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SUPPORTING DOCUMENTS (online only)
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SUPPLEMENTARY METHODS
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Method S1. The CCD966SK cells were cultured in minimum essential medium
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containing 10% fetal bovine serum, 1% non-essential amino acid, and 1 mM sodium
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pyruvate. Both NMFH-1 (Cancer Genet Cytogenet. 2005;161:28-35) and OH931
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(Cancer Genet Cytogenet. 1997;94:138-43) myxofibrosarcoma cell lines were
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cultured in RPMI 1640 supplemented with nutrients and antibiotics as previously
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described. All cultures were incubated at 37C in an atmosphere of 5% CO2 in air at
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100% humidity.
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Method S2. For immunoblotting analysis, equal amounts of total protein were
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extracted from cell lysates with RadioImmunoprecipitation Assay buffer (Cell
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Signaling), separated by 15% SDS-PAGE, and electroblotted into PVDF membrane
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(Millipore). The filters were probed with the polyclonal anti-p12CDK2AP1 (1:500; our
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previous study),19 anti-CDK2 (1:200, Santa Cruz), anti-CASP8 (1:1000; Cell
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Signaling), anti-CASP9 (1:1500; Cell Signaling) and monoclonal anti- actin (ACTB;
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1:3000; Chemicon; loading control) antibodies. Protein bands were detected by
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Chemiluminescent Reagent Plus kit (Perkin-Elmer) with horseradish
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peroxidase-labeled secondary antibody. The intensity of bands from immunoblotting
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assays was quantified by densitometry and normalized to that of ACTB in each lane.
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The active CASP3 was analyzed by using Caspase-3/CPP32 Fluorometric Assay Kit
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(K105-100, BioVision Inc).
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Method S3. CASP3 activity assay was performed using Caspase-3/CPP32
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Fluorometric Assay Kit (#K105-100, BioVision) according to the manufacture’s
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instruction. Briefly, 106 cells were transfected with pcDNA3.1/His (control) and
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pcDNA3.1/His-CDK2AP1 plasmids, respectively, for 48 h. Cells were resuspended in
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50 L of chilled Cell Lysis Buffer. After treatments with Reaction Buffer (2X) and
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DTT (10 mM final concentration), CASP3 substrate DEVD-AFC was added (50 M
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final concentration) and incubated at 37C for 2 h. The samples were then read in a
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Luminometer (EL800, BioTek) with 400-nm excitation and 505-nm emission. The
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resultant data were next normalized to the control to obtain relative
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7-amino-4-trifluoromethylcoumarin (AFC) activity.
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Method S4. The relative fold-change in mRNA was calculated by comparative
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threshold cycle (Ct) method through normalization to the housekeeping GAPDH
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transcript. Concentrations of primers were optimized to achieve equivalent PCR
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efficiencies among GAPDH and each target gene. Quantitative RT-PCR assay was
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performed in a total 15 μL of reaction mixture, including 7.5 μL of SYBR Green
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Mix (ABgene), 0.75 μL of forward and reverse primers, and 6 μL (12.5 ng/μL) of
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cDNA. The reaction was initiated by heat reactivation for the hot start polymerase at
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95C for 15 min, followed by 50 PCR cycles consisting of denaturation at 95°C for 10
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s, annealing at 60C for 5 s, and elongation at 72C for 12 s. The primers
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(concentration) used were CDK2AP1 (4 M): 5’-CAGGTGCCCCAAAGCAAAT-3’
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and 5’-CTGCGTACGTGGGTCTGATCT-3’; CDK2 (3 M):
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5’-GCTAGCAGACTTTGGACTAGCCAG-3’ and
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5’-AGCT.CGGTACCACAGGGTCA-3’; GAPDH (4 M):
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5’-GGTGGTCTCCTCTGACTTCAACA-3’ and
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5’-AGCTTGACAAAGTGGTCGTTGAG-3’. Relative expression folds of target
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mRNAs were calculated using the comparative cycle threshold (Ct) method. The fold
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change of target transcripts, after normalization to GAPDH, was then given by 2- ∆∆Ct,
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where ∆∆Ct = ∆Ct(target transcript)-∆Ct(GAPDH); ∆Ct represents the Ct after
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transfection subtracted from Ct before transfection.
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Method S5. The p12CDK2AP1-specific RNA interference from Taiwan National RNAi
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core facility, two clones [i.e., #4 (TRCN000002061) and #5 (TRCN000002062)] able
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to efficiently knockdown CDK2AP1 transcript and subsequent p12CDK2AP1 protein
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were identified and transfected into NMFH-1 cells for further incubation in culture
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medium for 72 h prior to subsequent analyses, with a non-specific clone targeting Luc
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gene (TRCN0000072243) serving as the control. Similar to transfection of the
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expression plasmids, 1 μg plasmid in 3 μL of TransFast reagent (Promega) was used
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for transfection of the shRNAi plasmids.
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Method S6. For apoptosis analysis, 5 x 105 NMFH-1 and OH931 cells were plated in
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60-mm Petri dishes for 24 h, harvested after transfection for 48 h, incubated with
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Annexin V-Fluorescein isothiocyanate (FITC) kit (Bender MedSystems) containing
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propidium iodide for 15 min, and then subjected to flow cytometric assays (IPICS
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XL-MCL, Beckman Coulter).
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Method S7. For trypan blue exclusion test, 5 x 105 NMFH-1 cells were plated for 24
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h, harvested after transfection of pEGFP (control) or pEGPF-CDK2AP1 for 24, 48
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and 72 h, respectively, and then resuspended in PBS (1 mL). An equal amount of cell
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suspension was added to 2X trypan blue stains (0.4%, Sigma-Aldrich) for 2 min and
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white cells were counted in a hemocytometer.
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Method S8. To attain a complete appraisal of the expression status of p12CDK2AP1
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protein, immunohistochemistry was applied to 102 cases arrayed in 4 TMA blocks.
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Among these, 80 cases included in 3 TMA blocks were immunostained for CDK2
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(1:200, #ab32147, abcam), active CASP3 (1:250, #1476-1, Epitomics) and Ki-67
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(1:100, #ab66155, abcam) to cross validate the functional roles of p12CDK2AP1 protein.
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ChemMate EnVision kit (DAKO) was used to detect immunoreactivity. Sections of a
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normal oral mucosal tissue known as reactive to p12CDK2AP1 were used as positive
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controls throughout. The immunolabeling index was then classified as (0 to < 5%), 1+
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(5 to < 25%), 2+ (25 to < 50%), 3+ (50 to < 75%), and 4+ (75% & above), 1+ and 2+
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are regarded as low, while 3+ and 4+ are high p12CDK2AP1 or CDK2
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immunoexpression. Since tumor cells seldom express active CASP3, 1+ staining and
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above were determined as high expression. The determination of Ki-67
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immunoexpression was based on our previous work.23