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SUPPLEMENTARY MATERIALS AND METHODS
Plasmids, site-directed and deletion mutagenesis, and lentivirus production
Vectors Flag- and HA-tagged pcDNA4T0, GST-tagged PGEX4-T-1, EGFP- and Cherry-tagged
3×Flag-PGK-Puro, and pLV-U6-EGFP-Puro were used in this study. HEK293T and HepG2 cells
cDNA libraries were used to amplify the appropriate sequences.
The following primers were used to perform site-directed and deletion mutations:
5'-GCGCTTCTCTCCTTTGCTCT-3' and 5'-TCTGCCCAGAGCAAAGGAGA-3' were used to
make
the
mSREBP1a
R321K
mutant;
5'-GGGGCCAGTGTGTCCTCCA-3'
and
5'-GATATCTGCAGAATTCTCAGCG-3' were used to make the mSREBP1a R321K/S430A
mutant; 5'-CCCTTGCTGCCAAGGGAC-3' and 5'-TTGGCAGCAAGGGCAGTG-3' were used to
make
the
mSREBP1a
R446K
5'-CAGAATTCTCAGCGGGAGCGGTCCAGCATGCCCTTGC-3'
mutant;
and
5'-TCGGATCCATGGCTGACTACAAGGACGACGATGAC-3' were used to make the mSREBP1a
R480K
mutant;
5'-GCTTCTCTCCTGCGCTCTGGGCAGA-3'
and
5'-CCCAGAGCGCAGGAGAGAAGCGCAC-3' were used to make the mSREBP1a R321A
mutant;
and
5'-CCAGGGCAGCACCATCAGTACCTGGACATTGG-3'
and
5'-CTGATGGTGCTGCCCCTGGTGAACGCTTCC-3' were used to make the PRMT5 ΔM mutant.
In lentivirus packaging, the sequence of the mSREBP1a WT or R321K mutant was
inserted into the viral skeleton plasmid EGFP-tagged 3×Flag-PGK-Puro. Annealing and
connection of the shRNAs were performed before insertion into the viral skeleton plasmid
pLV-U6-EGFP-Puro. All products were subsequently transformed into HEK293T cells using
lentivirus packaging reagents (GenePharma, Shanghai, China), according to the manufacturer’s
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instructions, to acquire the virus. The shRNA sequences used in our research are shown in
Supplementary Table S3.
Immunoprecipitation and immunoblotting analyses
Harvested cells were lysed in RIPA lysis buffer (Beyotime) at 4ºC. Immunoprecipitaion was
carried out by incubating cell protein lysates with antibody-conjugated protein A+G sepharose
beads (Thermo scientific), an anti-Flag M2 affinity gel (Sigma-Aldrich), or Pierce anti-HA agarose
(Thermo scientific) at 4ºC for 3 hours. Protein samples were resolved by SDS-PAGE, transferred
to polyvinylidene fluoride membranes (PVDF) (GE healthcare) and probed with the indicated
primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa
Cruz, CA, USA). The chemiluminescent HRP substrate (Millipore, Massachusetts, USA) was
used to visualize antibody binding. The protein levels of Flag-mSREBP1a in different samples
immunoprecipitated by anti-Flag M2 affinity gel were detected by immunoblotting analysis using
antibody against Flag, followed by quantification by the image analysis software Imagine J (NIH,
MD, USA), and then normalized to the same level. The antibodies used in our research are
shown in Supplementary Table S4.
Confocal immunofluorescence microscopy
HepG2 cells harvested on coverslips were washed with phosphate buffered saline (PBS) before
fixing in 4% formalin for 15 minutes. Cells were then treated with 0.25% Triton X-100 for 10 min,
followed by three washes with PBS. The cells were blocked with 1% bovine serum albumin
(BSA)/PBS for 1 h, followed by incubation with primary antibodies (anti-SREBP1a (1:100) (Santa
Cruz, sc-13551, mouse mono-antibody) and anti-PRMT5 (1:500) (Abcam, EPR5772, rabbit
mono-antibody)) for 2 hours at room temperature. After two PBS washes, the cells were
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incubated with a secondary antibodies mixture of goat anti-rabbit IgG H&L conjuncted with Alexa
Fluor® 488 (1:1000) (Thermo scientific) mixed with goat anti-mouse IgG H&L conjuncted with
Alexa Fluor® 568 (1:1000) (Thermo scientific) for 1 hour. The resulting signals were visualized
using a confocal laser-scanning microscope (Olympus BX61, TKY, JPN).
Gene expression analysis
Total RNA was isolated from cells using an E.Z.N.A total RNA Kit I (Omega bio-tek, GA, USA).
cDNA was synthesized using a PrimerScript RT reagent Kit (Takara, OSTU, JPN). The real-time
PCR reaction was performed according to the protocol of the SYBR Premix Ex Taq kit (Takara),
and using a StepOnePlus Real-Time PCR System (Applied Biosystems, USA). The fluorescence
data of the detected genes were normalized to cyclophilin B (CB) expression using the 2-ΔΔCT
method. Primers used are listed in Supplementary Table S5.
Luciferase reporter assay
A plasmid containing the human FASN gene promoter fused to the luciferase gene in the pGL3
vector (Promega, WI, USA) was used as a reporter vector. Cells were subcultured in 24-well
culture dishes (1.5×104/well) and transfected with the luciferase reporter vector plus a renilla
luciferase plasmid at a ratio of 10:1. The luciferase activity of the cell extract was analyzed using
a Dual-Luciferase Assay system (Promega), according to the manufacturer’s instructions. The
relative levels of FASN luciferase activity were normalized to the renilla luciferase activity levels.
Oil red O staining
Cells were washed with PBS before fixing in 4% formalin for 10 minutes at room temperature.
After rinsing each plate quickly using 60% isopropanol, the cells were stained by 0.3% Oil red O
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(Millipore) at room temperature for 1 hour. The plates were rinsed with 60% isopropanol and PBS
in turn before the Oil red O staining was observed under a microscope.
Cell growth assay
HepG2 cells were plated on 24-well plates in 1% CSFBS DMEM (5000 cells per well, three
parallel wells). Cells were harvested at different time points after transfection and the cell
numbers on each plate were counted. Trypan blue staining was used to identify dead cells when
counting.
Colony formation assay
Cells were suspended in 0.3% agarose (Sigma-Aldrich), and placed at 50 cells per well on the
top of solidified 0.6% agarose in 24-well plates. After three weeks of cultivation, the cells were
fixed in 10% formalin and stained with 0.1% crystal violet. The relative number of colonies was
calculated, where the number of the control group was designated as 1.
Immunohistochemistry
The experiment using human tissues was approved by Human Assurance Committee of Ren Ji
Hospital Shanghai Jiao Tong University School of Medicine. HCC tissue sections were obtained
by surgical resection at the Ren Ji Hospital with the agreement of the patients. Patients in our
research were followed-up until September 2013 (from 1 to 80 months) after surgery. All tumor
biopsies had a matched normal tissue sample from the same patient, and detailed
clinicopathological data were also obtained, which is shown in Supplementary Table S2. The
pathological types of paraffin-embedded slides were checked again by HE staining before
immunostaining. Subcutaneous tumor tissues of nude mice were fixed in 4% paraformaldehyde,
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dehydrated, paraffin embedded and cut into 4-µm sections. Cells grown on coverslips were fixed
in 4% paraformaldehyde after 48 hours of transfection. The endogenous peroxidase activity in
the tissue sections or fixed cells was blocked with 3% hydrogen peroxide solution (Sangon
biotech), the antigens were retrieved, and the nonspecific binding of the primary antibodies was
blocked by 4% normal goat serum (Gibco). Subsequently, tissue sections or cells coverslips
were incubated with different primary antibodies, followed by HRP-conjugated goat anti-rabbit
IgG (1:1000, Cell signaling, MA, USA) incubation. 3,3'-diaminobenzidine (DAB) chromogen
substrate solution was to visualize the results. The following antibodies were used in the
immunohistochemistry experiment: anti-Ki67 rabbit polyclonal antibody (1:500, Sigma-Aldrich),
an anti-321R(SDMA)-mSREBP1a rabbit polyclonal antibody designed and synthesized by
Hangzhou huaan biotechnology Co., Ltd of China (1:100 in HCC tissues, 1:200 in xenograft
tissues and cells fixed on coverslips).
321R(SDMA)-mSREBP1a expression in HCC tissues was scored blindly and
independently by two scientists. The scores for nucleus staining frequency (0 = 0–19%, 1 =
20–39%, 2 = 40–59%, 3 = 60–79%, 4 = 80–100%) and intensity (0 = negative, 1 = weak, 2 =
moderate and 3 = strong staining) were added to obtain an overall staining score (OSS). An OSS
of 0–2 was deemed low, 3–5 moderate and 6–7 high.
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