Supplementary Information
Supplementary Materials and Methods
Generation of Plasmid Constructs and Establishment of Cell Lines Stably or
Inductively Expressing SOX1
The following constructs were used: SOX1 cDNA (pCMV6-XL5) was
purchased from OriGene Technologies, Inc., and Gateway Cloning Technology
(Invitrogen) was utilized to subclone the SOX1 cDNA downstream of the N-terminal
His
6
tag
peptide
of
the
destination
vector
pT-REx-DEST31
(with
tetracycline-inducible promoter) to generate pT-REx-DEST-SOX1 vector. Cells were
transfected with pT-REx-DEST vector only or pT-REx-DEST-SOX1vector using the
liposome-mediated transfection method (Invitrogen) according to the manufacturer's
instructions to generate cells expressing SOX1 constitutively.
Two days
post-transfection, cells stably expressing SOX1 or empty vector were seeded into
DMEM containing 10% fetal calf serum and 0.9 mg/ml G418 solution (Roche) at a
low density. Cells were then incubated at 37°C in 5% CO2 for 15~20 days, and
several colonies were selected and validated for SOX1 expression by Western blot.
To build up an inducible system, pcDNA6/TR (containing the tetracycline
repressor
gene)
stable
clones
were
selected
and
then
transfected
with
pT-REx-DEST-SOX1vector to generate cells expressing SOX1 under doxycycline
(DOX) (Invitrogen) induction. Two days after transfection, 0.9 mg/ml G418 solution
and 7.5 μg/ml blasticidin (Invitrogen) were added to cells for selection of stable
clones as mentioned before. To validate the inducible stable clones, SOX1 expression
was induced for 48 hr with 1 μg/ml DOX prior to harvesting for Western blot. Stable
clones were selected and maintained in medium containing 0.3 mg/ml G418 w/o 2.5
μg/ml blasticidin antibiotics.
RNA Isolation, RT-PCR and Real-time Quantitative RT-PCR
RNA was isolated from each sample using a Qiagen RNeasy kit (Qiagen). An
additional DNase I digestion procedure (Qiagen) was included in the isolation of
RNA to remove contaminating DNA according to the manufacturer’s protocol. One
microgram of total RNA from every sample was used for cDNA synthesis using a
transcriptor first strand cDNA synthesis kit (Roche). Then, cDNA was amplified by
PCR with primers specific for SOX1 (1, 2) and the glyceraldehyde-3-phosphate
dehydrogenase gene (GAPDH) using a PCR Master Mix Reagents Kit (Applied
Biosystems). Quantitative RT-PCR analysis was performed on an ABI 7700 Sequence
Detector (Applied Biosystems). The matching primers and tagman probe were
obtained from commercial Applied Biosystems Tagman® Assay-on DemandTM Gene
Expression products. Glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH)
was used as an internal control. The relative gene expression was determined based on
our previous report (3).
Cell Proliferation Assay (MTS Assay)
The cell proliferation assay was performed using CellTiter 96 AQ One Solution
(Promega) according to the manufacturer’s instructions. Total cells were harvested at
the designated times after seeding. Briefly, reagent (20 l/well) was added to 100 l
of medium containing cells in each well of 96-well plates and incubated for 1 hr at
37C under humidified 5% CO2 in air. For colorimetric analysis, the absorbance at
492 nm was recorded using an ELISA reader. Each experiment was repeated at least 3
times.
Invasion Assay
The invasiveness of the SK-Hep-1 and HA22T cells, with SOX1 or vector-only
control, were examined using a Transwell invasion culture system. The standard
protocol was as described previously (4).
Colony Formation Assay (Anchorage-Independent Growth Assay)
Cells were trypsinized and resuspended in 1.5 ml of 0.35% agarose and poured
onto a 1.5 ml 0.5% agarose bed in 35-mm tissue culture dishes. After 4–5 weeks, cells
were stained with a solution containing 0.005% crystal violet, 1.9% formaldehyde,
and 0.15 M NaCl for 30 min. After washing and drying, colonies larger than 1 mm
were counted.
Western Blot
A standard protocol was used. The primary antibodies used and dilutions were
as follows: goat anti-SOX1 (R&D systems, 1:2000), mouse anti-Flag (Sigma, 1:2000),
mouse anti--catenin and anti-lamin A/C (BD Biosciences, 1:2000), mouse
anti--actin (Abcam, 1:2000), mouse anti-TCF3/4 (Abcam, 1:2000), rabbit anti-GST
(BETHYL, 1:2000), rabbit anti-c-Myc (Cell Signaling, 1:500) and a cell cycle
regulation sampler kit (Cell Signaling). The second antibodies used and dilutions were
alkaline phosphatase-conjugated anti-mouse IgG, anti-goat IgG and anti-rabbit IgG.
To detect alteration in -catenin levels, cytosolic and nuclear extracts were prepared
and examined as described previously (3).
GST Pull-down
We subcloned SOX1 cDNA into pGEX-4T-1 (GE Healthcare), carrying genes
encoding a glutathione S-transferase (GST), and expressed as GST-SOX1 fusion
protein in Escherichia coli (DH5α strain) by IPTG induction. Bacterial protein
extracts in the binding buffer (140 mM NaCl, 2.7 mM KCl, 10mM Na2HPO4, 1.8 mM
KH2PO4, pH 7.3) containing GST-SOX1 or GST alone were immobilized on a
glutathione-Sepharose column (GE Healthcare, GSTrap HP, 1 ml) by injecting with a
syringe. After washing with binding buffer, an equal amount of the total protein
lysates from Huh6 cells was injected into the column and incubated with fusion
protein-glutathione-Sepharose at 4°C for 1 h. The column was then washed several
times with binding buffer, and the remaining proteins were eluted using elution buffer
(50 mM Tris-HCl, pH 8.0) with 10 mM reduced glutathione. We then ascertained
what protein was pulled down by GST-SOX1.
Flag-tag Co-immunoprecipitation
Whole-cell lysates (800 μg of protein) of COS7 cells transfected with plasmids
expressing SOX1, Flag-β-cateninΔ45 (S45Y) alone, or both Flag-β-cateninΔ45 and
SOX1 were subjected to mixing with anti-FLAG-M2 affinity gel (Sigma) and
incubated at 4°C overnight with continuous mixing followed by at least 3 washes with
IP buffer. The protein complex was eluted by competition with 3X FLAG peptide
(final concentration: 5 μg/μl) in elution buffer and checked subsequently by Western
blot.
Co-immunoprecipitation
Total cell extracts with or without SOX1 expression from Hep3B cells were
lysed with IP buffer (0.5% Triton, 10 mM Tris pH 7.5, 145 mM NaCl, 5 mM EDTA,
2 mM EGTA) (5) under addition of proteinase inhibitor cocktail Complete 6 (Roche).
An equal amount of a total of 800 μg lysates was used for co-immunoprecipitation
(Co-IP) experiments performed using Magna chip Protein G magnetic beads
(Millipore) according to the modified manufacturer’s protocol for direct
immunoprecipitation. First, 3 μg of the monoclonal β-catenin or mouse IgG antibody
(Millipore) were mixed with the beads and incubated at 4°C for 1 hr with continuous
mixing followed washing with IP buffer. Second, the antibody-immobilized beads
were incubated with protein lysates overnight at 4°C with continuous mixing. The
beads were washed with IP buffer at least three times and the protein complexes
bound to the antibody were eluted using an appropriate amount of elution buffer [0.2
M glycine (pH 2.5)]. Subsequent Western blot analyses were performed as described
before.
Immunocytochemistry
Hep3B cells were grown on coverslips and cultivated in the absence or presence
of doxycycline for 3 days. Cells were fixed with 4% paraformaldehyde in
phosphate-buffered saline (PBS) for 20 minutes and then permeabilized by incubation
with 0.6% Triton X-100 in PBS for 5 minutes at room temperature. Nonspecific
binding sites were saturated using a blocking solution of 3% bovine serum albumin
(BSA) in PBS. For double immunofluorescence staining, cells were incubated with
anti-β-catenin and anti-SOX1antibodies overnight at 4°C, and FITC-conjugated
anti-mouse IgG and rhodamine-conjugated anti-goat IgG secondary antibodies were
applied. Primary and secondary antibodies were diluted in 0.3% BSA/PBS. Finally,
cells were stained with TOTO-3 iodide (Invitrogen) to visualize the nuclei. Confocal
fluorescence microscopy was performed using a Zeiss LSM510 confocal laser
scanning microscope equipped with a two-photon argon laser at 488 nm (Cy2), 543
nm (Cy3), or 633 nm (Cy5, Alexa Fluor 647), respectively.
Senescence-associated β-galactosidase Staining
Hep3B cells were plated in a 12-well plate and treated with or without DOX
(1μg/ml) for 7 days to induce SOX1 expression. For senescence-activated
β-galactosidase (SA-βGal) staining, we used a Senescence-βGal Staining Kit (Cell
Signaling) according to the manufacturer’s instructions. Stained slides were
subsequently detected the location of SOX1 and nucleus following the
immunocytochemistry as described before.
Statistical Analysis
The results are expressed as the mean ± standard error (SE). The SOX1
expression levels in the primary HCC tissue and the non-tumor parts were compared
using the paired sample t-test. The statistical significance was determined using the
Mann–Whitney U test for the variables of the two sample groups. The statistical
significance was determined using the Kruskal Wallis Test for the variables of the
three sample groups. P < 0.05 was considered statistically significant.
Supplementary Figure Legends
Fig.
S1.
(A)
Increased
SOX1
expression
dramatically
suppressed
anchorage-independent growth (AIG) of HepG2, Huh7 and SK-Hep-1 cells. The
representative photographs were taken after 4 weeks of incubation and the number of
colonies was measured. (B) Elevated SOX1 expression inhibited the invasion ability
of SK-Hep-1 and HA22T cells. A representative photograph of invasive cells on the
lower surface of the insert is shown.
Fig. S2. Tumors harvested on day 38 after implantation were used to verify the level
of SOX1 expression by Western blot. β-actin was used as a loading control.
Fig. S3. Time- and dose-dependent induction of SOX1 protein expression by
doxycycline. (A) DOX (0, 0.01, 0.1,1 and 10 μg/ml) was applied to cells for 48 hr. (B)
Hep3B cells were treated with DOX (1 μg/ml) for 0, 12, 24, 48 and 72 h. Cell lysates
were subjected to Western blot analysis. β-actin was used as a loading control.
Fig.
S4.
(A)
Inducible
SOX1
expression
dramatically
suppressed
anchorage-independent growth of HepG2, Huh7 and SK-Hep-1 cells. The
representative photographs were taken after 4 weeks of incubation and the numbers of
colonies were measured. (B) A Matrigel invasion assay was performed in SK-Hep-1
cells inductively expressing SOX1 by treatment with doxycycline 1 μg/ml for 3 days.
The cells were then placed in a Matrigel-coated Boyden chamber and allowed to
invade for 24 hr. A representative photograph of invasive cells on the lower surface of
the insert is shown.
Fig. S5. (A) The SOX1 protein levels expressed inductively by 1 μg/ml DOX for 7
days (DOX+) and then withdrawal of DOX for a further 7 days (DOX withdrawal)
were investigated by Western blot. (B) A representative photograph of the AIG assay
from cells treated as described in (A) is shown.
Fig. S6. GST pull-down assay. Purified GST only or GST-SOX1 proteins were
immobilized on a glutathione-sepharose column and mixed with lysate from Huh6
cells. After washing and elution, the remaining proteins were analyzed by Western
blot using anti-β-catenin and anti-GST antibodies.
Fig. S7. Truncated Flag-SOX1 suppresses β-catenin-mediated TCF/LEF signaling. (A)
Full-length Flag-SOX1 and truncated Flag-SOX1 as indicated were established and
shown by Western blot from COS7 cells. (B) Huh6, Hep3B and HepG2 cells were
transfected with Flag-tagged plasmids as indicated and the TOPFLASH reporter gene
and dual-luciferase activities were measured as described above. The data showed that
full-length Flag-SOX1, Flag-ΔN-SOX1 and Flag-ΔC-SOX1 could suppress
β-catenin-mediated TCF/LEF signaling with an efficiency comparable with that of
Flag-Vector, whereas Flag-C-SOX1 could not suppress it. The luciferase activity was
normalized to the Renilla luciferase activity. The results are presented as the mean ±
SE. Experiments were performed in triplicate. Significant differences are indicated by
asterisks; (*) for P < 0.05 and (**) for P < 0.01.
References
1. Lai HC, Lin YW, Huang TH, Yan P, Huang RL, Wang HC, Liu J, et al.
Identification of novel DNA methylation markers in cervical cancer. Int J Cancer
2008;123:161-167.
2. Su HY, Lai HC, Lin YW, Chou YC, Liu CY, Yu MH. An epigenetic marker panel
for screening and prognostic prediction of ovarian cancer. Int J Cancer
2009;124:387-393.
3. Shih YL, Hsieh CB, Lai HC, Yan MD, Hsieh TY, Chao YC, Lin YW. SFRP1
suppressed hepatoma cells growth through Wnt canonical signaling pathway. Int J
Cancer 2007;121:1028-1035.
4. Su HY, Lai HC, Lin YW, Liu CY, Chen CK, Chou YC, Lin SP, et al. Epigenetic
silencing of SFRP5 is related to malignant phenotype and chemoresistance of ovarian
cancer through Wnt signaling pathway. Int J Cancer 2010;127:555-567.
5.
Kan L, Israsena N, Zhang Z, Hu M, Zhao L-R, Jalali A, Sahni V, et al. Sox1 acts
through multiple independent pathways to promote neurogenesis. Developmental
Biology 2004;269:580-594.