Lu et al
mPGES-1 promotes HCC growth via activation of EGR1/-catenin
SUPPLEMENTARY INFORMATION
SUPPLEMENTARY METHODS
Immunofluorescence localization. Following each treatment, the cells grown on
coverslips were washed twice with cold PBS and fixed in 4% paraformaldehyde for
10 min. The cells were treated with 0.3% H2O2 in methanol for 30 minutes to abolish
endogenous peroxidase activity and then treated with 0.1% Triton and 5% DMSO.
After blocked with 5% BSA at 37℃ for 1 hour, the cells were incubated with 2 µg/ml
primary antibody at 37℃ for 1 hour or 4℃ overnight. Then the cells were incubated
with TRITC-linked IgG (Santa Cruz) or FITC-linked IgG (Santa Cruz) with
counterstaining with DAPI (diluted 1:1000 in ddH2O). Coverslips were applied with
mounting medium and fluorescence images were captured with Olympus FV1000II
Laser Scanning Confocal Microscope (Olympus) by FV10-ASW1.7 software or
fluorescence microscope (Olympus).
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).
The homogenate was centrifuged at 12,000 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) under rotating slowly at least 40 min at 4°C and then sonicated to lyse the
nuclei. 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). Nuclear proteins were obtained for
EMSA by using the gel shift assay system (Promega, Madison, WI). Consensus
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Lu et al
mPGES-1 promotes HCC growth via activation of EGR1/-catenin
oligonucleotides for TCF/LEF binding site were biotin labled [TCF/LEF-site: 5’AGATCAAAGGG-3’and 5’-CCCTTTGATCT-3’]. Each binding reaction was carried
out with 100 fmol of biotinylated dsDNA probe and 2µl of purified nuclear protein
(5µg/µl) in 20 μl of binding buffer containing 0.5mg/ml poly(dI:dC) (25 mM HEPES
at PH8.0 with 50 mM KCl, 0.1% Triton-X100, 2 mM MgCl2, 3 mM DTT, and 5%
glycerol). Twenty-five pmol unlabeled cold DNA motifs (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 samples were
loaded onto 6% TBE polyacrylamide gels and separated in 0.5%×TBE at 100v on ice.
The separated protein-DNA complexes were then transferred to nitrocellulose
membranes, followed by Western blotting using anti-biotin antibody.
Culture plate colony formation assay. 1 x 103 cells were plated on 10-cm culture
plates and cultured in complete medium for 14 days. For visualization, colonies were
stained with crystal violet in 50% methanol and 10% glacial acetic acid for counting.
Data were obtained from three independent experiments.
Soft agar colony formation assay.
1 x 103 cells were plated on 10-cm plates
containing 0.5% and 0.35% double layer soft-agar. The plates were incubated at 37°C
in humidified incubator for 21 days and the culture medium was changed 1-2 times
per week. The colonies were visualized by staining with 0.005% Crystal Violet for 1
hour and counted using a dissecting microscope equipped with the MacBiophotonics
Image J.
Cell cycle analysis. Cell cycle distribution was determined by measuring the cellular
DNA content using flow cytometry. In brief, the cells were synchronized in G0 phase
by serum deprivation for 48 h; the cells then were released from growth arrest by
reexposure to 10% fetal bovine serum for 24 h. The cells were then collected by
trypsinization and washed with PBS. The collected cells were fixed in 70% ethanol
in 50 mM glycine buffer (pH 2.0) overnight at -20°C. After the incubation with 100
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Lu et al
mPGES-1 promotes HCC growth via activation of EGR1/-catenin
μg/ml RNase A for 30 minutes at 37°C, the cells were resuspended in 0.5 ml 100
µg/ml propidium iodide solution (PI) for staining. The stained cells were monitored
by a FACScan cytometer (Becton Dickinson) or BD LSRII FACS D1VA. The
percentage of cells in the S, G0/G1, and G2/M phases of the cell cycle was
determined using the cell cycle analysis software EXPO32 Cell Quest or MODFIT.
All experiments were performed in triplicates.
Cell invasion assay. Cell invasion assay was performed using 24-well Transwell
chambers (8.0 μm pore size polycarbonate membrane) according to the
manufacturer's instructions (BD Biosciences).
Briefly, 0.5 ml cell suspension
(2.5x104 cells) were added to the upper chamber and the complete medium with 10%
FBS was added to the bottom well. After 24 hours of incubation, the non-migrated
cells were removed by a cotton tip from the upper side of the chamber and the
migrated cells were fixed and stained with hematoxylin. The average number of
migrated cells was determined by counting from 5 to 10 high power fields (HPFs)
under the microscope at either 40 X or 200 X magnifications depending on cell
density. All the experiments were repeated at least three times.
Wound healing assay. Cells grown in 10-cm dishes with 90% confluence were
starved in low serum medium (0.5% - 0.1% serum) overnight. A line was drawn with
a sterile 200 μl pipette tip on the bottom of the dish; the cells were rinsed with PBS
and cultured in the same medium for indicated time points before photography.
BrdU staining. Cells at approximate 80% confluence were incubated with BrdU
(Roche) for 4 hours; immunofluorescence staining was performed using a mouse
monoclonal anti-BrdU antibody. In brief, BrdU treated cells were fixed with 4%
paraformaldehyde for 30 min at 4ºC. Following fixation, the cells were washed in
PBS with 1% TritonX-100 (3 x 5 min) and incubated on ice in 1N HCl for 10 minutes
to break DNA structure of the labeled cells. The cells were incubated at room
temperature with 2 N HCl for 10 minutes followed by incubation at 37°C for 20
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Lu et al
mPGES-1 promotes HCC growth via activation of EGR1/-catenin
minutes. After acid washing, the cells were buffered with 0.1M borate buffer at room
temperature for 12 minutes. The cells were then washed with PBS containing 1%
TritonX100 ( 3 x 5 min) and incubated with PBS containing 1% TritonX100 + 1M
Glycine + 5% normal goat serum at room temperature for 1 hr, followed by
incubation with anti-BrdU antibody for overnight. After washing three times with
PBS containing 1% Triton X-100, the cells were treated with anti-mouse TRITC
fluorescent conjugated secondary antibodies to visualize anti-BrdU labeled cells.
BrdU positive cells were counted in ten random chosen fields from three independent
samples.
Luciferase reporter assay. Cells were transiently transfected with either LEF/TCF
luciferase construct (Panomics) or pGL3-Luc control vector with LipofectiamineTM
2000 (Invitrogen). After 24-36 h of transfection, the cells were harvested and cell
lysates were prepared with 1 X cell lysis buffer (Promega). Luciferase activities of
cell extracts were measured with the luciferase assay system (Promega) according to
manufacturer's instructions.
Statistical analysis. Results are presented as mean ± standard error (SEM) from a
minimum of three replicates. The significant difference between different groups was
evaluated by SPSS12.0 statistical software or Student’s T-test. P<0.05 was considered
as statistical significance.
SUPPLEMENTARY FIGURE LEGENDS
Figure S1. Western blotting for mPGES-1 in Hep3B (A) and Huh7 (B) cells stably
transfected with four different vectors: GFP control (pCMV6-AC-GFP); GFPmPGES-1 overexpression (pCMV6-AV-GFP-mPGES-1); RNAi control (pGFP-VRS); and mPGES-1 RNAi (pGFP-V-RS-mPGES-1 RNAi). Cell lysates were obtained
and subjected to SDS-PAGE and western blotting analysis as described in the method
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Lu et al
mPGES-1 promotes HCC growth via activation of EGR1/-catenin
section. β-action was used as the loading control.
Figure S2. Soft-agar colony formation assay. Hep3B (A) and Huh7 (B) cells were
placed in 10 cm soft agar dishes (1 x 103 cells per dish) and incubated in a humidified
atmosphere of 5% CO2 incubator at 37°C for 21 days. The colonies were visualized
by staining with 0.005% Crystal Violet (Sigma) for more than 1 hour. Representative
photographs of colony formation from different stable cell lines are shown at the left
panels. The bar graphs at the right panels represent the colony formation rate (%)
which was calculated by dividing the numbers colonies divided by 1 x 103 plated
cells. The data (mean ± SEM) were obtained from three independent experiments.
Figure S3. Representative flowcytometry graphs for Hep3B (A) and Huh7 (B) cells
with altered expression of mPGES-1. The cells were synchronized in G0 phase by
serum deprivation for 48 h and then released from growth arrest by reexposure to 10%
fetal bovine serum for 24 h. The cells were subsequently collected for flowcytometry
analysis as described in the Supplementary Methods.
Figure S4. Histopathological analysis of the xenograft tumors recovered from the
SCID mice 4 weeks after inoculation with the Hep3B (A) and Huh7 (B) cell lines.
Representative photographs of the hematoxylin and eosin stain (H&E) and
immunostain for PCNA are shown at the left panels. The percentage of PCNA
positive cells (the numbers of positively stained cells divided by the total number of
cells in randomly selected magnification fields) are shown at the right panels (the data
are presented as mean ± SEM, n = 5-6).
Figure S5. Immunofluorescence for EGR1 in Hep3B (A) and Huh7 (B) stable cell
lines (with TRITC staining and DAPI counterstaining; original magnification×200;
scale bar 10μm). The level of EGR1 was increased in mPGES-1 overexpressed cells
but decreased in mPGES-1 knockdown cells.
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