Supplemental Information by pW9tEG9T


									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.

  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,

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

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 “***”

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

considered statistically significant.

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

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

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

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.

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.

Supplemental Tables

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


           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


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

chain reactions, cloning, and site-directed deletion mutagenesis

Reaction       Gene      or        Sequence
Gene Cloning
                               AS 5′-CTGGGTACCTCAGTTGATTAGGGCAGA-3′
               SIK3            S: 5′-ATTGAATTCTCCCCGCCCGTATCGGCTAC-3′
                               AS 5′-TATGAATTCTTACACGCCTGCCTGCTC-3′
               △7-mer              ATGCAT-3′
                               AS 5′-ATGCATCAGTGCCGGCTGCGGGGTGGGTGA
               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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