Supplementary Methods
Retroviral Infection and Generation of Stable Cell Lines- To generate Smad3 expressing
retroviruses, Pheonix E packaging cells were plated at 106 cells/60-mm-diameter tissue culture
dishes and transfected with the LPCX retroviral vector (empty or containing Flag-WT Smad3) by
the calcium phosphate method. In the case of TRII expressing retrovirus, AVOSC packaging cells
were transfected with the MFG-CAT retroviral vector (empty or containing TRII). At 6 h posttransfection, the medium was replaced with fresh DMEM containing 10% FBS, and cells were
grown for an additional 24 h. The conditioned medium containing recombinant retroviruses was
collected and filtered through 0.45 m-pore-size polysulfonic filters. Samples of these
supernatants were applied immediately to SNU484 cells or SNU638 cells, which had been plated
18 h before infection at a density of 105 cells/60-mm-diameter tissue culture dishes. Polybrene
(Sigma, St. Louis, MO, USA) was added to a final concentration of 8 g/ml, and the supernatants
were incubated with the cells for 12 h. After infection, the cells were placed in fresh growth
medium and cultured as usual. Selections with 1 g of puromycin/ml or 400 g of neomycin/ml
were initiated 24 h after infection. After about 15 days, individual clones picked from plates of the
recombinant retrovirus-infected cells were transferred to 24 microtiter wells and expanded to
generate cell clones stably expressing Smad3 and TRII.
Immunohistochemical Staining- Tissues were fixed in 10% neutral buffered formalin, mounted in
paraffin, and 5 m sections were deparaffinized. Endogenous peroxidase was blocked with 3%
H2O2 in methanol for 30 min. Non-specific protein binding was blocked with a solution containing
1% bovine serum albumin (BSA) and 5% goat serum for 30 min. Sections were incubated for 2hr
with the primary antibodies anti-Smad3 (I-20; Santa Cruz, CA, USA), anti-CEA (A0115) in
TBS/1% BSA. Rabbit IgG at 4 g/ml was used as a negative control. Antigen-antibody complexes
were detected using the Vectastatin Elite avidin biotin complex (ABC) peroxidase kit from Vector
Laboratories (Burlingame, CA, USA) according to the manufacturer’s instructions. After a 30-min
incubation with ABC reagent, a 5-min reaction with 3,3’-diamino benzidine/H2O2 (BioGenex, San
Ramon, CA, USA) was used to detect the bound peroxidase. Slides were counterstained with
Carazzi haematoxylin.
Isolation of RNA and RT-PCR- Total RNA was isolated from human gastric cancer cells using
Trizol (Gibco BRL, Gaithersburg, MD, USA). RT-PCR was performed according to the
manufacturer’s instruction using a TITANIUMTMOne-Step RT-PCR kit (BD Biosciences Clontech,
Palo Alto, CA, USA). The primers used were as follows: CEA (CEACAM5) (sense/anti-sense), 5’GAAATGACACAGCAAGCTAC-3’/ 5’-GGACAGCTGCAGCCTGGGAC TGAC-3’ (product length:
496
bp),
CEACAM6
(sense/antisense),
5’-GTCACAGCAGCCCTGACCA
GAGC-3’/5’-
GTTGCTTCTTCATTCACAAGATCTG-3’ (product length: 481 bp), CEACAM8 (sense/antisense),
5’-GACAGCACAGCTGACAGCCGTGC-3’/
5’-CCAGTTGTAGCCACGAGGGTC-3’,
(product
length: 286 bp) and human glyceraldehydes-3-phosphate dehydrogenase (GAPDH) (sense/antisense), 5’-GCAGGGGGGAGCCAAAAGGG-3’/ 5’-TGCCAGCCCCAGC GTCAAAG-3’ (product
length: 566 bp). PCR was performed with a 20 l volume. The amplification cycle (denaturation
step at 94C for 30 sec, and annealing step at 65.0C for 30 sec, and an extension step at 68C for
1 min) was repeated 30 times and followed by a final extension for 2 minutes at 68C.
Northern Blot Analysis- Ploy(A)+ RNAs of human gastric cancer cells were isolated with Trizol.
RNA was electrophoresed on 1.0% agarose-formaldehyde gel and transferred to nylon membrane,
and cross-linked using UV Stratalinker (Stratagene, La Jolla, CA, USA). Blots were hybridized in
Rapid-hyb buffer (Amersham Pharmacia Biotech, Piscataway, NJ, USA) at 65C for 20 h.
32
P-
labeled CEACAM1, CEA and CEACAM6 probes, generously provided by Dr. C. P. Stanners
(McGill University, Canada) and 1.5-kb fragment of human TRII gene were prepared using
PRIME-IT random primer labeling kit (Stratagene).
Generation of mice and histological examination- Smad3ex8/ex8 mice were generated by
targeted disruption of the Smad3 gene by homologous recombination. Targeted embryonic stemcell clones were microinjected into C57BL/6 blastocysts to obtain germline transmission. Mice
heterozygous for the targeted distruption were intercrossed to produce homozygous offspring
(Yang et al., 1999). All animals were killed under deep CO2 anethesia at the 10th week from the
birth date and laparotomized with excision of their stomachs. The stomachs were opened along the
greater curvature and only the glandular stomach was harvested. Tissues were fixed in 10% neutral
formalin in PBS, processed by standard method, and embedded in paraffin. Tissues were sectioned
at 4 m for histological examinations.