Supplementary Materials and Methods
Cell culture
All colorectal cancer cell lines were purchased from ATCC (Manassas, VA,
USA). Leptomycin B was purchased from Wako (Tokyo, Japan). Cells were grown at
37C and 5% CO2 in Iscove’s Modified Dulbecco’s Medium (IMDM) or RPMI 1640
(Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen) and 1%
penicillin-streptomycin (Invitrogen). Leptomycin B (Wako, Tokyo, Japan) was added to
the medium at a concentration of 200 nM, and cells were incubated for 3 h before
analysis.
Transient and stable transfection
Transient and stable transfection was performed using Lipofectamine 2000
(Invitrogen) according to the manufacturer’s instructions. Briefly, RKO or HCT116
cells were plated in 6-well plates in IMDM containing 10% FBS without antibiotics, 1
day before transfection, such that they were 70-90% confluent at the time of transfection.
On the day of transfection, 4 µg of each plasmid and 10 µL of Lipofectamine 2000 were
incubated separately in 250 µL of Opti-MEM I Reduced Serum Medium (Invitrogen).
After 5 min of incubation at room temperature, the diluted plasmids and Lipofectamine
2000 were combined and incubated for an additional 20 min at room temperature. The
DNA-Lipofectamine 2000 complexes were then added to each well, and the cells were
incubated for the indicated time at 37°C in a CO2 incubator.
For stable transfection, 5 × 104 cells transfected with annexin A2-FLAG
plasmid containing a geneticin resistance gene were transferred to 10-cm dishes 48 h
after transfection, and 600 µg/mL geneticin was added to the complete medium
containing IMDM, 10% FBS, and 1% penicillin-streptomycin. The complete medium
with geneticin was replaced every 4 days until geneticin-resistant colonies began to
appear. At least 30 clones were screened by immunoblotting and immunostaining with
anti-FLAG and annexin A2 antibodies to isolate annexin A2-FLAG -expressing clones.
RNA interference experiments
siRNA duplexes were purchased from Sigma-Aldrich (Santa Clara, CA,USA).
The target sequences of siRNA are listed in Supplementary Table 1. Control siRNA
used Mission Negative control SIC-001, confidential sequence (Sigma-Aldrich).
Transient siRNA transfection was carried out using Lipofectamine 2000 (Invitrogen)
according to the manufacturer’s instructions. Transfected cells were cultured for 72 h at
37C in a CO2 incubator.
Agarose 2D-DIGE
First, 50 µg of nuclear extracts from each CIN cell line (HT29, SW480, SW837,
CaCO2) and MIN cell line (HCT116, RKO, DLD-1, SW48) were labeled with 400
pmol of Cy5 or Cy3 (GE Healthcare UK Ltd., Buckinghamshire, England), respectively.
An internal standard created by pooling aliquots of all samples was labeled with Cy2.
Mixed labeled extract (50 µg each) was applied to the agarose 2D-DIGE gel. These
spots were detected and quantitated with DeCyder imaging analysis software, and then
statistical analysis was performed across the 8 gels. All of the samples were examined
in duplicate or triplicate. To identify proteins in each spot, 500 g of nonlabeled nuclear
extract was separated by conventional agarose 2-DE.
GeLC-MS analysis and protein identification
Gel lanes were excised from SDS-PAGE gels using razor blades and sliced into
5-mm slices. In-gel tryptic digestion of proteins was performed as follows. The gel
slices were cut in small pieces, destained in 50% acetonitrile/50 mM NH4HCO3, and
washed with deionized water. The gel pieces were dehydrated in 100% acetonitrile for
15 min and then dried in a SpeedVac evaporator (Wakenyaku, Kyoto, Japan) for 45 min.
The gel pieces were rehydrated in 10-30 l of 25 mM Tris-Cl (pH 9)/20% acetonitrile
containing 25 ng/l trypsin (Trypsin sequence grade; Roche, Basel, Switzerland) for 45
min. After removal of the unabsorbed solution, the gel pieces were incubated in 10-20
l of 50 mM Tris-Cl (pH 9)/20% acetonitrile for 20 h at 37°C. The solution containing
digested fragments of proteins was transferred to a new tube, and the peptide fragments
remaining in the gel also were extracted in 5% formic acid/50% acetonitrile for 20 min
at room temperature. Digested peptides equivalent to a maximum of 1 pmol of protein
were injected onto a trap column (0.3 × 5 mm L-trap column) connected to an analytical
column (0.1 × 150 mm L-column2) (Chemicals Evaluation and Research Institute,
Saitama, Japan), which was attached to a NanoSpace HPLC pump (Shiseido Fine
Chemicals, Tokyo, Japan) and a Magic 2002 splitter (AMR, Tokyo, Japan). The flow
rate of the mobile phase was 400 nL/min. The solvent composition of the mobile phase
was programmed to change in 120-min cycles with varying ratios of solvent A (2%
(v/v) CH3CN and 0.1% (v/v) HCOOH) to solvent B (90% (v/v) CH3CN and 0.1% (v/v)
HCOOH) in the following manner: 5-45.5% B over 95 min, 45.5-90% B over 4 min,
90% B for 0.5 min, 90-5% B over 0.5 min, 5% B for 20 min. Purified peptides were
introduced from HPLC to a LTQ XL (Thermo Scientific, San Jose, CA, USA). Peptide
mass data were matched by searching the Human International Protein Index database
(IPI, July 2009, 72 079 entries, European Bioinformatics Institute) using the MASCOT
engine.