation assays were performed as per the
manufacturer's instructions (17–295, Upstate Biotechnology, Lake Placid, NY). Briefly,
prostate cancer cells (~1×106 cells/100 mm dish) were fixed by adding formaldehyde at a
final concentration of 1% and incubating for 10 min at 37°C. The cells were washed
twice with ice-cold PBS containing protease inhibitors (1 mM phenylmethylsulfonyl
fluoride, 1 µg/mL aprotinin and 1 µg/mL pepstatin A), harvested, and treated with SDS
lysis buffer for 10 min on ice. The resulting lysates were sonicated to shear the DNA to
fragment lengths below 1000 bp (amplitude 60%, 4x10 s, Fisher Sonic Dismembrator 60,
Pittsburgh, PA). From each sonicated sample, 5% was used as the input control for
immunoprecipitated fragments. The complexes were immunoprecipitated with antibodies
specific for MeCP2 (no. 07–013) from Upstate Biotechnology. In total, 10 µL of
antibody were used for each immunoprecipitation according to the manufacturer's
instructions. Antibody controls were also included for each ChIP assay; no precipitation
was observed. The antibody/protein complexes were collected using salmon sperm
DNA/protein A agarose slurry and washed several times as per the manufacturer's
instructions. The immune complexes were eluted with 1% SDS and 0.1 M NaHCO3, and
the crosslinks were reversed by incubation at 65°C for 4 h in the presence of 200 nM
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NaCl. The samples were treated with proteinase K for 1 h, and the DNA was purified by
phenol/chloroform extraction, ethanol precipitation, and resuspended in 30 µL of H2O.
Initially, PCR was performed with different numbers of cycles or dilutions of input DNA
to determine the linear range of the amplification; all results shown fall within this range.
Following 30 cycles of amplification, PCR products were run on 2% agarose gels and
analysed by ethidium bromide staining.
Reverse transcription-PCR analysis. Total RNA was isolated from prostate cell lines
and tissue samples using RNeasy mini kit (Qiagen, Valencia, CA) and reversetranscribed using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA)
according to the manufacturer’s instructions. The primers used for PCR were as follows:
TIMP-2 sense 5'- CCG
AAT TCT GCA GCT GCT CCC CGG TGC ACC CG-3'
and antisense 5'-GGA AGC TTT TAT GGG TCC TCG ATG TCG AG-3'; GAPDH sense
5’-CGG AGT CAA CGG ATT TGG TCG TAT-3’ and antisense 5’AGC CTT CTC CAT
GGT GGT GAA GAC-3’. Real-time PCR was conducted in 50 L volumes containing
1 L cDNA, 25 L SYBR Green, 2.5 L of 5 mol/l of each of the specific primers and
the probe, and 19 L RNA-free water. All of the reactions were performed in triplicate in
an iCycle iQ system (Bio-Rad, Hercules, CA). Optical system software version 3.1 was
used to detect the fluorescent level of TIMP-2 and GAPDH for 40 cycles. The PCR
conditions were as follows: 95°C for 5 min, followed by 40 cycles at 95°C for 1 min,
58°C for 1 min and 72°C for 1 min. The final extension was at 72°C for 5 min.
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Matrigel invasion assay. We used 6.5 mm-diameter Transwell inserts (Costar,
Cambridge, MA) with the 8 m-pore membranes coated with matrigel (Becton
Dickinson, Bedford, MA) to assess the invasive potentials of prostate cells before and
after treatment with 20 M 5-aza for 5 days and 50 nM TSA for an additional 24 h. In
some experiments, cells were treated in combination with 5-aza/TSA plus human TIMP-2
IgG. Cells were detached, washed twice in PBS and resuspended in serum-free DMEM.
A total of 5×105 cells in 0.2 mL were placed in the upper chamber of a Transwell and the
lower chamber was filled with 400 L of DMEM/10% fetal bovine serum. After a 24 h
incubation period, the cells in the upper chamber that did not migrate were gently scraped
away and adherent cells present on the lower surface of the insert were stained with
Hema-3 and photographed.
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