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The supplement materials of the MATERIALS AND METHODS
Cell culture. RWPE2-W99 cell lines (prostate epithelial cells lines) and DU-145 cell lines
(prostate carcinoma cell lines) were purchased from the American Type Culture
Collection (ATCC). RWPE2-W99 cells were cultured in Keratinocyte Serum Free
Medium (K-SFM) kit (Invitrogen Corporation, Carlsbad, CA, USA) supplemented with
10% fetal calf serum (Invitrogen Corporation, Carlsbad, CA, USA). DU-145 cells were
cultured under the same conditions.
PhoenixTM packaging cells (Orbigen, Inc., San
Diego, CA, USA) were a kind gift from Dr. Amy S. Yee and were cultured in Dulbecco’s
modified Eagle medium (DMEM) (Invitrogen Corporation, Carlsbad, CA, USA )
supplemented with 10% fetal calf serum.
Prostate samples The seventeen pairs of tissue samples (S1-S17) from seventeen patients
were obtained from a tissue bank at the Institute of Urology, Peking University, China.
Another five pairs of tissue samples (S18-S22) from five patients were obtained from the
Department of Urology，Sir Run Run Shaw Hospital. These tissue samples were
operative samples of radical prostatectomy and were diagnosed by pathology. All tumor
specimens was graded by Gleason’s grading system. The Gleason score of six tumor
specimens was 6（3+3）, and sixteen tumor specimens was 7（3+4 or 4+3），the details
of the specimens are presented in supplementary table 1. Every pair of samples included
one prostate carcinoma tissue sample and one normal prostate tissue sample away from
the tumors. All samples were conserved in liquid nitrogen.
Vectors for retroviral-mediated gene expression
The HBP1 vector was
constructed by cloning the human HBP1 fragment into pBabe (EcoRI). The pmHMG
vector, which contained three point mutations in the high-mobility group [HMG]
DNA binding domain of HBP1, was generated by overlapping PCR using the HBP1
vector as a template. Point mutations were introduced at positions 434, 435, and 437
in the pmHMG vector, which changed lysine-434 to glutamic acid (AAA to GAA),
arginine-435 to glutamic acid (AGA to GAA) and methionine-437 to threonine (ATG
to ACG). The 218-314 vector was designed for expression of a deleted form of
HBP1, which lacked residues 218-314. All of these expression vectors included the
HA tag.
Retroviral gene transduction was carried out using PhoenixTM packaging cells
(Orbigen, Inc.). DU-145 cells were infected with retrovirus, and were grown in
selective media containing 1 g/ml puromycin at one day post-infection. We
generated stable cell lines expressing pBabe, HBP1, or pmHMG after 14 days of
selection. The expression of these vectors in DU-145 cells was detected by
immunoblot analysis using an anti-HA monoclonal antibody(05-904, Millipore.USA)
or an anti-HBP1 polyclonal antibody (sc-25390，Santa Cruz Biotechnology Inc.
USA).
Chromatin immunoprecipitation (ChIP) assays. Immunoprecipitations were
performed overnight at 40℃ with anti-HBP1 antibodies or IgG alone (as a negative
control). 1-5 μl of the 50 μl of eluted DNA were used in 28 cycles of PCR
amplification. A PCR primer set specific for the HBP1 binding sequence within the
MIF promoter region corresponded to: sense 5’-TGCTGCCTTGTCCTCTTCCTG-3’
and antisense 5’-GTGTATGGCCTCTCATAGAGC-3’. The control PCR primer set
specific for the MIF promoter fragment from -1,930 to -1,754 lacking the HBP1
binding site included: sense 5’-GGTTTTGGAGGGGGTTCTTAT-3’ and antisense
5’-CAGCCCGAAAATATCAGC-3’. PCR products were resolved using agarose gel
electrophoresis and ethidium bromide staining.
Electrophoretic mobility-shift assays (EMSA). A double-stranded oligonucleotide
probe
(5’-CCCATTCATTCATTCATTCATTCAGCAG-3’) corresponding to bases
-815 to -788 in the MIF promoter Was used, which encompasses the HBP1 binding site.
Nuclear extracts from DU-145 cells(designated as D), HBP1-transfected DU-145
cells(designated as H), pmHMG-transfected DU-145 cells (designated as Pm) and
RWPE2-W99 cells (designated as R)were prepared. For each super-shift reaction (20 μl),
2 μl anti-HBP1 or anti-Foxo3a antibody (as a negative control) (Santa Cruz
Biotechnology Inc. USA) were incubated with nuclear extracts from cells at 4°C for
30 min, followed by an additional incubation for 30 min at room temperature with
labeled probe. After the incubation, samples were electrophoresed and exposed to film .
Protein lysate preparation and immunoblot analysis. Whole cell lysates from cultured
cells were prepared by homogenization in a lysis buffer. The samples were separated on
15% SDS-polyacrylamide gels (Promega Corporation). Anti-HBP1 polyclonal antibody
(sc-8488，Santa Cruz) anti-MIF polyclonal antibody(sc-20121，Santa Cruz) or anti-actin
polyclonal antibody(sc-1616-R，Santa Cruz)was used as the primary antibody. After the
primary antibody incubation, the blots were then incubated with secondary horseradish
peroxidase-conjugated secondary antibodies that recognize the appropriate species of
primary antibody (1:1,000) for 2 h. Immunoreactive bands were visualized using an
enhanced chemiluminescence method, followed by exposure to X-ray film (Kodak).
HBP1 knockdown. RNAi-mediated HBP1 knockdown was accomplished by short
hairpin RNA (shRNA) produced by the DNA-based shRNA-expressing retroviral vector
(pSuper-Retro).The vectors were kind gift from Dr. Amy S. Yee. The knockdown
experiment was performed as described (Paulson et al., 2007). The HBP1
shRNA target
sequence is ACTGTGAGTGCCACTTCTC.
MTT assays. Cells (1 ×104 cells/well) were plated in 96-well dishes and were cultured
under normal conditions. Purified recombinant human MIF (rMIF 289-MF R&D systems)
was added to the HBP1-transfected DU-145 cell culture media (100 ng/ml). MTT assays
were performed at 24 h, 48 h, 72 h, 96 h, and 120 h using 50 μl of 5 mg/ml MTT
(Promega Corporation) per well. The absorbance at 490 nm was determined using an
automated microplate reader (Bio-Rad). The MTT data represent the mean + SE of two
independent experiments done in triplicate. Statistical significance was assessed by the
two-sample Student’s t test using SPSS; P values < 0.05 were considered statistically
significant.
Colony formation assays. For colony formation assays, 1× 104 cells were suspended
in 1.5 ml of 0.35% agar in DMEM media containing 10% FBS and were plated in 6-well
dishes with a 0.7% agar bottom layer. The cells were cultured at 37oC with 5% CO2.
rMIF (100 ng/ml) was added to the HBP1-transfected DU-145 cell culture media. After
14 days, the colonies were stained using crystal violet and were counted microscopically
within the field of a 40×objective lens (Nikon Eclipse TE 2000-U). Colonies with a
diameter greater than 200 μm were counted and analyzed; the colony numbers are
represented as means + SE of two independent experiments done in triplicate. Statistical
significance was assessed by the two-sample Student’s t test using SPSS; P values < 0.05
were considered statistically significant.
Matrigel invasion assays. Matrigel invasion assays were conducted in 24-well transwell
culture plates containing microporous (8.0 μm) membranes coated with Matrigel (BD
Biosciences, San Jose, CA, USA) diluted 1:3 with DMEM. Cells (5 x 104 cells/well) were
added to the upper chamber containing 200 l basal medium (DMEM containing 0.1%
BSA); rMIF (100 ng/ml) was added to the upper chamber containing HBP1-transfected
DU-145 cells. The lower chambers contained 500 l DMEM with 10% FBS. Following a
36 h incubation, the nonmigrating cells were removed from the upper chamber using a
cotton swab, the adherent cells on the lower surface of the insert were stained with crystal
violet, and the number of invading cells stained by crystal violet was counted in nine
representative fields at 100×magnification. The data were expressed as the average
number of cells per treatment per 100×field + SE and were analyzed by the two-sample
Student’s t-test using SPSS; P values < 0.05 were considered statistically significant.
Statistical analysis. For the clinical correlations to relapse, the Oncomine was used to
compile a data set with Oncomine-normalized HBP1 and MIF for each patient.
Pearson correlation was used to analyze the correlation between HBP1 and MIF.
Kaplan-Meier curves for disease free interval (DFI) were created for each group of
normalized HBP1 and MIF expression levels and statistically compared between
groups using the log-rank test. SPSS 13.0 was used for data analysis throughout this
article. An
level of 0.05 was used to determine statistical significance when
interpreting results.