Supporting Data:
Generation of HBx-expressing HepG2 cells
The generation of HBx-expression HepG2 cells (HepG2-X) was as followed: The
coding region of HBx was amplified from CH-Hep-1, a HCC cell line established
from surgically removed tissue of HCC by our laboratory, using PCR with sense
primer 5’-AAAGCTTGCCGCCACCATGGCTGCTCGGGTGTG-3’ and antisense
primer 5’- AGAATTCTCATTAGGCAGAGGTGAAA-3’. The resulting 491-bp PCR
product was cloned into pGEM-T-easy vector (Promega) and sequenced. The results
confirmed that the HBx gene was adr strain (data not shown). The HBx fragment was
excised by HindIII and EcoRI digestion, and subsequently cloned into the same sites
of pcDNA3, yielding pc-HBx, which directed the expression of HBx controlled by the
CMV promoter. HBx plasmid and control vector were transfected into HepG2 cells
and screened under 400 g/mL G418 (Merck, Whitehouse Station, NJ). The stably
transfected cell clones were obtained by limiting dilution and confirmed by Western
blot analysis using anti-HBx antibody (Santa Cruz Biotechnology).
PCR Analysis of ASPP1 and ASPP2 Expression
Primers
used
were
as
5’-GCCTCAACCATAAGCGACAT-3’,
5’-CCACAGAGCATACGCCACA-3’;
5’-GAAGACTCGGTGAGCATGCG-3’,
followed:
ASPP1
ASPP1
ASPP2
ASPP2
Forwards:
Rewards:
Forwards:
Rewards:
5’-GCGATACGCTCTGAGCCAGT-3’. The PCR conditions were as follows: 94°C
for 1 min, then 28 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a
final elongation at 72°C for 10 min. PCR products were electrophoresed in 2%
agarose gel.
Construction of Lentiviruses Encoding shASPP1 and shASPP2
After annealing, double-strand oligos were inserted to the liner vector pHsCXW
plasmid, which had an U6 promoter at ClaI site before CMV promoter. The resulting
plasmids were sequenced, designated as pshASPP1, pshASPP2, and pshNon, and then
transfected into HepG2 cells. Quantitative reverse transcription PCR was used to
validate gene silencing efficiency induced by shRNA at 72 h post-transfection. The
sequences targeting human ASPP1 and ASPP2 for RNA interference in this study
were
5’-
and
GCAAGCCACACCACCTAAGAA-3’
5’-GCTGAGGGAGAAAGAGAAGAA-3’, respectively. For negative control, we
used
sequence
targeting
none
of
the
known
genes
(5’-AATTCTCCGAACGTGTCACGT-3’). The sense and antisense sequences of the
siRNA target sites of interest were separated by a hairpin loop sequence CTCTTGA.
To produce lentivirus containing shRNA, HEK-293T cells were co-transfected with
the resulting vector described above and ViraPower Packaging Mix (Invitrogen,
Carlsbad, CA) using Lipofectamine 2000 according to the manufacturer’s guidelines.
Infectious lentiviruses were harvested at 48 and 72 h post-transfection and filtered
through 0.45μm PVDF filters, designated LV-shASPP1, LV-shASPP2, and LV-shNon
respectively.
Recombinant
lentiviruses
were
concentrated
100-fold
by
ultracentrifugation (2 h at 50,000 g). The virus-containing pellet was dissolved in
DMEM, aliquoted and stored at -80 °C. The infectious titer was determined by
counting the blue-stained colonies after crystal violet staining in 293 cells.
Immunohistochemistry Staining
The expression of ASPP1 and ASPP2 was analyzed with EnVision system (Changdao
Bio-tech, Shanghai, P.R.China) in formalin-fixed, paraffin-embedded sections of
primary tumors. The slides were fixed and the endogenous peroxidase activity was
quenched by incubation in methanol with 3％ hydrogen peroxide for 30 min. The
slides were then washed with phosphate-saline (PBS) and treated in 10 mmol/L citrate
buffer (pH 6.0) by a temperature-controlled microwave oven with a power of 700 W
for 15 minutes. Nonspecific binding was blocked by 10% normal goat serum in PBS
for 30 min. The monoclonal antibody against ASPP1 (LX54.2) or ASPP2 (DX54.10)
was diluted at 1:100.After incubation with the primary antibody overnight at 4°C,
slides were rinsed with PBS and the working solution of horseradish
peroxidase-labeled goat anti-rabbit or anti-mouse was applied for 60 minutes at 37°C.
After rinse with PBS for three times, staining was visualized using the peroxide
substrate solution diaminobenzidine. Counterstained by hematoxylin, the slides were
dehydrated in graded alcohol and mounted. Negative controls were prepared in the
absence of primary antibody.
Evaluation of immunostaining was independently performed by two experienced
pathologists. The intensity of immunostaining was semi-quantitatively estimated
according to the signal intensity and distribution. Briefly, a mean percentage of
positive tumor cells was determined in at least five areas ×400 magnification and
assigned to one of the five following categories: 0, <5%; 1, 5-25%; 2, 25-50%; 3,
50-75% and 4, >75%. The intensity of immunostaining was scored as follows: 1,
weak; 2, moderate and 3, intense. For tumors that showed heterogeneous staining, the
predominant pattern was taken into account for scoring. The percentage of positive
tumor cells and the staining intensity were multiplied to produce a weighted score for
each case. Tissues with immunohistochemical scoring ≤4 were considered as low
expression and 5-12 as high expression.
Analysis of p53 Gene Status
A cDNA-based sequencing method was used to determine the status of the p53 gene
in HCCs. RNA was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany);
First-strand cDNA was generated using the Reverse Transcription System Kit
(Promega). The single stranded cDNA was subsequently amplified by the polymerase
chain reaction using 2 overlapping sets of p53-specific primers covered the entire
protein-coding
region.
Primer
5’-TGCTCAAGACTGGCGCTAA-3’,
CTCATAGGGCACCACCACA-3’;
sequences
Rewards
Forwards
were:
Forwards
1:
1:
5’-
2:
5’-
TGCAGCTGTGGGTTGATTC-3’, Rewards 2: 5’-GCAAGCAAGGGTTCAAAG-3’.
PCR products that have given clear bands of expected length in the ethidium bromide
stained agarose gel were purified with the AxyPrep DNA Gel Extraction Kit (Axygen
Biosciences, Union City, CA). The purified products were sequenced by Shanghai
DNA Bio Technologies Company (Shanghai, P.R.China). The sequences were aligned
to the wild-type p53 sequence using the software program Vector NTI Suite 9
(Invitrogen).
Inhibition of DNMTs Expression
siRNAs designed against DNMT1 (5’-CAAUGAGACUGACAUCAAATT-3’) and
DNMT3A (5’-GGAUAGCCAAGUUCAGCAATT-3’), or control siRNA
(5’-UUCUCCGAACGUGUCACGUTT-3’) were transfected in HepG2 cells by
Lipofectamine 2000 (Invitrogen). The expression of DNMT1 and DNMT3A was
examined by Western Blotting or qRT-PCR using primers as followed: DNMT1:
Forward:5’- CCTGAGGCCTTCACGTTCAA-3’, Rewards:5’ACTTGTGGGTGTTCTCAGGC-3’; DNMT3A:Forward:5’TATTGATGAGCGCACAAGAGAGC-3’, Rewards:5’GGGTGTTCCAGGGTAACATTGAG-3’.
Figure Legend
Figure S1. (A) qRT-PCR and Western blotting analyzed DNMT1 and DNMT3A
expression in HepG2 cells with HBx or pcDNA3 stable transfection. (B) siRNA
designed against DNMT1 and DNMT3A reduced the expressions of DNMT1 and
DNMT3A in HepG2 cells, as indicated by Western blotting.