Supplemental Information by pW9tEG9T

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									Supplemental Materials and Methods

  Screening of phage library. Serological screening of phages displaying peptides

recognized by malignant ascitic antibodies was performed using the Ph.D.-7 phage


display peptide system (New England BioLabs, Beverly, MA). A biopanning


procedure was carried out according to the manufacturer’s instructions with some


modifications. Briefly, after removal of binders recognized by a mixture containing


healthy control IgG, the remaining phages (about 1011 pfu) were incubated with


immobilized individual acetic IgG (10 μg/100μL per well) in 96-well plates for 1 h at


room temperature. Unbound phages were discarded by washing 10 times with TBST


solution [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween-20]. The bound


phages were eluted with an elution buffer [0.2 M Glycine-HCl (pH 2.2), 1 mg/mL


BSA] and propagated by infecting their host cells (Escherichia coli ER 2738 strain) at


37 °C with vigorous agitation for 4–5 h. After centrifugation, collection of the phages


in supernatant, resuspension of the phages in TBS buffer, and determination of the


phage titers were conducted according to the manufacturer’s instructions. This


biopanning procedure was repeated three more times to enrich the antibody-bound


phages. Fifteen blue plaques were randomly selected, amplified, and collected. The


single-chain phage DNA of the resultant cell pellets was purified and directly


sequenced to reveal the displayed peptides of the antibody-bound phages.


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  Immunohistochemistry (IHC). IHC was performed using a conventional method

as described previously (Chang et al., 2006). Formalin-fixed, paraffin-embedded


tissue samples were obtained from cooperating hospitals and stained with GST


control antibody or antibody specific to CA125 (Dako, Carpinteria, CA) or SIK3.


Briefly, the sections were serially dewaxed, rehydrated, and washed with PBS. After


treatment for 20 min with a heat-mediated antigen retrieval process in 10 mM sodium


citrate (pH 6.0), sections were rinsed three times in a wash buffer [10 mM Tris-HCl


(pH 7.4) and 150 mM sodium chloride] followed by treatment with 3% hydrogen


peroxide for 5 min to block endogenous peroxidases. Following PBS washes, the


samples were incubated with primary antibody (1:20 for CA125 or 1:4000 for SIK3


antiserum) for 1 h at room temperature or overnight at 4 °C. The bound primary


antibodies were detected using the LSAB kit (Dako), and slides were counterstained


with hematoxylin. The expression statuses of SIK3 and CA125 were independently


graded at a final magnification of 200× by at least two pathologists in a blinded


manner. Conflicting scores were resolved at a discussion microscope. Correlation


between the expression of CA125 and SIK3 and patient-related clinical variables was


determined using Fisher’s exact test.


  Northern blot analysis. Total RNA was extracted from SK-OV3, TOV-112D,

OC109, and OVCAR3 cells using the Qiagen RNA isolation kit (Qiagen, Hilden,


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Germany). Total RNA was resolved in a 1.2% agarose gel containing formaldehyde


and transferred to a Nytran nylon membrane (Whatman, Keene, NH). A 782 bp


amplicon containing the sequence coding for the PPHGYAH peptide was generated


by polymerase chain reaction (PCR) using SIK3-specific primers (Supplemental


Table 1), which served as the template for probe labeling. The 32P-labeled probe was


prepared using a random primer labeling system (Amersham Pharmacia Biotech,


Piscataway NJ) and hybridized with the membrane at 65 °C for 18 h. After three


washes under stringent conditions (Shih and Floyd-Smith, 1996), the membrane was


exposed to X-ray film (Fujifilm, Tokyo, Japan).


  Gene construction and antiserum generation. The FJ10213 plasmid (a gift

kindly provided by Dr. Nagase from Kazusa DNA Research Institute) served as a


template for the KIAA0999 gene constructs in this study. Based on its published gene


sequence (NCBI GenBank; access number: AB023216), we constructed the


Myc-tagged plasmid, pBS-myc-CT-0999, encoding a C-terminal KIAA0999 protein,


by cloning a PCR fragment containing nucleotides 2715–3773 inserted into


pBlueScript at an EcoRI site. Moreover, the plasmid, pBS-myc-CT-0999 del-7-mer,


which lacked the sequence encoding the PPHGYAH peptide, was also generated by


site-directed deletion mutagenesis using a pair of primers listed in Supplemental


Table 2. The gene constructs or pBlueScript control vector were transfected into HeLa


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cells using Lipofectamine (Invitrogen, Carlsbad, CA), and the corresponding protein


expression was driven by infection of the vaccinia-T7 viruses (Shih et al., 2001).


Recently, the KIAA0999 gene product was named as SIK3 that belongs to the


AMPK-related kinase (AMPK-RK) family (Katoh et al., 2004). To study the function


of SIK3 in cells, the plasmid pcDNA-myc-SIK3 was constructed by generating a


PCR fragment containing the nucleotides 441–4229 of FJ10213 inserted into the


pcDNA3.1 vector at an EcoRI site.


  To raise antiserum specific to SIK3, the PCR amplicon containing the nucleotides


3300–3773 was enzymatically digested, inserted into a pGEX-KG vector at 5′-Xba I


and 3′-Xho I sites, and expressed in Escherichia coli (JM109 strain) cells. The


GST-tagged CT-SIK3 recombinant protein was purified as previously described


(Chang et al., 2006) and served as an immunization antigen for rabbit. The resultant


antisera were purified with the Melon Gel IgG Purification System (Thermo Fisher,


Rockford, IL) according to the manufacturer’s instructions.


  Statistics. Sensitivity and specificity of SIK3 and CA125 to ovarian cancers were

calculated using SPSS software (Chicago, IL). Univariate analysis was performed


using Fisher’s exact test. The statistic differences between SIK3 transfectants and


their corresponding controls in tumor size, cell proliferation, and cell populations of

each cell cycle stage were determined by t-test analysis. “*”, “**”, and “***”


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indicate P < 0.05, P < 0.01 and P <0.001, respectively”. A P value < 0.05 was


considered statistically significant.




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Supplemental Figure Legends

Supplemental Figure 1.     The absence of CRBN expression in ovarian cancer.

Panel A, CA502 ascitic antibodies immunoreactive to the autologous tumor lysates.


The total lysates were prepared from normal-like ovarian epithelial cells, IOSE385,


and the tumor cells purified from the ascitic fluids of patient CA502 with


serous-subtype ovarian cancer using Ficoll-Plaque Plus and Percoll gradient


centrifugations. Panel B and C, Detection of CRBN expression in ovarian cancer cells


and tissues, respectively. The total lysates of CA502 ascitic tumor cells, IOSE385,


OVCAR3, and SK-OV3 cells were prepared. For Western blotting analysis, 80 μg of


each cell lysates were resolved in 7.5% or 10% SDS-containing polyacrylamide gel,


blotted on nitrocellulose membranes, and probed with protein A/G-purified CA502


ascitic antibodies (32 μg/mL) for Panel A, or with CRBN-specific antibody for Panel


B, respectively. The immunocomplexes were visualized with SuperSignal


chemiluminescence. A protein band with approximate molecular mass 150 kDa


recognized by CA502 ascitic antibodies in the autologous tumor lysates was indicated


with an arrow in Panel A. The commercially-available CRBN recombinant protein


fused with GST tag was loaded as a positive control in Panel B. β-Actin was used as


an internal control for both panels. For IHC study in Panel C, transverse serial


sections obtained from 38 ovarian cancer samples were stained with home-made SIK3


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or CRBN antibodies and visualized using the LSAB system. The CRBN


immunoreactivity did not be found in the ovarian tissues, either. One representative


pair of the serial sections of a sample stained for SIK3 [(a)] or CRBN [(b)] is shown


here. Magnifications, 100×. Scale bar, 100 μm.


  Supplemental Figure 2.       Association of SIK3 expression with cell growth.

Panel A, MTT assay. SK-OV3, TOV-112D, OVCAR3, and OC-109 cells (2 × 104 cells


per well) were seeded onto 24-well plates and cultured at 37 °C for 24, 48, 72, or 96 h,


as indicated. The cell number was determined by a standard MTT assay. Data are the


mean  SD of three experiments and represent as fold increases in cell growth of

individual cell lines at 24 h. Panel B, Northern blot analysis. Total RNA of the cells

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was extracted, resolved, and transferred to a nylon membrane. A           P-labeled probe


aligning the 3′-coding sequence of the SIK3 gene was generated using a random


primer labeling system. Its transcripts, with lengths of about 4.6 kb, were visualized


by autoradiography (arrow). The 28S ribosome RNA acted as a loading control. Panel


C, Western blot analysis. One hundred micrograms of cell lysates was prepared,


resolved in a 7.5% SDS-containing polyacrylamide gel, and probed with purified


anti-SIK3 antibody. The major immunoreactive protein is indicated with an arrow.


β-Actin was used as a loading control.


  Supplemental Figure 3.       Positive immunostaining for SIK3 and CA125 in


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adenomyosis. Transverse serial sections obtained from 20 adenomyosis samples were

examined immunohistochemically using antibody specific to SIK3 or CA125. The


immunocomplexes were detected and visualized using the LSAB system. Sections


from only two samples showed positive staining for SIK3 [(a)]. One representative


sample is demonstrated here. On the other hand, its serial tissue section was also


stained positive for CA125 [(b)]. Magnification, 200×. Scale bar, 50 μm.




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Supplemental References


Chang GC, Liu KJ, Hsieh CL, Hu TS, Charoenfuprasert S, Liu HK et al (2006).

Identification of alpha-enolase as an autoantigen in lung cancer: its overexpression is

associated with clinical outcomes. Clin Cancer Res 12: 5746-54.



Katoh Y, Takemori H, Horike N, Doi J, Muraoka M, Min L et al (2004).

Salt-inducible kinase (SIK) isoforms: their involvement in steroidogenesis and

adipogenesis. Mol Cell Endocrinol 217: 109-12.



Shih NY, Floyd-Smith G (1996). Protein kinase C-delta mRNA is down-regulated

transcriptionally and post-transcriptionally by 12-O-tetradecanoylphorbol-13-acetate.

J Biol Chem 271: 16040-6.



Shih NY, Li J, Cotran R, Mundel P, Miner JH, Shaw AS (2001). CD2AP localizes to

the slit diaphragm and binds to nephrin via a novel C-terminal domain. Am J Pathol

159: 2303-8.




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Supplemental Tables

Supplemental Table 1. Peptide sequences of phages bound by the CA502 ascitic


antibodies

           Peptide Sequence                      Putative Gene
 Q     P      H   H    Y    S   L        Rat CL3AA or CL3BA protein
 N     P      H   S    Y    P   H                      —
 Q     P     H    H    Y    P   L                   —
 S    P      H    H    Y    P   H                   —
 T    P      H    H    Y    M   H                   —
 Q    P      H    H    Y    F   K                   —
 W     P      H    H    F   P   H                   —
 V    P      H    G    Y    F   L      Murine RIKEN cDNA 0610007L05
 T    P      H    G    Y    A   H         Human KIAA0999 or SIK3
 V     P     H    S    Y    P   H                      —
 Q    P      H    H    Y    P   F                      —
 A    P      H    H    Y    P   M                      —
 L    A       I   N    I    K   S                      —
 V    P      H    S    Y    P   H                      —
 S    P      H    S    Y    P   R                      —
  x    P      H   x    Y    x   x             Consensus sequence

Note: “—“ represents no putative genes found in NCBI GenBank; “x” represents any

possible amino acid residues. Six out of seven displayed residues identical to the


protein sequences of known genes in the NCBI database are highlighted with grey


background.




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Supplemental Table 2. List of primers used for reverse transcriptase-polymerase


chain reactions, cloning, and site-directed deletion mutagenesis

Reaction       Gene      or        Sequence
               Construct
Gene Cloning
               Myc-CT-SIK3     S   5′-CGGGAATTCTCTCAGCAACATGCCAGGC-3′
                               AS 5′-CTGGGTACCTCAGTTGATTAGGGCAGA-3′
               SIK3            S: 5′-ATTGAATTCTCCCCGCCCGTATCGGCTAC-3′
                               AS 5′-TATGAATTCTTACACGCCTGCCTGCTC-3′
Mutagenesis
               Myc-CT-SIK3     S   5′-GCTTCCTCACCCACCCCGCAGCCGGCACTG
               △7-mer              ATGCAT-3′
                               AS 5′-ATGCATCAGTGCCGGCTGCGGGGTGGGTGA
                                   GGAAGC-3′
RT-PCR
               SIK3            S   5′-CCAGCAGCTACAGCCCTTCAA-3′
                               AS 5′-ACTGAATGCAGCAGTTGGCTGATGA-3′
               β-Actin         S   5′-GGTCACCCACACTGTGCCCATCTA-3′
                               AS 5′-GAAGCATTGCGGTGGACGATGGAG-3′

S, sense primer; AS, antisense primer




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