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 37C 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. 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